On the role of treml6 in B-1a B cell development And ICOS co-stimulation in adaptive immune responses against M. tuberculosis [original]

On the role of

treml6 in B-1a B cell development

And

ICOS co-stimulation in adaptive immune responses

against M. tuberculosis

vorgelegt von

Diplom-Ingenieurin

Geraldine Nouailles

Von der Fakultät III - Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktor der Ingenieurwissenschaften

- Dr.-Ing. -

genehmigte Dissertation

Promotionsausschuss:

Vorsitzender: Prof. Dr. Ulf Stahl

Berichter: Prof. Dr. Roland Lauster

Berichter: Prof. Dr. Jens Kurreck

Berichter: Prof. Dr. Stefan Kaufmann

Tag der wissenschaftlichen Aussprache: 12.02.2010

Berlin 2010

D83

TABLE OF CONTENTS

1 INTRODUCTION ..................................................................................................................................................................... 6

1.1 Immune System ................................................................................................................................................................. 6

1.2 Innate Immunity and its receptors ............................................................................................................................ 6

1.2.1 Triggering receptors expressed on myeloid cells (TREM) receptor family................................. 7

1.2.2 Antigen presentation .......................................................................................................................................... 9

1.3 Adaptive Immunity .........................................................................................................................................................10

1.3.1 T cell activation and co-stimulation ...........................................................................................................10

1.3.2 Effector T cells .....................................................................................................................................................11

1.3.2.1 Role of ICOS co-stimulation in immune responses ......................................................................13

1.3.3 Development of peripheral B cell subsets ...............................................................................................14

1.4 Tuberculosis ......................................................................................................................................................................18

1.4.1 Epidemiology and disease ..............................................................................................................................18

1.4.2 Murine immune response to Mtb infection .............................................................................................20

2 AIMS OF THIS STUDY ........................................................................................................................................................23

3 MATERIALS AND METHODS ..........................................................................................................................................25

3.1 Mice .......................................................................................................................................................................................25

3.2 Materials .............................................................................................................................................................................25

3.3 Methods ...............................................................................................................................................................................25

3.3.1 Animal work .........................................................................................................................................................25

3.3.1.1 Breeding of mice ........................................................................................................................................25

3.3.1.2 Construction of gene-deficient mice ..................................................................................................25

3.3.1.3 Genotyping of treml6

+/+

(WT) and treml6

-/-

mice .........................................................................27

3.3.1.4 Infection of mice with Mtb .....................................................................................................................28

3.3.1.5 Tissue and organ isolation .....................................................................................................................29

3.3.1.6 Isolation of leukocytes from tissues and organs ..........................................................................30

3.3.1.7 Blood samples .............................................................................................................................................32

3.3.1.8 In vivo cytotoxicity assay .......................................................................................................................32

3.3.1.9 Irradiation of mice and cell transplantation ..................................................................................32

3.3.1.10 Bromodeoxyuridine (BrdU) in vivo proliferation assay .........................................................33

3.3.1.11 Thymus independent-type-2 (TI-2) immunizations with trinitrophenol (TNP)-Ficoll

..........................................................................................................................................................................................33

3.3.2 mRNA expression profile of treml6 ............................................................................................................33

3.3.2.1 Preparation of ribonucleic acid (RNA) from isolated tissues or cell suspensions .........33

3.3.2.2 Reverse transcription of RNA and qRT-PCR ..................................................................................34

3.3.2.3 RT-PCR analysis .........................................................................................................................................34

3.3.3 Flow cytometry ...................................................................................................................................................35

3.3.3.1 Standard staining protocol and MHCI tetramer staining ..........................................................35

3.3.3.2 Immunological characterization of immune cell types ..............................................................36

3.3.3.3 In vitro re-stimulation and intracellular cytokine staining (ICS)...........................................36

3.3.3.4 Fluorescence activated cell sorting (FACS) ....................................................................................37

3.3.3.5 In vitro B cell stimulation and proliferation assay .......................................................................38

3.3.3.6 Calcium (Ca

)- Signaling in B-2 and B-1a B cells.........................................................................38

3.3.3.7 Intranuclear BrdU staining ....................................................................................................................40

3.3.3.8 Measuring early apoptosis by AnnexinV stain ..............................................................................40

3.3.3.9 Flow cytometric data analysis ..............................................................................................................40

3.3.4 Cell culture ............................................................................................................................................................41

3.3.4.1 Cell culture of stromal cell lines and preB-I B cells .....................................................................41

3.3.5 Enzyme-linked immuno sorbent assay (ELISA) and Multiplex analysis.....................................42

3.3.5.1 TNP-specific ELISA....................................................................................................................................42

3.3.5.2 Multiplex analysis of antibody isotypes in sera ............................................................................42

4 RESULTS .................................................................................................................................................................................43

4.1 Generation and immunological analysis of treml6 deficient mice ..............................................................43

4.1.1 Treml6 protein product TLT-6 is a predicted ITIM-carrying receptor ........................................43

4.1.2 Treml6 mRNA expression pattern and initial immunological characterization of treml6

deficient mice ..................................................................................................................................................................45

4.1.3 Lack of treml6 reduces numbers of B-1 B cell precursors in BM and fetal liver ......................46

4.1.4 Treml6 deficiency results in B cell subset changes in peritoneal cavities ..................................48

4.1.5 Functional analysis of B-2 and B-1a B cells from treml6 deficient mice .....................................49

4.1.5.1 IL-4 evoked in vitro proliferation is enhanced in treml6 deficient mice ............................50

4.1.5.2 BCR-dependent Calcium-signaling is not affected by the lack of treml6 ............................51

4.1.5.3 Impact of treml6 deficiency on B cell maintenance and homing capacities ......................52

4.1.6 Immune responses of WT and treml6

-/-

mice to TI-2 antigens .......................................................54

4.1.7 Unimpaired reconstitution of Rag1

-/-

mice with treml6

-/-

BM .........................................................55

4.1.8 Concluding remarks on the role of treml6 in immunity .....................................................................57

4.2 Impact of ICOS on T cell responses and protection against Mtb infection ..............................................58

4.2.1 ICOS expression by CD4

and CD8

T cells during Mtb infection ...................................................58

4.2.2 Impact of ICOS deficiency on Mtb burden and pathology .................................................................60

4.2.3 Impact of ICOS deficiency on generation of CD4

Th1 responses during Mtb infection ......62

4.2.4 Impaired maintenance of Mtb-specific effector CD8

T cells in ICOS

-/-

mice ............................64

4.2.4.1 Reduced killing by PepA-specific CD8

T cell in ICOS

-/-

mice ..................................................66

4.2.5 Impaired CD8

effector memory maintenance during chronic Mtb infection in ICOS

-/-

mice

..............................................................................................................................................................................................67

4.2.6 Reduced frequencies and numbers of Treg in ICOS

-/-

mice during Mtb infection ...................68

4.2.7 Concluding remarks on the role of ICOS during murine Mtb infection .......................................69

5 DISCUSSION ...........................................................................................................................................................................71

5.1 Treml6 is a positive regulator of B-1a B cell development ............................................................................71

5.2 ICOS co-stimulation shapes T cell responses against Mtb .............................................................................79

6 SUMMARY ..............................................................................................................................................................................83

6.1 Treml6 regulates B-1a B cell development ...........................................................................................................83

6.2 ICOS co-stimulation shapes T cell responses against Mtb .............................................................................84

7 ZUSAMMENFASSUNG ........................................................................................................................................................85

7. 1 Treml6 reguliert die Entwicklung von B-1a B Zellen ......................................................................................85

7.2 Kostimulation durch ICOS prägt die T Zellantwort gegen Mtb ....................................................................86

8 REFERENCES ........................................................................................................................................................................87

9 ACKNOWLEDGEMENTS ...................................................................................................................................................98

10 ABBREVIATIONS ..............................................................................................................................................................99

APPENDIX 1: MATERIAL .................................................................................................................................................. 103

APPENDIX 1.1: Buffers and solutions ................................................................................................................ 103

APPENDIX 1.2: Media and cell culture reagents ........................................................................................... 104

APPENDIX 1.3: Reagents ......................................................................................................................................... 105

APPENDIX 1.4 Plastic ware .................................................................................................................................... 107

APPENDIX 1.5: Antibodies ..................................................................................................................................... 108

APPENDIX 1.6: Primers ........................................................................................................................................... 110

APPENDIX 1.7: Enzymes ......................................................................................................................................... 110

APPENDIX 1.8: Kits .................................................................................................................................................... 111

APPENDIX 1.9: Machines ........................................................................................................................................ 111

APPENDIX 1.10: Software....................................................................................................................................... 112

APPENDIX 1.11: Online programs and databases ........................................................................................ 112

APPENDIX 1.12: Suppliers...................................................................................................................................... 112

1 INTRODUCTION

1.1 Immune System

The mammalian immune system has evolved to ensure protection against various pathogens. It

is organized by an array of cells and molecules with specialized roles in the defense against

infection. All cells of the immune system develop from pluripotent stem cells in the bone

marrow (BM). Among these cells are lymphocyte precursors that mature to naïve cells in BM

and/or thymus, the generative lymphoid organs, and respond to antigen in the peripheral

secondary lymphoid organs such as spleen, lymph nodes, and mucosal and cutaneous lymphoid

tissues.

Pathogens entering the organism have to overcome the surface barriers of the immune system,

such as the epidermis and mucosa and are then confronted with two different types of immune

responses, the innate and the adaptive immune response. Innate immune responses occur to

the same extent regardless of how many times the infectious agent is encountered, whereas

acquired immune responses improve on repeated exposure to a given infection. Both immune

responses are tightly interconnected and function by cooperation (1-3).

1.2 Innate Immunity and its receptors

The innate immune system consists of all immune defense mechanisms that lack immunologic

memory and is phylogenetically ancient. Beside various soluble antimicrobial proteins of the

complement system, it comprises phagocytic cells such as monocytes, dendritic cells (DCs),

neutrophils and macrophages, cells that release antimicrobial and inflammatory mediators

(basophils, mast cells, and eosinophils), and cells that kill infected cells (natural killer cells). In

addition to antimicrobial effector mechanisms, some of the cells of the innate immune system

also play a crucial role in antigen presentation, thereby linking the innate and the adaptive

immune response (4).

In contrast to the adaptive immune response (for details see 1.3), the innate immune response

is mediated by germ-line encoded receptors e.g. pattern recognition receptors (PRR), with

genetically predetermined specificity. The best-known class of innate immune receptors are the

toll-like receptors (TLRs). Other classes include the nucleotide oligomerization domain (NOD)-

like receptors (or recently defined as nucleotide-binding domain and leucine-rich repeat

containing molecules [NLRs]), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs) (5,

6). Although more limited in their diversity, compared to adaptive immune receptors, cells

equipped with PRR are able to respond faster to various microbial products, be they bacterial,

viral, fungal, or parasitic (7). The replication of the invading pathogen can thus be controlled

within hours. The recognized pathogen-associated molecular patterns share three aspects: a)

they are produced only by pathogens, and not by the host, b) the structures are usually

essential for the survival or pathogenicity of the microorganism, and c) they are usually

invariant structures shared by entire classes of pathogens (e.g. lipopolysaccharides (LPS) shared

by virtually all gram-negative bacteria). Recognition of these structures by the innate immune

system induces expression of costimulatory molecules, secretion of cytokines and chemokines,

which recruit other cells of the innate immune system and activate antigen-specific

lymphocytes and initiate the adaptive immune response (8, 9).

1.2.1 Triggering receptors expressed on myeloid cells (TREM) receptor family

TREM receptors belong to a surface receptor family of NON-TLR innate immune receptors that

participate in immunological processes. The TREM gene cluster is localized on human

chromosome 6p21 and murine chromosome 17C3; it comprises the gene sequences of e.g.

TREM1 (gene: trem1), TREM2 (gene: trem2), TREM-like transcript- (TLT-)1 (gene: trem-like

(treml)1) and, TLT-2 (gene: treml2) all of which are conserved between mice and men. Genes

encoding TREM or TREM-like (TREML) receptors have been identified in several species, such as

chicken, pig, and cow (10-12). In these species, the number of trem and trem-like genes is

weakly conserved, and for some trem and trem-like genes, it is not possible to define orthologs

between mice and men (10-12). Murine trem5, pdc-trem, trem3 and treml6 have no human

ortholog (Fig. 1) (10-12).

Structurally TREM and TREML receptors share an extracellular, variable (V-type)

immunoglobulin (Ig) domain and a transmembrane region. TREM receptors have a short

cytoplasmic tail and couple with DNAX activating protein of 12kDa (DAP-12) that carries an

immunoreceptor tyrosine-based activating (ITAM) motif (green in Fig. 1). TREML or TLT

receptors have a longer cytoplasmic tail that contains, except for treml2 (grey in Fig. 1), one or

more immunoreceptor tyrosine-based inhibitory (ITIM) motifs (red in Fig. 1) (12).

FIGURE 1. TREM family of

receptors. Localization of TREM

genes on mouse chromosome 17C

(left) and human chromosome

6p21.1 (right). Genes shown in

green are either known or predicted

to associate with DAP12. Those

shown in red contain an ITIM

motif(s). Treml2, shown in gray,

neither associates with DAP12 nor

has an ITIM. Where known, TREM

expression is indicated. pDC

(plasmacytoid dendritic cells); PMN

(polymorphonuclear leukocytes); M

(monocytes); Mφ (macrophages);

BMDC (bone marrow-derived

dendritic cells); B (B lymphocytes);

DC (dendritic cells); iDC (immature

dendritic cells); T (T lymphocytes);

Mi (microglia); OC (osteoclasts);

Plts (platelets); Meg

(megakaryocytes). Ligand identity

or lack thereof is listed in

parentheses underneath the cell

type(s) expressing TREM. Diagram

is representative of TREM

chromosomal location but is not

drawn to scale. C and T define the

centromeric and telomeric ends of

the chromosome, respectively.

Figure and legend taken from Ford

and McVicar (12)

ITAM containing receptors induce cell activation through protein tyrosine kinase dependent

signaling pathways. Ligand binding induces a phosphorylation cascade that results in cell

activating processes such as actin polymerization, calcium mobilization and activation of

transcription factors. Inhibitory receptors, e.g. ITIM-containing receptors (ITIM-Rs), attenuate

such activation signals (13). Inhibitory receptors mediate this function normally only upon their

clustering with an activating counterpart on the cell surface. When both activating and

inhibitory receptors are coengaged by their respective ligands, the net outcome is determined

by the relative strength of these opposing signals. In general, inhibition exerted by ITIM-Rs is

only local and transient. It does not induce a cell-wide or sustained non-responsiveness but

abrogates activation signals when and where they occur (13).

TREM receptors are expressed on a variety of cells, including monocytes, macrophages, DCs, T

cells, B cells, microglia, thrombocytes, and osteoclasts (Fig. 1). The functions of TREM receptors

are well described for a few members only and have mainly been studied by in vitro or blocking

antibody experiments. TREM1 was the first member to be described; it functions through the

ITAM-domain bearing adaptor molecule DAP12 as an amplifier of TLR signaling (14, 15). TREM2

mice are so far the only published knock-out animals within the family (16). TREM2 is primarily

described as an attenuator of macrophage activation and cytokine secretion in response to TLR

ligands LPS, zymosan, and CpG (16, 17). More recent data suggest an additional role in negative

regulation of autoimmunity (12). PDC-TREM is expressed on murine plasmacytoid DCs (pDCs)

upon stimulation with CpG and enhances type 1 interferon production (18). TLT-1 and TLT-2 also

promote cellular activation, although neither couples to DAP12 for signaling. TLT-1 inhibits

platelet aggregation in vitro and enhances FcεRI-mediated calcium signaling in platelets (19, 20).

TLT-2 is expressed on peritoneal and alveolar macrophages, and neutrophils and TLT-2 is the

only TREM receptor that is expressed on lymphocytes (CD8

T cells, activated CD4

T cells and B

cells). It plays a role in leukocyte activation and might confer co-stimulation to T cells by binding

to B7-H3. Notably, TLT-2 does not carry ITIM motifs, but contains a src homology 3 (SH3)-

binding domain (21). No report has investigated functions of treml6, the subject of this thesis.

1.2.2 Antigen presentation

Antigen presenting cells (APCs) are capable of antigen processing, presentation and T cell

activation. DCs have long been regarded to be the most potent APCs due to their enhanced

ability to initiate T cell responses. Nevertheless, B cells and macrophages are also capable of

antigen presentation and evidence accumulates that doubts the unique role of DCs versus

macrophages in the initiation of T cell responses (22). Moreover, recent studies showed that

basophils also act as APCs and are able to initiate Th2 type immune responses (23-25).

Regardless which cell acts as the APC, for an antigen to be presented, proteins first have to be

fragmented by proteolysis. The resulting peptides are then associated with major

histocompatibility complex (MHC) molecules and the MHC-peptide complexes are expressed at

the cell surface where they can be recognized by the T cell receptor (TCR) on a T cell. However,

the pathway leading to the association of peptides with MHC molecules differs for MHC class I

(MHCI) and MHC class II (MHCII) molecules. MHCI molecules present degradation products

derived from cytosolic (endogenous) proteins and present those to CD8

T cells. MHCII

molecules present fragments derived from extracellular (exogenous) proteins that are located in

an intracellular compartment and present those to CD4

T cells (26, 27).

1.3 Adaptive Immunity

Defense against microbes is mediated by early reactions of the innate immune system and

subsequently by the responses of the adaptive immune system. Adaptive immunity is

characterized by specificity for distinct molecular entities and the ability to “remember” and

thus to respond faster and more vigorously upon repeated exposure to the same pathogen.

It is based on the activation and clonal expansion of B and T lymphocytes following antigen

recognition through the TCR or B cell receptor (BCR) (2, 3). The lymphocyte receptors are

responsible for the great diversity and specificity of antigen recognition. In contrast to the

receptors of the innate immune system, they are not genetically predetermined but are

generated through somatic rearrangement of germ-line encoded gene segments. This

rearrangement results in 10

to 10

possible BCRs and TCRs even though the human genome

consists of only 75,000 to 100,000 genes (28).

The adaptive immune response can be divided into humoral and cell-mediated immunity. The

first is accomplished by B cells and helper T cells type 2 (Th2 cells) and is directed against

extracellular bacteria, viruses, parasites and their toxins. Cell-mediated immunity is mediated by

helper T cells type 1 (Th1 cells) directed against viruses and intracellular bacteria, e.g.

Mycobacterium tuberculosis (Mtb) (2, 3, 29). B lymphocytes are antibody-producing cells. After

recognizing extracellular antigens through the BCR, a membrane-bound form of an antibody, B

cells differentiate into antibody-secreting effector cells, called plasma cells. A subset of the

activated B cells differentiates into memory B cells (2, 3).

1.3.1 T cell activation and co-stimulation

The classical activation of naïve T lymphocytes is the result of a two-cell interaction between the

T cell and an APC during which the T cell receives two signals from the APC. Signal one is the

recognition of antigenic peptides presented by MHC molecules by the TCR (for details see 1.2.2).

Signal two is e.g. provided by the triggering of CD28 on the T cell by CD80 (B7-1) and CD86 (B7-

2) molecules on the APC (30). Notably, CD28 is only one member of the CD28 family of co-

stimulatory molecules (30, 31). The latter family consists of several members which differ with

regard to their expression profiles, their ligands, and consequently their immunological

functions. CD28-coreceptors bind to their respective ligands of the diverse B7-family of

molecules and either amplify, such as CD28 and ICOS, or hinder, such as PD-1 and CTLA-4,

signals of the TCR complex (30-34). Together with distinct cytokines that provide signal three

these signals can lead to the activation and proliferation of the T cells resulting in the generation

of antigen specific effector T cells that act during the acute phase of infection. In addition

activation of lymphocytes also leads to the generation of memory cells. In contrast to effector

cells, memory cells require fewer signals for activation and proliferation and therefore act faster

upon re-infection. Memory T cells can be divided into central memory T cells (T

), which reside

preferentially inside secondary lymphoid organs and have the ability to proliferate upon

activation to become effector cells, and effector memory T cells (T

), which reside within the

periphery and quickly produce effector cytokines upon TCR trigger without having the ability to

proliferate (35, 36).

1.3.2 Effector T cells

The differentiation of CD4

effector T cells initiated by the interaction with APCs in the presence

of cytokines secreted from pathogen-activated cells of the innate immune system is a hallmark

of adaptive immunity. Dependent on the cytokine milieu in which the APC T-cell interaction

takes place, CD4

effector T cells can differentiate into different types of effector cells, such as

regulatory T cells (Treg), Th1 cells, Th2 cells, and helper T cells type 17 (Th17 cells). These

lineages differ in their cytokine expression profile and immune-regulatory functions (29).

The generation of Th1 effector cells is coupled to the sequential actions of interleukin-12 (IL-12)

produced by innate immune cells and interferon-gamma (IFN-γ) secreted by NK cells and

possibly the binding of Notch receptor ligands of the DLL family. Transcription factors involved

in the polarization towards the Th1 cell differentiation are signal transducer and activator of

transcription (STAT) 4, STAT1 and T box transcription factor (T-bet) (37, 38). The differentiated

Th1 cells are characterized by their production of IFN-γ and are involved in cellular immunity

against intracellular pathogens. They exert their effector function through IFN-γ which induces

the classical activation of macrophages, comprising the up-regulation of MHC-II, secretion of

pro-inflammatory cytokines IL-6, tumor necrosis factor (TNF), and IL-1, as well as nitric oxide

(NO) production and respiratory burst, and thereby they improve the killing of intracellular

microbes by macrophages (39, 40). CD4

Th1 cells also provide help to CD8

T cells, by

promoting both their activation through APCs and their proliferation (41-43).

The development of Th2 cells depends on the presence of IL-4, possibly as well on the binding of

Notch receptor ligands of the Jagged family, and the sequential activation of STAT6 and GATA3

transcription factors (38, 44). Th2 cells produce IL-4, IL-5 and IL-13 and are required for humoral

immunity to control parasites and other extracellular pathogens. Their main effector function is

to provide B cell help. The secretion of typical type 2 cytokines such as IL-4, IL-5 and IL-13

promotes antibody secretion and antibody class switching by B cells (40, 45). The antibodies

secreted into the blood and body fluids by B cell can in turn target extracellular pathogens. Th2

type response can also induce alternative activation of macrophages, including upregulation of

mannose receptor, up-regulation of MHCII, and expression of arginase. Alternatively activated

macrophages contribute to clearance, presentation of antigens and parasite-induced granuloma

formation in Th2 type immunity (39, 40).

Recently, Th17 cells have been described as a distinct lineage of CD4

effector T cells

characterised by IL-17A and IL-17F production and the expression of the transcription factor

retinoic acid-related orphan receptor γ expressed in T cells (RORγt) (46-48). Their development

from naïve CD4

T cells depends on CD28/ICOS co-stimulation (48) and is driven by transforming

growth factor-beta (TGF-β) and IL-6 (49, 50). Although initially thought to be the inducing

cytokine, it is now evident that IL-23 is necessary for long-term survival of Th17 cells (47, 50).

The role Th17 cells play during infection is not yet fully clarified. Mangan et al. showed that

Th17 cells are required for host protection against the extracellular bacterium Citrobacter

rodentium. However, Th17 cells were discovered in the context of autoimmune diseases, where

they induce tissue inflammation through IL-17 secretion (48, 51). In particular, IL-17 has been

linked to neutrophil recruitment into tissue sites through the induction of granulocyte colony-

stimulating factor and IL-8 (52, 53). Therefore, Th17 cells are thought to regulate tissue

inflammation (54).

In contrast, Treg inhibit autoimmunity and protect against tissue injury. The developmental

pathways of Treg and Th17 cells have been described to be reciprocal. The peripheral

development of Treg depends on TGF-β while the presence of IL-6 inhibits the differentiation

into Treg (49, 50). Treg can be classified into two major populations, naturally occurring Treg

(nTreg) and induced Treg (iTreg). Both express the forkhead-box transcription factor P3 (FoxP3).

The nTreg mature in the thymus while the iTreg derive from CD4

T cells that acquired their

suppressive activity upon TCR stimulation in the periphery (55, 56). Initially characterized by

their ability to control autoimmune disease in mice (57), Treg have now been shown to

influence immune responses in a wide variety of microbial infections (58-60), e.g. Leishmania

major (61), Listeria monocytogenes (62), Plasmodium falciparum (63) and Mtb (64-66).

Noteworthy, the differentiation of naïve CD4

T cells into the different effector lineages is no

longer considered to be necessarily irreversible. With the discovery of Treg and Th17 cells and

the investigation of their development, data accumulates that suggest that at least iTreg and

Th17 remain more plastic than expected, and that under specific circumstances they can

acquire another effector phenotype (67).

The signals that stimulate differentiation of naïve CD8

T cells into CD8

effector T cells (also

named cytotoxic T lymphocytes (CTLs)) are antigens presented by MHCI, co-stimulatory

molecules, and Th cells secreting IFN-γ and IL-2. Activated CTLs are effector T cells that

recognize and kill target cells expressing MHCI-antigen complexes, e.g. infected host cells

containing bacteria or viruses. The killing occurs through release of granules with perforin and

granzymes. Cytotoxic T lymphocytes (CTL), like CD4

T cells of Th1 type (CD4

Th1 cells), are

capable of secreting cytokines, predominantly IFN-γ, and TNF-α (36).

1.3.2.1 Role of ICOS co-stimulation in immune responses

A prominent member of the CD28 receptor family and object of this thesis is ICOS (33). ICOS

expression

is restricted to activated T cells, and ICOS binds to ICOS-ligand (ICOS-L) (33, 68-70) a

member of the B7 protein

family. ICOS-L is highly expressed

on professional APCs; however,

endothelial and epithelial cells have also been shown to express ICOS-L (71, 72). In vitro, ICOS

co-stimulation increases T cell

proliferation, and production of both Th1 and Th2 type cytokines,

but it only induces marginal IL-2 secretion (33, 68). T cells from ICOS-deficient

mice exhibit

reduced IL-4 and IL-5 production, but normal or

increased IFN-γ production (73-75).

Consequently the biological role of ICOS was studied in Th1 and Th2 type autoimmune and

infection models (76-84). During infection with Listeria monocytogenes, the inhibition of ICOS

signaling impaired both listeria-specific CD4

, as well as CD8

T cell responses, and resulted in

higher susceptibility of mice (76). A reduction of CD4

T cell responses was observed after

infection of mice with other intracellular pathogens (e.g. Leishmania mexicana, Nippostrongylus

brasiliensis, Toxoplasma gondii and vesicular stomatitis virus (77, 79, 81)). In contrast, genital

tract infection with Chlamydia trachomatis resulted in an increased CD4

Th1 response in ICOS

-/-

mice (78). Although phenotypes varied with the specific model employed, in general, the

absence or blockage of ICOS led to either unaffected or reduced effector CD4

or CD8

T cell

responses (76-84). Moreover, a recent study observed that ICOS regulates the expansion and

pool size of effector T cells, T

, and regulatory T cells (Treg) in steady state (85). These results

suggest a biological role of ICOS as a co-stimulatory molecule that confers survival signals to a

variety of T cells (85).

1.3.3 Development of peripheral B cell subsets

In the mouse, B cells are generated from pluripotent hematopoietic stem cells (HSCs) in the liver

during mid-to-late phase fetal development and in the bone marrow after birth. Different

nomenclatures for the developmental stages of B cells exist and the two major ones shall be

introduced here, namely those of Hardy (86) and Melchers-Rolink (87). The classifications of the

differentiation stages are based on cell surface markers and recombination events. Both

classifications use CD45R/B220 to identify B-lineage cells, and IgM and IgD to identify immature

and mature B cells. CD19 was described later and then integrated into these classifications. It

was found to be the most specific marker of B cell commitment because it is regulated by the B-

cell exclusive transcription factor Pax5 (88). Melchers-Rolink proposed the expression of the

cytokine receptors CD25 and c-kit (CD117) to subdivide B cell precursors.

The following description refers to the stages of B-2 B cell development from HSCs in adult BM

(Melchers-Rolink nomenclature). Common lymphoid progenitors (CLPs) emerge from HSCs.

They still retain some T-cell developmental potential, but they express recombination-activating

gene (Rag) proteins. Their immediate downstream progeny are referred to as pro-B cells in

which immunoglobulin heavy chain rearrangements have begun. Pro-B cells in turn generate

pre-B1 cells. These two populations express B220 and c-kit, but differ in CD19 expression: pro-

B cells (CD19

) and pre-B1 cells (CD19

). Pre-B1 cells in which immunoglobulin heavy chain gene

rearrangement was successful mature into pre-B2 cells that express µ heavy chain protein in

their cytoplasm. CD25 is a specific marker for pre-B2 cells within the B lineage (87). Following

the productive rearrangements of immunoglobulin light chain genes and the expression of light

chain proteins, pre-B 2 cells mature into B-2 cells distinguished by the expression of surface IgM.

In this thesis developing B cells were named and identified according to the Melchers-Rolink

classification, pro-B (B220

CD19

c-Kit

CD25

), pre-B1 (B220

CD19

c-Kit

dim

CD25

), pre-B2

(B220

CD19

c-Kit

CD25

), immature (CD19

IgM(µ-chain)

kappa-light-chain

IgD

) and mature B

cells (CD19

IgM(µ chain)

kappa-light-chain

IgD

). The pro-B cells in this thesis correspond to

Hardy fraction A, and some of Hardy fraction B. Pre-B1 cells correspond to Hardy fraction B and

C (pro-B). Pre-B2 cells correspond to Hardy fraction C’ and D (large and small pre-B cells).

Immature and mature B cells correspond to Hardy fractions E and F, respectively (Fig. 2).

FIGURE 2. Nomenclature of stages in B cell development and expression of differentiation markers according

to Melchers-Rolink classification and corresponding fractions and nomenclature by Hardy classification.

Marker expression is indicated by lines.

Immature B cells that have escaped negative selection leave the bone marrow and migrate as

transitional B cells to secondary lymphoid organs. Only a minority of transitional B cells will

survive and be selected into one of the three long-lived mature B cell subsets, namely B-1,

follicular (FO) or marginal zone (MZ) B cells (89). FO and MZ B cells are the most abundant B cell

populations found in the spleen and are also referred to as B-2 B cells.

FO B cells are re-circulating small resting cells with an average life span in excess of several

months (90). Naïve FO B cells reside in the ‘follicular niche’ of spleen, lymph nodes or Peyer’s

Patches. They generate Thymus-dependent immune response to protein antigens and are

mediators of adaptive immunity. TLR ligation induces the proliferation of FO B cells, but in

contrast to MZ and B-1 B cells they lack the intrinsic ability to differentiate into antibody-

secreting cells if stimulated only by TLR ligands (91).

MZ B cells are a rather sessile population, in mice they preferentially reside in the vicinity of the

marginal sinus. They have a lower threshold for TLR activation and respond without priming to

blood-borne pathogens. MZ B cells bear BCR biased toward bacterial components (92, 93). In

addition to their important role in Thymus-independent (TI) immune responses, MZ B cells may

also participate in Thymus-dependent immune responses to protein antigen, as well as in

responses to lipid antigens (94).

The main habitats of murine B-1 B cells are pleural and peritoneal cavities, but B-1 B cells can

also be found at lower numbers in the spleen and intestine. B-1 B cells are a persistent, self-

renewing population. They have a less diverse immunoglobulin repertoire than B-2 B cells and

their BCR specificities are biased toward microbial components, as well as self-antigens (95). B-1

B cells can be further subdivided into B-1a and B-1b B cells, which show functional differences

during the immune response. B-1a B cells participate in innate immune responses by

spontaneously secreting IgM and thereby providing a first line of defense against certain

encapsulated bacteria, such as Streptococcus pneumoniae, whereas antibody production by B-

1b B cells is induced and has a role in the ultimate clearance of the pathogen and in providing

long-term protection (96).

The mechanisms of selection of newly formed B cells into B-1, FO or MZ B cell compartments

are not completely unraveled yet as well as the developmental differences between B-2 and B-1

B cells. Two main models are under debate, the ‘lineage model’ was proposed by Hayakawa,

Hardy, and Herzenberg and claims that the B-1 phenotype is genetically predetermined (97, 98).

This model was based on the observation that B-1 B cells possess unique features, such as the

disposition to express self-reactive autoantibodies, their self-replenishment capacity, the

expression of CD5 and their preferential occurrence in pleural and peritoneal cavities that

distinguish them phenotypically and functionally from B-2 B cells. In addition, cell transfer

studies showed that fetal liver progenitors are more effective in giving rise to B-1 B cells than

bone marrow progenitors in irradiated recipient mice (86, 99). It was thus concluded that two

types of B cell developmental pathways exist, one being fetal and generating B-1 B cells and one

being adult and generating B-2 B cells (97).

The ‘induced differentiation model’ was propagated first by Wortis and Clarke and suggests that

all B cells are derived from a common progenitor called B-0 cell. In their model the instruction in

the B-1 lineage is driven by the encounter of B-0 cells with naturally occurring TI-2-like antigens

and depends on BCR signaling strength (100, 101). This model was based on the observation

that B-1 phenotype - CD5 expression - can be induced in B-2 B cells after BCR cross-linking with

αIgM in the presence of IL-6, notably this conversion was independent of innate antigen, T cell

help, and CD40L (102). Further experimental evidence for the importance of BCR specificity for

B-1 B cells fate came from different studies showing that transgenic mice expressing genes for

autoantibodies generate preferentially B-1 B cells, e.g. mice expressing transgenic

autoantibodies specific for red blood cells (103) and transgenic mice expressing

antiphosphatidylcholine autoantibodies (104-106). The correlation that BCR signaling per se is

necessary for B-1 B cell development came from studies with mice that carried mutations or

transgenes inhibiting BCR signaling strength, these mice had reduced numbers of B-1 B cells

(107-109), while mice carrying mutations or transgenes that enhanced BCR signaling, had

increased numbers of B-1 B cells (110-115).

Obviously, both models aim to convert certain arguments brought by the other camp. Followers

of the ‘induced differentiation model’ claim that the different capacities of fetal versus adult

precursors to generate B-1 B cells simply origin in differences of the BCR repertoire in the fetal

or adult mice, the fetal BCR repertoire is skewed towards TI-2 specificities, while the adult BCR

repertoire seldom generates such specificities (100). Supporters of the ‘lineage model’ suggest

that the observed differences in the BCR repertoire between mature B-1 and B-2 B cells are

caused by antigen-driven positive selection for survival of B-1 B cells and not by the acquisition

of B-1 B cell phenotype by B-0 cells with such BCR specificities (116).

FIGURE 3. Unified model for B cell development adapted from Hardy (95) and Casola (117)

The recent identification of a B-1 B cell progenitor (B-1P) in fetal and adult bone marrow and

studies showing that cytokine requirements for B-1 B cell and B-2 B cell development differ,

provided new evidence for the lineage model (118, 119). In an attempt to combine all findings

of the past years two recent reviews proposed a ‘unified model’ that comprises both, distinct B-

1 and B-2 progenitors and different requirements of immature B cells in BCR signaling strength

to be positively selected into the mature B cell pool (95, 117). In this model BCR signal strength

is not driving the lineage type of the B cells, but influencing the positive or negative selection of

the immature B cells into the different B cell subsets, strong BCR signal drives the cells in the B-1

lineage, while cells receiving weak BCR signals become B-2 B cells. Intermediate BCR signals

favor the development of B cells along the MZ fate (117).

1.4 Tuberculosis

1.4.1 Epidemiology and disease

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is one of the most

threatening human pathogens worldwide with the highest prevalence in developing countries

(Fig. 4). It was first described on March 24, 1882 by Robert Koch who received the Nobel Prize in

physiology or medicine for the discovery in 1905 (120). The bacterium is a slowly growing

obligate aerobe. Mycobacterium species are classified as acid-fast bacteria due to their cell wall

impermeability to certain dyes. The composition of its cell wall is dominated by mycolic acids

that make up more than 50% of its dry weight (121). The genome of Mtb was sequenced in

1998. It has a size of 4.41Mb and consists of about 4000 protein-coding genes of which 52%

have a known function (122, 123).

FIGURE 4. Territories are sized in proportion to the absolute number of people who died from tuberculosis in

The World Health Organization (WHO) estimates that up to one third of the world population is

infected with Mtb. Annually 9 million people develop TB of whom 2 million die (124-126).

However, morbidity and mortality figures are only one facet of TB. Ninety percent of infected

individuals will never develop active TB disease, indicating that the human immune system

controls Mtb infection effectively, even though sterile eradication of Mtb is normally not

achieved.

Infection with Mtb generally occurs via inhalation of Mtb-containing droplets expelled by a

patient with pulmonary TB. The lung is the typical site of infection although all organs can be

affected. Within the lung alveolar space, bacilli are engulfed by alveolar macrophages and

transported into the lung parenchyma where they are contained by tissue macrophages within

granulomatous lesions (125, 127-129). A chronic infection develops and a balance between

pathogen replication and immune response is maintained, with the inherent risk of reactivation

at a later times (124, 125, 127). Notably, the risk of reactivation especially upon immune

suppression, e.g. during human immunodeficiency virus (HIV) infection increases 100-fold (128,

130).

The Bacille Calmette-Guérin (BCG) vaccine only protects from severe forms of pediatric TB, but

not from pulmonary TB in adults. Moreover, the available therapeutic options are poor.

Antimycobacterial drug treatment is lengthy and expensive leading to poor regimen

compliance. Another alarming observation is the rise of multi-drug resistant strains e.g. the

genotype family Beijiing/W and extensively drug-resistant (XDR) TB. For these strains, the

treatment costs are 100-fold higher if drug treatment is possible at all (129, 131).

1.4.2 Murine immune response to Mtb infection

Mouse models aided profoundly to understand the immune response against Mtb. To mimic the

natural infection route mice are usually infected with low doses (100-200 colony forming units

(CFU)) Mtb via aerosol.

After aerosol infection the invading bacilli are phagocytosed by alveolar macrophages and

probably interstitial DCs. Phagocytosed mycobacteria are able to block their delivery to the

lysosome and can therefore escape lysosomal destruction (132). The surviving bacteria are then

either transported to the draining lymph nodes where antigen-specific T cells are primed or

reach deeper lung tissues where they reside within macrophages. Infected macrophages initiate

an inflammatory response that results in the recruitment of different cells to the sites of

infection, beginning with monocytes, neutrophils, DCs, and later lymphocytes (133).

Notably, Mtb can first be detected in lung draining lymph nodes at 8 to 9 days post infection

(p.i.) (134, 135) and only then T cell activation occurs (134, 136). The mechanisms that restrict a

faster migration of infected DCs to the lymph nodes are incompletely understood. In

consequence, murine T cell response following a low-dose Mtb aerosol infection are relatively

slow, when compared with responses to viral or other bacterial infections (127). By day 9 p.i. T

cell priming occurs in the draining lymph nodes and IL-12 secretion by activated macrophages

and DCs drives the generation of Th1 type effector cells (137, 138). Since Mtb resides in

endosomes, antigen-specific T cell priming in the draining lymph node is mainly MHCII

restricted. However, MHCI priming also occurs. It is likely that crosspriming plays a role in CD8

T cell activation. Mycobacteria induce apoptosis in macrophages causing the release of

apoptotic vesicles that carry mycobacterial antigens to APC which in turn present them through

CD1 (glycolipids) or MHCI (peptides) (139).

FIGURE 5. Scheme of classical granuloma structures and proposed local immune responses; reprinted from

Ulrichs & Kaufmann (137)

Activated effector Th1 CD4

and CD8

T cells reach the lung by 3 weeks p.i.. T cells, notably CD4

Th1 cells, dominate in protective immunity against Mtb (125, 127-129). Upon activation, CD4

cells secrete multiple cytokines including IFN-γ and TNF-α, which in turn activate anti-

mycobacterial mechanisms in mononuclear phagocytes (124, 125, 127). Besides CD4

T cells,

other T cell subsets, such as CD8

T cells, gamma delta (γδ) T cells, CD1-restricted T cells and

Treg, participate in the immune response against Mtb and consequently influence disease

outcome (64, 66, 124, 127, 140-142).

Only with the arrival of effector Th1 CD4

and CD8

T cells, the so far steady growth of

mycobacteria in lungs and spleen of infected mice will be halted. Subsequent to this acute

infection phase, a chronic phase ensues where bacterial numbers remain stable at a level of

approximately 10

CFU in C57BL/6 mice for many months until progressive pathology results in

death.

During the chronic phase of infection structures resembling human tuberculous granuloma form

in the lungs of infected mice. As for humans, murine ‘granuloma’ are thought to ensure the

containment of mycobacteria and to focus the immune response to the site of infection. The

morphology of human solid granuloma is characterized by a necrotic center surrounded by

concentric layers of macrophages, epitheloid cells, multinucleated Langerhans giant cells, and

lymphocytes (Fig. 5). In contrast, murine ‘granuloma’ consist of loosely aggregated cells and

usually lack necrosis. Development of the granuloma is mediated by chemokines and cytokines

produced by local tissue cells and infiltrating leukocytes. CD4

T cells and macrophages produce

TNF-α and lymphotoxin alpha 3, which are required for the formation of the wall surrounding

the granuloma (121, 129, 130, 137, 138, 143)(Fig. 5).

2 AIMS OF THIS STUDY

This thesis has two independent aims. The first aim is to gain knowledge about the function of a

so far uncharacterized receptor that was predicted to be part of the innate immune system,

namely murine TLT-6 (gene: treml6), an ITIM-containing receptor of the TREM protein family.

The second aim is to investigate the role of ICOS, a well described co-stimulatory molecule

known to participate in adaptive immune responses, in the context of murine Mtb infection.

Immunological characterization of treml6 via the analysis of treml6

-/-

mice

TREM receptors form a newly identified family of NON-TLR surface receptors (12). Some of their

members have already been characterized, while the functions of others are still undefined (12).

TREM are of great interest in infection immunology since most characterized members have

been described to participate in inflammatory immune responses by either amplifying or

dampening TLR-derived signals (14, 17, 18, 144). In this context, treml6 was predicted to carry

an ITIM motif and was therefore proposed to be of inhibitory nature (12). Since nothing was

known about treml6, the first aim was to determine the expression profile of treml6 in WT mice.

The second aim was to phenotype our in-house generated treml6

-/-

mice with the focus on the

immune system. As the result of an initial analysis, I discovered that treml6 influences the

frequencies of B-1a B cells and their precursors. B-1 B cells were discovered in the early 1980’s

(145-147), and since then both their role in the immune system and their development have

been under debate (86, 148-150). Since treml6 appeared to be an important player in this

context, I further expanded the aim of this project and investigated the impact of treml6 on the

development, function and, maintenance of B cells.

Analysis of the influence of ICOS signaling on T cell responses against Mtb

The World Health Organization (WHO) estimates that up to one third of the world population is

infected with Mtb and annually 9 million people develop TB of whom 2 million die (124-126).

These numbers make Mtb, the causative agent of TB, one of the top three microbial killers

(126). The primary goal of this research was to expand the current knowledge about the host

response during Mtb infection. Protective immunity to tuberculosis depends on CD4

Th1 cells

in humans and in mice (151-154), but the adaptive immune response fails to eradicate Mtb and

to achieve sterile immunity. Nevertheless, a prerequisite for successful vaccine or drug design is

the understanding of this immune response and especially its weaknesses.

In the search of putative immune modulators, I analyzed Th1 responses against Mtb during my

diploma project and observed that ICOS expression correlated with Th1 effector phenotype. In

line with our observation, other groups reported that the amount of IFN-γ secretion by T cells

from patients with active TB disease correlates with their ICOS surface expression, and that ICOS

signaling can increase IFN-γ secretion (155). Moreover, Urdahl and colleagues showed that

pulmonary Treg express elevated levels of ICOS during murine Mtb infection (66). These data

pointed to ICOS as an important player in the formation of any kind of T cell response against

Mtb. Therefore, I aimed clarifying the role of ICOS co-stimulation in the generation and

maintenance of T cell responses against Mtb.

3 MATERIALS AND METHODS

3.1 Mice

Abbreviation Full strain Designation Notes

wild-type (B6 or WT) C57BL/6NCrl Charles River Laboratories

ICOS

C57BL/6.ICOS

Rag1

C57BL/6J-Irf2

tm1

Rag1

tm1

Treml6

C57BL/6.treml6

WT C57BL/6.treml6

+/+

3.2 Materials

See APPENDIX 1: MATERIALS

3.3 Methods

3.3.1 Animal work

3.3.1.1 Breeding of mice

Mice were bred and maintained under special pathogen free conditions with a 12 hour light

cycle; food and water were provided ad libidum. B6 mice were purchased from Charles River

Labs (Germany). ICOS

-/-

mice were a gift from Andreas Hutloff (RKI, Berlin). Rag1

-/-

, treml6

-/-

and

wild-type (WT) control mice were bred at the Max Planck Institute for Infection Biology animal

breeding facility (Berlin-Marienfelde, Germany). Infected mice were kept in a biosafety level 3

facility and non-infected ones in a biosafety level 2 facility. Mice were sacrificed by cervical

dislocation. The experiments were conducted according to the German animal protection law.

3.3.1.2 Construction of gene-deficient mice

Treml6 deficient mice were generated by Dr. M. Kursar in the laboratory of Bernard Malissen at

the Centre d'Immunologie de Marseille-Luminy, France. In the targeting construct depicted in

Fig. 6A, a deoxyribonucleic acid (DNA) fragment that includes exon 1 (starting from the ATG

translational start codon), 2, 3 and 4 of the treml6 gene was replaced by a beta-galactosidase

(lacZ) gene followed by a self-excising ACN-cassette (156) bearing a locus of X-ing over (loxP)-

sequence flanked neomycin resistant gene and a testes-specific promoter from the angiotensin-

converting enzyme gene (tACE)-promotor driven cre-gene. Briefly, the C57BL/6 BAC-DNA clone

RP23-32N6 – containing TREM-genes trem1, trem3, treml4, treml2, treml6, trem2, and treml1-

was purchased from RZPD Deutsches Ressourcenzentrum für Genomforschung GmbH. BAC-DNA

was used as template to poly chain reaction (PCR)-amplify a 5`-homology arm (HA-5`) and a 3`-

homology arm (HA-3`) using primers P1 (5’-ttggcgcgccgggaacagcctgtagctattga-3’) and P2 (5’-

acgcgtcgaccactggggagagcaggtatg-3’), and P3 (5’-acgcgtcgacgaggagaccactgtaagtaaaaatgac-3’) and

P4 (5’-ataagaatgcggccgctaaactattatttctgtgtaaaacataaggcagga-3’). In addition to sequence

homologous to the BAC-DNA, P1 contains a AscI-site, P2 and P3 a SalI-site, and P4 a NotI-site

(bold sequences). The lacZ-ACN sequence was excised by SalI digestion from vector pTK-lacZ-

Neo-Cre, which was engineered and kindly provided by Dr. Markus Koch. HA-5`, lacZ-ACN, and

HA-3` were cloned into the thymidine-kinase (TK) containing vector pTK_MK (engineered and

kindly provided by Dr. Markus Koch). The resulting targeting vector was ScaI-linearized and

electroporated into Bruce4 mouse embryonic stem (ES) cells (origin B6). Colonies resistant both

to G418 (300 mg/mL) and to gancyclovir (2 mM) were screened by PCR for homologous

recombination using primers P5 (5’-tgcctggttttctccttactt-3’) and P6 (5’-

aatgggataggttacgttggtgtag-3’) – for homologous recombination of HA-5`, and P7 (5’-

atcgatgagttgcttcaaaaatc-3’) and P8 (5’-tccttctccttctccttcttgtt-3’) – for homologous

recombination of HA-3`. One recombinant ES clone (M1_E1) was found positive for both

screening PCRs (Fig. 6B).

Production of Mutant Mice Mutant embryonic stem cells (ES cells) were injected into (white)

B6 blastocysts and were capable of germline transmission. The ACN cassette was self-excised

during male germline transmission.

FIGURE 6. Generation of treml6

-/-

mice. A, Cloning strategy. B, Screening PCRs of 5’ and 3’ homology arms

on Bruce4-B6 (WT) ES cells and mutated ES clone M1_E1. C, Mouse genotyping; WT PCR (primer P11 and P12

in Fig. 6A), LacZ PCR (primer P9 and P10 in Fig. 6A). In white, tail DNA from WT mice (WT), treml6

+/-

(HET),

and treml6

-/-

(knockout (KO)) mice.

3.3.1.3 Genotyping of treml6

+/+

(WT) and treml6

-/-

mice

Genomic DNA isolation from tail biopsies Five mm tail biopsies were kept in 1.5-ml

microcentrifuge tubes at -20°C until genomic DNA isolation. Tails were lysed overnight with 500

µl tail buffer supplemented with 0.2 mg/ml Proteinase K under shaking at 55°C and 900 rpm.

Next day samples were centrifuged for 10 min at 20,000x g and 4°C. Supernatants contained

DNA, and were collected in fresh 1.5-ml microcentrifuge tubes. DNA was precipitated by adding

500 µl isopropyl alcohol, shaking of the mixture and centrifugation for 10 min at 20,000x g and

4°C. Supernatants were discarded and DNA pellets washed with 500 µl 70% ethanol. To re-

sediment the DNA pellets, they were centrifuged for 5 min at 20,000x g and 4°C. Ethanol

supernatants were removed and DNA pellets were allowed to dry before resuspending them in

500 µl double distilled (dd) H

O. To improve dissolving, DNA-water samples were shaked for 15

min at 60°C and 600 rpm.

PCR amplification and analysis of wildtype and LacZ gene PCR products PCRs were performed

on tail DNA amplifying either the wild-type gene (WT PCR product ~400 bp; with primer B4 Tail

WT fw1 (P11) and B4 Tail WT rev1 (P12)) or the inserted LacZ gene (KO PCR product ~500 bp;

primer TP_B4_KO-sHA_ fw (P9) and TP_B4_KO-lacZ-rv (P10)). PCR mix contained 5 µl tail DNA,

16.9 µl dH

O, 0.2 µl dNTP’s (10 mM each), 0.1 µl forward primer (100 pmol/µl), 0.1 µl reverse

primer (100 pmol/µl), 0.2 µl Taq polymerase and 2.5 µl BioTherm buffer (10x). PCR cycles

comprised 3 min of initial denaturation at 94°C, followed by 30 cycles of 1) 40 sec at 94°C, 2) 40

sec at 60°C and, 3) 1 min at 72°C and a final elongation step for 5 min at 72°C. PCR samples were

stored at 4°C. 5 µl of DNA loading buffer were added to 25 µl PCR products and samples were

run for 40 min on a 1% agarose gel. Gels were photographed with a UV-light photometer and

resulting bands were analyzed to determine the genotype (Fig. 6C).

3.3.1.4 Infection of mice with Mtb

Mtb strain H37Rv NY was grown in Middlebrook 7H9-broth at 37°C with shaking until bacterial

growth reached an OD

600

=0.7, corresponding to an approximate cell density of 10

cells/ml.

These mid-logarithmic cultures were harvested by centrifugation, washed with 1x phosphate

buffered saline (PBS), re-suspended in 10% glycerol, aliquoted and stored at -80°C until use. All

mycobacteria stocks were titrated prior to use by plating serial dilutions onto 7H11-agar plates

and counting the CFU after three weeks of incubation at 37

C. Aliquots were homogenized prior

to use by repeated transfer through a syringe with a 26G needle. Infection of mice was

performed using a Glas-Col inhalation exposure system. An aliquot of frozen Mtb stock culture

was thawed and diluted (as determined in titration experiments performed for every infection

stock) with water. The next day, five mice were sacrificed, their lungs removed, homogenized

and plated onto 7H11-agar to verify the initial infection dose.

Determination of bacterial titers Mice were sacrificed at distinct time points after infection

by cervical dislocation. Lungs and spleens were transferred into sterile sample bags containing 1

ml PBS/Tween solution (PBST). For experiments in which T cell analysis was performed

simultaneously with CFU determination, the small lower right lung lobe was used for CFU and

estimated to represent 1/5 of the total lung for CFU calculations. Similarly, half of the spleen

was used for CFU determination. Organs were homogenized in the sample bags by smashing

and after serial dilution in PBST, 50 µl were plated on 7H11-agar plates containing ampicillin and

cyclohexamide which were sealed with PARAFILM® and wrapped in aluminum foil. After 3-4

weeks of incubation at 37°C, CFU were counted. Statistical significance was determined using

the Mann-Whitney test.

Histology Tissues were fixed in 4% paraformaldehyde (PFA) in 1x PBS for 24 h at 4°C,

transferred into 1x PBS and stored at 4°C until embedding. Histology samples were dehydrated,

embedded in paraffin blocks and allowed to harden. Five μm sections were cut, mounted on

glass slides and dried overnight at 37°C. Sections were re-hydrated and stained with

Hematoxylin and Eosin Y (H&E). Re-hydration was performed as follows: 2x 10 min xylene, 2x

10 min 95% ethanol, 2x 10 min 80% ethanol, 10 min 70% ethanol and finally 10 min de-ionized

water. For H&E staining, slides were incubated for 1 min in concentrated Hematoxylin solution,

dipped in acid ethanol (1% HCL in 95% ethanol), rinsed with tap water for 10 min, rinsed with

de-ionized water, and followed by Eosin Y staining for 20 sec. Slides were subsequently rinsed

with water and dehydrated for 3 min in 95% ethanol followed by 2 min in xylene. Samples were

mounted using cover slip slides and Mercor glass and were photographed at 2.5, 10, 20, and 40x

magnification. For experiments in which T cell analysis was performed simultaneously with

histological analysis, the entire large left lobe of lungs was used.

3.3.1.5 Tissue and organ isolation

Peritoneal Cavity Peritoneal cavities were flushed twice with each 5 ml 1x PBS using a 1.20

x 40 mm needle and 10-ml syringe, the resulting cell suspension was collected in 15-ml tubes.

Mesenteric lymph nodes (MLN) Abdomen was opened with an incision made with surgical

tweezers. MLN were removed carefully from mesenteric tissues and collected in 5 ml PBS/BSA

(bovine serum albumin) solution in a 15-ml tube.

Spleen A small incision at the left of the peritoneal wall was made with surgical scissors.

The spleen was grasped with tweezers and pulled free of the peritoneum and the connective

tissues. It was placed in 5 ml PBS/BSA solution in a 15-ml tube.

Liver Livers were removed from the abdomen by disconnecting them from the diaphragm.

They were placed in 5 ml PBS/BSA in 50-ml tubes.

Peyer’s Patches The small intestine was isolated from the peritoneal cavity and carefully

searched for white-shimmering Peyer’s Patches. Peyer’s Patches were removed from the small

intestine with surgical scissors and tweezers.

Lungs Lungs were isolated by an incision in the chest, beginning at the xiphoid and extending

to the neck with surgical scissors. The ribs were cracked left and right of the ribcage and lifted.

To diminish the numbers of blood lymphocytes lungs were perfused through the right heart

ventricle with cold 1x PBS. Excised lungs were placed in 5 ml PBS/BSA in 50-ml tubes.

Thymus Once lungs were removed, thymi could be isolated by grasping them with

tweezers.

BM To isolate BM, first the skin of mice was removed around the leg and ankles. Then flesh

and tissues were cut away from femur and tibia. Femur was cut above the hip joint and tibia at

foot joint. Femur and tibia were separated and cut open on both ends. BM was extracted by

flushing the bones with 20 ml complete Roswell Park Memorial Institute (cRPMI) media using a

0.55-mm needle and 20-ml syringe. BM cell suspensions were collected in 50-ml tubes.

Bones Remaining bones after BM isolation were used as bone samples.

3.3.1.6 Isolation of leukocytes from tissues and organs

Lung Individual lungs were placed in Petri dishes and cut into 5 mm sized pieces with scissors.

Typically, the right lung lobes were used for lymphocyte analysis. Lungs from 1-2 mice were

pooled in order to obtain enough cells. Freshly prepared Collagenase medium (10 ml) was

added to the diced lungs and incubated for 30 min at 37°C, 7% carbon dioxide (CO

). The

digested lung pieces were pressed through iron mesh sieves into the lids of the Petri dishes

using plungers of 10-ml syringes. The cell suspensions were collected in 50-ml tubes. The sieves

and Petri dishes were washed twice with 10 ml PBS/BSA added to the 50-ml tubes and

centrifuged for 6min in a Heraeus centrifuge at 400x g. The pellets were re-suspended in 10 ml

40% Percoll/RPMI solution at room temperature (RT). Five ml of this cell suspension were

layered over 3 ml 70% Percoll/RPMI in a 15-ml tube. The resulting gradients were centrifuged

for 25 min at 600x g without brake at RT. The cells from the interface were transferred to new

50-ml tubes and washed with 30 ml RPMI. The pellets were re-suspended in red blood cell lysis

buffer and incubated for 2 min to remove remaining erythrocytes. After an additional washing

with PBS/BSA, cells were filtered through 70-μm filters and re-suspended in RPMI complete

media and kept on ice.

Spleen, MLN and thymus Freshly removed organs were placed in 5 ml PBS/BSA

solution in 15-ml tubes. Normally, half of the spleen was used for lymphocyte analysis. Spleens

from 2 mice were pooled, in order to be consistent with lung cell preparations. Spleens or MLN

or thymus were pressed through 70-µm filters with plungers of 10-ml syringes into Petri dishes.

The suspensions were collected in 15-ml tubes. The filters and Petri dishes were washed with 10

ml PBS/BSA solution and the washes were added to the 15-ml tubes. Next the suspensions were

centrifuged for 6 min in a Heraeus centrifuge at 400x g. To remove erythrocytes, the pellets

were re-suspended in 2 ml red blood cell lysis buffer and incubated for 2-3 min with occasional

shaking. 10 ml PBS/BSA solution were added and the tubes were centrifuged. The resulting

pellets were re-suspended in RPMI-10 complete medium and filtered through 70-μm filters.

Peritoneal Cavity Cell suspensions were centrifuged for 6 min in a Heraeus centrifuge at

400x g. Pellets contaminated with erythrocytes were excluded from analysis. Pellets were then

re-suspended in RPMI-10 complete medium and filtered through 70-μm filters.

BM Tibia and femur from dissected mice were cut and flushed with RPMI-10

complete medium to remove leukocytes from the bones. Cell suspensions were collected in 50-

ml tubes. To remove erythrocytes, the pellets were re-suspended in 2 ml red blood cell lysis

buffer and incubated for 2-3 min with occasional shaking. 10 ml PBS/BSA solution were added

and the tubes were centrifuged. The resulting pellets were re-suspended in RPMI-10 complete

medium and filtered through 70-μm filters.

The viability of the cells was determined by Trypan blue exclusion. Twenty μl of each cell

suspension were diluted in 180 μl 1x Trypan blue solution and counted in a haemocytometer.

Cell numbers were calculated with the following formula: cell number from 16 quadrants x

chamber factor (10

) x dilution factor x volume of cell suspension = total cell number. Cells were

re-suspended in RPMI complete medium at 10-40x 10

cells/ml.

3.3.1.7 Blood samples

Blood samples were taken from the tail vein. The blood was directly collected in serum

separator tubes, let stand for a minimum of 30 min at RT, followed by centrifugation at 12,000x

g for 3 min and finally the serum was stored at -20°C until analysis.

3.3.1.8 In vivo cytotoxicity assay

Splenocytes target cell suspensions from naive B6 mice were prepared as described above and

evenly

split into two fractions. One fraction was pulsed with 10

-4

M Mtb32A-derived peptide

(PepA: GAPINSATAM) for 1 h at 37°C and then labeled with a high

concentration (2 µg/ml) of

Carboxy-fluorescein diacetate succinimidyl ester (CFSE) (CFSE

high

population), and

the other

fraction was incubated for 1 h at 37°C with 10

-5

M listeriolysin

91-99

(LLO

91-99

: GYKDGNEYI) control

peptide and labeled with a low concentration (0.2 µg/ml) of

CFSE (CFSE

low

population). CFSE

low

and CFSE

high

-labeled

cells (2x 10

cells in total) were mixed at a 1:1 ratio and adoptively

transferred in 200 µl 1x PBS into Mtb-infected B6 and ICOS

-/-

mice via tail vein injection.

Twenty

hours later, recipient spleen cells were analyzed by

flow cytometry. Percent specific lysis was

determined by loss of the

peptide-pulsed CFSE

high

population compared to the control CFSE

low

population in infected mice relative to loss of the

peptide-pulsed CFSE

high

population compared

to the control CFSE

low

population in naïve mice using the formula (1 – (AVG r

naive

infected

) x 100),

r: % CFSE

low

/ % CFSE

high

3.3.1.9 Irradiation of mice and cell transplantation

Rag1

-/-

recipient mice were sublethally γ-irradiated (400 rad) 24 h before transplantation. For

transplantation either donor mouse BM cells, or in vitro cultivated pre-B I cells kept on IL-

7/stromal cells (OP9) were used. The harvested cells were washed twice in cell culture-tested 1x

PBS and were concentrated to 5x 10

cells/ml. The remaining cell clumps were removed by

straining the cell suspension through a MACS® Pre-Separation Filter with 30-µm mesh. For

intravenous (i.v.) transplantation mice were warmed by infrared irradiation for vasodilatation of

the tail veins. 100 µl with 5x 10

cells were injected into the lateral tail vein. The condition of

transplanted mice was controlled regularly.

3.3.1.10 Bromodeoxyuridine (BrdU) in vivo proliferation assay

Mice received drinking water supplemented with 0.8 mg/ml BrdU and 1% (wt/vl) Sucrose for 4,

8 or 12 days. BrdU drinking water was protected from light with aluminum foil. At day 4, 8 or 12

of treatment mice were sacrificed and in vivo B cell proliferation was analyzed as described in

3.3.3.7.

3.3.1.11 Thymus independent-type-2 (TI-2) immunizations with trinitrophenol

(TNP)-Ficoll

Mice were immunized with TNP-Ficoll by intraperitoneal (i.p.) injection of 10µg TNP-Ficoll in

100µl 1x PBS. At days 0, 5, 8 and 14 serum samples were taken and analyzed for TNP-specific

antibodies via enzyme-linked immuno sorbent assay (ELISA).

3.3.2 mRNA expression profile of treml6

3.3.2.1 Preparation of ribonucleic acid (RNA) from isolated tissues or cell

suspensions

Tissue isolation B6 mice were sacrificed and tissues (BM, bones, spleen, MLN, peritoneal

cavity, Peyer’s patches, thymus, lung, liver, fetal liver) were removed. Fetal liver was obtained

by mating the mice overnight and intercepting the pregnancy at day 18. RNA was isolated by the

Trizol Reagent RNA preparation method. Briefly, about 100 mg tissue was homogenized in 1 ml

of Trizol Reagent with an Ultra Turrax T8 tissue homogenizer. Cell samples were collected in 1.5-

ml microcentrifuge tubes in 1 ml Trizol and homogenized by pipeting. Trizol samples were

stored at -80°C until RNA extraction.

RNA extraction RNA was extracted as recommended by the manufacturer. Homogenized

tissue samples were centrifuged at 500x g for 5 min to remove insoluble material. The cleared

homogenate solution was transferred into a fresh 1.5-ml microcentrifuge tube. All samples were

incubated for 5 min at RT, before 0.2 ml chloroform were added and mixed vigorously for 15

sec. Following 2-3 min incubation at RT, the suspensions were centrifuged for 20 min at 14,000x

g and 4°C to allow phase separation. The upper colorless phase, containing the RNA, was

removed and transferred into fresh microcentrifuge tubes. To precipitate the RNA 0.5 ml

isopropyl alcohol were added to the samples, mixed and incubated for 10 min at RT. After 20

min centrifugation at 14,000x g and 4°C the RNA precipitate formed gel-like pellets.

Supernatants were removed and pellets were washed with 1 ml 75% ethanol, vortexed and

centrifuged for 5 min at 7,500x g at 4°C. Again, supernatants were removed and pellets were

allowed to dry briefly before resuspending them in 20 µl RNase free water. Purified RNA

samples were stored at -80°C.

3.3.2.2 Reverse transcription of RNA and qRT-PCR

Reverse transcription of purified RNA 10 µl of the initially purified RNA samples

(maximum 5 µg) were used for reverse transcription. For that 1 µl random hexamer primers

(200 µg/ml) were added, the mixture was incubated for 10 min at 65°C and then placed on ice.

After 5 min on ice, a reaction mix containing 4 µl 5x first strand buffer, 1 µl 10 mM dNTPs and 2

µl 0.1 M DTT were added and after 10 min incubation at RT 1 µl of superscript reverse

transcriptase was added. This mixture was immediately incubated for 50 min at 42°C and finally

incubated for 15 min at 70°C in order to inactivate the reverse transcriptase and to stop the

reaction.

qRT-PCR on complementary DNA (cDNA) To compare the amount of cDNA used in each

reaction, beta-actin (β-actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) primers

were included. PCRs were either run in standard mode for 40 cycles with 20 sec 95°C and 60 sec

60°C in the ABI Prism 7000 Sequence Detection System (Applied Biosystems) using ABI PRISM

optical 96-well plates (Applied Biosystems) or in FAST mode for 40 cycles with 1 sec 95°C and 15

sec 60°C with MicroAmp™ Fast Optical 96-Well Reaction Plates (Applied Biosystems). To control

specificity of primers, in both standard and FAST mode, dissociation stages with 15 sec 95°C, 15

sec 60°c and 15 sec 95°C were added to the runs.

3.3.2.3 RT-PCR analysis

Amplifying primers were designed to span exon-exon junctions (EEJ) to avoid amplification of

genomic DNA and to generate products of 100 – 300 bp size. Reaction mixtures were set up in

30 µl final volume using 15 pmol of each primer, 5 µl template cDNA and 15 µl 2x (Fast) SYBR-

Green PCR Master mix (Applied Biosystems). Quantifications were performed at least twice with

independent cDNA samples and in triplicates for each cDNA and primer pair. Data analysis was

performed using the ABI Prism 7000 SDS Software, REST-MCS© beta (Pfaffl &Horgan) and

Microsoft Excel. The threshold cycle (c

) was determined for each sample and fold differences

relative to the expression level in one of the analyzed cDNA samples or a virtual value (for

treml6 expression in naïve organs or cells) was calculated for each cDNA sample and primer pair

(fold-difference=2

-∆Ct

). Differences in amount of transcribed cDNA were normalized to the

expression of housekeeping genes gapdh and β-actin.

3.3.3 Flow cytometry

Single and multicolour stainings were analysed on a LSR-II flow cytometer equipped with a (L1)

argon laser (488 nm), a (L2) Helium neon laser (633 nm), a (L3) UV laser (355 nm) and a (L4)

violet laser (405 nm) or in the biosafety level 3 facility on a FACS Canto II equipped with a (L1)

argon laser (488 nm), a (L2) HeNe laser (633 nm), and a (L4) violet laser (405 nm). If necessary,

fluorescence minus one (FMO) staining was performed to adjust photo multiplier tubes (PMT)

voltages and to compensate manually between the channels.

3.3.3.1 Standard staining protocol and MHCI tetramer staining

Standard staining protocol All stainings were carried out in U-bottom 96-well-plates, which

were kept on ice or at 4°C during the whole procedure. Unnecessary light exposure was

avoided. The centrifugation was done for 3 min at 300x g and 4°C. Generally, 2-4x 10

cells were

incubated in 100 μl PBS/BSA solution with rat serum, anti (α)CD16/αCD32 monoclonal

antibodies (mAbs) to block non-specific antibody binding. After 5 min primary antibodies were

added at a previously titrated optimal dilution and cells were incubated for 15 min at 4°C. Cells

were then centrifuged, washed with 180 µl PBS/BSA, re-centrifuged, and re-suspended in 100 μl

of PBS/BSA solution. If a primary antibody was biotin-conjugated, the staining procedure was

repeated with streptavidin conjugates. Stained cells were kept at 4°C protected from light until

measurement by flow cytometry the same day.

PepA tetramer staining Identification of PepA specific CD8

T cells in organs of Mtb

infected mice was performed using MHCI tetramers constructed with peptide from the Mtb-

derived antigen PepA and labeled with the fluorochrome PE. About 2-4x 10

cells were

incubated in 100 μl PBS/BSA solution with rat serum, αCD16/αCD32 mAb and streptavidine to

block non-specific tetramer and antibody binding. After 5 to 10 min, cells were stained with

PepA tetramers as well as several T cell surface antigens including αCD8, αCD4, αCD62L, αCD69,

αCD44, and αCD27 mAb. After 45 min at 4°C, stained cells were washed with PBS/BSA and re-

suspended in 100 μl of PBS/BSA solution. Stained cells were kept at 4°C protected from light

until measurement by flow cytometry the same day.

3.3.3.2 Immunological characterization of immune cell types

Leukocytes from peritoneal cavity, spleen, MLN, thymus and BM were isolated and numbers

determined as described in 3.3.1.6. Frequencies and absolute numbers of a) T cells, b) B cells, c)

APCs, and d) neutrophils were determined by flow cytometric analysis as described in 3.3.3.1. T

cells were identified as helper T cells by the surface expression of CD4

and CTLs were identified

by the surface expression of CD8α and absence of CD11c or CD11b expression. B cells were

defined as B220 expressing cells and representative for APCs were DCs defined by the surface

expression of CD11c and macrophages defined by the intermediate expression of CD11b.

Neutrophils were defined as high GR-1 and CD11b surface expressing leukocytes.

3.3.3.3 In vitro re-stimulation and intracellular cytokine staining (ICS)

Mtb-protein-derived peptides used for restimulation Antigen 85A (Ag85A) and Antigen 85B

(Ag85B) The MHCII presented peptides Ag85A (QDAYNAGGGHNGVFDFPDSG) and Ag85B

(FQDAYNAAGGHNAVFNFPPNG) are derived from proteins Ag85A and Ag85B, respectively which

are secreted by Mtb in early infection stages and which are encoded by the respective genes are

fbpA and fbpB.

Six Kilodalton Early Secretory Antigenic Target (ESAT6) The MHCII presented ESAT6 peptide

(MTEQQWNFAGIEAAASAIQG) is derived from the ESAT6 protein and encoded by the gene esxA.

ESAT6, together with culture filtrate protein 10 (CFP10) are exported through an alternative

secretion pathway encoded by genes of the region of deletion-1 locus. ESAT6 and CFP-10 are

important antigens in diagnosis of TB, where they are used to detect the presence of Mtb

specific T cells in patients.

Mtb32A-derived Peptide (PepA) The MHCI presented peptide PepA (GAPINSATAM) is

derived from Mtb32A protein which was isolated from Mtb culture filtrate and was identified as

a putative serine protease. The open reading frame of Mtb32A corresponds to “pepA” Rv0125.

Mtb32A is rapidly processed and exported from the bacilli after synthesis.

In vitro restimulation To measure antigen-specific cells by intracellular cytokine staining, 2-4x

cells were cultured for 6 h in a total volume of 200 µl of RPMI-10 complete medium

containing 10 μg/ml of Brefeldin A, and the following Mtb derived peptide antigens at 10

-4

concentrations: Ag85A

241-260

, Ag85B

240-260

, ESAT6

1-20

, and PepA peptides. As a control, 2-4x 10

cells were also incubated in parallel in a total volume of 200 µl of RPMI-10 complete medium

containing 10 μg/ml of Brefeldin A and no peptides.

Intercellular cytokine staining To determine frequencies of cytokine producing cells

intracellular cytokine staining was performed. The restimulated cells (see above) were washed

with PBS/BSA and blocking solution containing rat serum and αCD16/αCD32 mAb were added

and incubated on ice for 10 min. Surface marker antibody solutions containing fluorochrome

conjugated αCD4 and αCD8 mAb were then added and cells were incubated on ice for 20 min.

Cells were then washed with 1x PBS and fixed for 15 min at RT with 2% PFA in 1x PBS. Cells were

washed with PBS/BSA solution, permeabilized with saponin buffer and incubated in this buffer

with rat serum and αCD16/αCD32 mAb for 10 min. Intracellular antigen antibody solutions

containing αIFN-γ, αTNF-α, αIL-2, and αIL-17 mAb conjugated to different fluorochromes (see

materials table) were added and incubated for 20 min on ice. After washing in 1x PBS, cells were

re-suspended in 200 μl 1% PFA in 1x PBS and were kept protected from light at 4°C overnight.

The next day, cells were washed with PBS/BSA and kept protected from light at 4°C at until

analysis by flow cytometry.

3.3.3.4 Fluorescence activated cell sorting (FACS)

Cells were isolated and stained as described in 3.3.1.6 and 3.3.3.1, respectively. Cells were

concentrated to 2x 10

/ml and passed through a 30-µm MACS® Pre-Separation Filter directly

before sorting. Prior to sorting BD FACS tubes used to collect the sorted cells were incubated for

2-3 h with cRPMI to avoid unspecific binding of single cells to the plastic surface and contained

500 µl cRPMI to aid in collecting the sorted cells. Sorting was conducted by members of the

FACS facility of the DRFZ/MPIIB, Berlin using a FACSAria

cell sorter. After sorting, cells were

centrifuged at 300x g for 6 min and cell pellets were re-suspended in 300 µl to 500 µl cRPMI and

let rest for 2-4 h in a humidified incubator at 37°C and 5% CO

before cell counting and further

manipulations.

3.3.3.5 In vitro B cell stimulation and proliferation assay

Peritoneal B-1a and B-2 B cells from WT and treml6

-/-

mice were stained with αB220 mAb and

αCD5 mAb as described in 3.3.3.1 and cells were sorted as B-1a B cells (B220

dim

CD5

dim

) and B-2 B

cells (B220

CD5

) as described in 3.3.3.4.

CFSE staining of sorted B cell populations Sorted cells were re-suspended at a concentration

of 1x 10

/ml in prewarmed PBS/0.1% BSA. CellTrace

CFSE (5 mM in dimethyl sulfoxide

(DMSO)) was added to a final concentration of 5 µM and samples were vortexed immediately

after addition and incubated in a humidified incubator at 37°C, 5% CO

for 10 min. The staining

was quenched by adding 5 volumes of ice-cold cRPMI and incubation to the cells on ice for 5

min. Cells were pelleted by centrifugation at 300x g for 5 min and washed with cRPMI. Pelleting

and washing was repeated two times. Cells were resuspend at 1x 10

/ml (B-2 B cells) and 5x 10

/ml B-1a B cell cells in cRPMI.

In vitro stimulation of CFSE-labelled B cell populations Triplicates of 1x 10

B-2 B cells and

5x 10

- 1x 10

B-1a B cells from WT and treml6

-/-

mice were cultured for 2.5 days in the

presence of a) 10 ng/ml IL-4, b) 10 ng/ml IL-4 and 3 µg/ml αCD40, c) 10 ng/ml IL-4 and 15 µg/ml

F(ab’)

fragment goat anti-mouse IgM (F(ab’)

αIgM), d) 10 ng/ml IL-4 and 1.5 µg/ml F(ab’)

αIgM,

e) 10 ng/ml IL-4 and 2 µg/ml LPS, and f) 10 ng/ml IL-4 and 1 µM CpG ODN (Oligodinucleotides

containing CpG motifs) and conditions a) to f) without IL-4 supplementation.

Measurement of proliferating CFSE-labeled B cell populations At day 0, CFSE labeling was

controlled. After 2.5 days plates were spun down and triplicates re-suspended in 50 µl cold

PBS/BSA each. 10 min prior to measurement 2.5 µl 7-amino-actinomycin D (7-AAD) were added

and CFSE and 7-AAD intensity were measured on the LSRII.

3.3.3.6 Calcium (Ca

)- Signaling in B-2 and B-1a B cells

Leukocytes from spleen and peritoneal cavity cells were isolated as described in 3.3.1.6, surface-

stained with αB220 mAb and αCD5 mAb as described in 3.3.3.1 and ‘loaded’ with Indo-1.

Isolation, staining and loading was conducted at RT. Calcium movement was assessed after

stimulation of cells with F(ab’)

αIgM. Peritoneal B-1a B cells were gated as B220

CD5

dim

and

splenic B-2 B cells were gated as B220

CD5

cells. Unstained cells were used as a control to

exclude the influence of extracellular antibody binding on calcium flux.

Indo- 1 loading of cells All samples were protected from light throughout Indo-1 loading

and measurement of cells. Indo-1 staining solution was prepared by adding 1 µl 10% Pluronic

F127 and 0.7 µl 1 mM Indo-1 to 200 µl 5% fetal calf serum (FCS)-RPMI, followed by vortexing for

5 sec. Stained and unstained (control) leukocytes from spleen or peritoneal cavity were re-

suspended at 2x 10

cells in 500 µl 5% FCS-RPMI, 200 µl of Indo-1 staining solution were added,

samples were vortexed and incubated at 30°C under mild agitation (~300 rpm) for 25 min. Cells

were centrifuged for 5 min at 300x g at RT and washed twice in 800 µl Krebs-Ringer solution +

CaCl

. After the final centrifugation, cells were re-suspended in 1 ml Krebs-Ringer solution and

kept under mild agitation at 20°C until measurements.

Setting up the flow cytometer Increases in free intracellular calcium in gated B cell

populations were measured as changes in ratio of Indo-violet (bound Ca

) to Indo-blue

(unbound Ca

) signals in real time. Indo-signals were detected with the UV (L3) of the LSRII.

Indo-blue peaks at 450-500nm and Indo-violet at 405nm. A violet bandpass filter (BP) centered

at 405 +/- 20nm BP and a blue bandpass filter centered at 530 +/- 30nm and 505LP as long

bandpass (LP) filter were used, the violet laser was turned off. Light scatter gates were set and

photomultiplier tube gain settings were optimized by placing the mean blue fluorescence in the

upper half of the histogram channels and the violet fluorescence in the lower half of the

histogram channels. Linear amplification was used. The instrument setup and cellular loading

was controlled by treating 1x 10

Indo-1 loaded cells with ionomycin at 1 µg/ml final

concentration. If an immediate response in 100% of cells occurred, loading and set-up were

successful. Remaining ionomycin was carefully removed by flushing the lines 2 min with FACS

Clean, then 2 min with FACS Rinse and 2 min with dH

Measurement of Indo-1 loaded samples Samples were acquired at a flow rate of 200-500

events/sec and with linear amplification. First unstimulated samples were measured for 30 sec

to determine the baseline, tubes were removed without stopping the acquisition, quickly the

stimulating agent (10µg/ml or 1µg/ml F(ab’)

αIgM) was added, samples were vortex and re-

placed into the sample injection port for a total of 7 min. Raw data files were transferred to

FlowJo software, analyzed and are presented as a median and in comparative overlay analyses

using Graph Pad Prism software.

3.3.3.7 Intranuclear BrdU staining

WT and treml6

-/-

mice were fed BrdU as described in 3.3.1.10 and spleen and peritoneal cavity

leukocytes isolated as described in 3.3.1.6. Isolated cells were surface stained with B-2, B-1a and

B-1b B cell markers as described in 3.3.3.1. Surface stained cell were washed with 1x PBS and

fixed for 20 min at RT in 170 µl Cytofix/Cytoperm buffer (BD Pharmingen). Fixed cells were

centrifuged, washed with 170 µl BD Perm/Wash buffer, re-suspended in 170 µl BD Cytoperm

Plus buffer, and incubated on ice for 10 min. Permeabilized cells were then centrifuged and

washed twice with 170 µl Cytofix/Cytoperm buffer and re-suspended in 170 µl

Cytofix/Cytoperm buffer and incubated for 5 min at RT. Re-fixed cells were centrifuged, washed

with 170 µl 1x BD Perm/Wash buffer and re-suspended in 100 µl of diluted DNase (300µg/ml in

1xPBS) and incubated for 1 h at 37°C. Cells were then washed twice in 1x BD Perm/Wash buffer.

BrdU staining Washed cells were re-suspended in 50 µl 1x BD Perm/Wash buffer containing 1

µl BrdU-APC antibody. Cells were stained for 20 min at RT, centrifuged, washed with 170 µl 1x

BD Perm/Wash buffer and re-suspended in 100 µl PBS/BSA. Cells were stored at 4°C until

analysis by flow cytometry.

3.3.3.8 Measuring early apoptosis by AnnexinV stain

Leukocytes from spleen and peritoneal cavity cells were isolated as described in 3.3.1.6, surface-

stained with αB220 mAb, αCD19 mAb, αCD43 mAb and αCD5 mAb as described in 3.3.3.1.

Stained cells were washed twice with cold 1x PBS and resuspended in 1x AnnexinV Binding

Buffer (BD) at a concentration of 1x 10

cells/ml. Cy

5 AnnexinV and 7-AAD were added (1:20)

and samples incubated at RT for 15 min. Staining was stopped by adding 400 µl 1x AnnexinV

Binding Buffer to 100µl sample. Samples were analysed within 1 h by flow cytometry. Viable

cells were defined as AnnexinV

and 7-AAD

, early apoptotic cells were defined as AnnexinV

and

7-AAD

and, end stage apoptotic or dead cells as AnnexinV

and 7-AAD

cells.

3.3.3.9 Flow cytometric data analysis

Data were exported as FCS 3.0 files and analyzed with FlowJo or as experiments and analyzed

with BD FACSDiva software in combination with FACS-Analyser v0.9.9.

3.3.4 Cell culture

3.3.4.1 Cell culture of stromal cell lines and preB-I B cells

Cell culture of stromal cell lines The adherent stromal cell line Puromycine-Hygromycine-

resistant OP9 cells (OP9-PH) were cultured in minimum essential medium (MEM) alpha medium

supplemented with 2% FCS in a humidified incubator at 37°C, 10% CO

. The stromal cell lines

were passaged every 3-4 days after reaching a density of approximately 1,6x 10

cells/145 cm

dish, with 10 ml trypsin/ethylenediaminetetraacetic acid (EDTA) (0.5%) per dish to detach the

cells from the plastic (5-10 min incubation at 37°C). OP9-PH cells were replated at 1x 10

cells/75 cm

-flask for the co-culture with lymphocyte progenitors and at 1-2x 10

cells/145 cm

dish for the maintenance of the stromal cell lines. After three to four days of culture at 37°C,

10% CO

, in 75 cm

flasks the semi-confluent stromal cell layer could be γ-irradiated with 3000

rad, which prevents further cell division during co-culture with pre-B I cells.

Cell culture of stromal cell/IL-7 dependent pre-B cell lines Fetal liver derived Pre-B I

cells were grown on a 70%-80% confluent layer of 3000 rad γ-irradiated stromal cells in Iscove's

modified Dulbecco's medium (IMDM)-based SF medium supplemented with 2% FCS.

Additionally the medium contained about 50-100 U/ml mouse recombinant (r)IL-7 which was

added as a culture supernatant from IL-7 cDNA transfected J558L cells (a kind gift from Szandor

Simmons). Pre-B I cells were removed from the adherent stromal cell layer by gentle clapping of

tissue culture flasks with a flat palm or by aspirating the medium several times up and down.

The pre-B I cell cultures were removed every 3-4 days and replated on a new 3000 rad γ-

irradiated stromal cell layer with 2-5x1 0

cells/T75 flasks depending on the growth behaviour of

each cell line.

Establishment of pre-B cell lines Cell suspensions from fetal liver (day 18 of gestation) of WT

or treml6

-/-

mice were plated as mass cultures on a semi-confluent layer of γ-irradiated (3000

rad) OP9 stromal cells in the presence of IL-7. The IL-7-containing medium consisted of IMDM

supplemented with 2% fetal calf serum, 0.03% (wt/vol) primatone RL, 50 µM 2-

mercaptoethanol, 1 mM glutamine, and 1% conditioned supernatant of rIL-7-producing J558L

cells

After 1 week of in vitro culture, the growing pre-B I cells were further propagated as

polyclonal or clonal cell lines.

Freezing and thawing of tissue culture cells Cells which were intended for storage were

re-suspended in freezing medium (90% FCS, 10% DMSO) at a density of about 2-5x 10

cells/ml.

Aliquots of 1 ml were transferred into 1.5 ml freezing vials and were frozen slowly in styrofoam

boxes at -80°C (approximate cooldown -1°C/min) for at least 24 h before transferring them into

a liquid nitrogen storage tank. Thawing of frozen cells was accomplished by incubating the vials

in a 37°C water bath. The cell suspension was immediately diluted in 10 ml medium and washed

once to remove traces of DMSO. Thereafter, cells were transferred into appropriate culture

medium and grown in a humidified CO

-incubator at 37°C, with 5% or 10% CO

3.3.5 Enzyme-linked immuno sorbent assay (ELISA) and Multiplex analysis

3.3.5.1 TNP-specific ELISA

To detect TNP-specific antibodies 96-well Nunc Maxisorb plates were coated in a humid

chamber either 2 h at 37°C or overnight at 4°C with 50µl 10 µg/ml TNP-BSA coating solution.

Plates were then blocked with 200µl ELISA blocking solution and incubated in a humid chamber

either for 2 h at 37°C or overnight at 4°C. Plates were washed 3 times with 1x PBS. Meanwhile

sera dilutions were prepared in ELISA dilution buffer. Starting dilution was 1:20, the following

dilutions were prepared as 1:3 dilutions from the previous, 8 dilutions were made from 1 serum.

As standard, pooled sera from day 8 were used. The same samples were used as standard on all

analyzed plates to allow comparison. 50µl of serum dilution were added per well and incubated

in a humid chamber either for 2 h at 37°C or overnight at 4°C. Plates were washed 3-times with

1x PBS. IgG3-alkaline phosphatase (AP) and IgM-AP antibodies were diluted 1:1000 in ELISA

dilution buffer, 50µl were added to each well and plates were incubated in a humid chamber

either for 2 h at 37°C or overnight at 4°C. Plates were washed 3-times with 1x PBS. 100µl of

1mg/ml p-Nitrophenylphosphate substrate in Tris buffer (Sigma) were added to each well and

OD was measured at 405nm on an ELISA reader. Standard curves were plotted on a

semilogarithmic scale, and arbitrary concentrations of samples were determined.

3.3.5.2 Multiplex analysis of antibody isotypes in sera

Antibody isotype composition in naïve WT and treml6

-/-

mice was determined with the Millipore

Mouse Immunoglobulin Isotyping Kit, according to the manufacturer`s instructions.

4 RESULTS

4.1 Generation and immunological analysis of treml6 deficient mice

TREM and TLT proteins form a novel surface receptor family of NON-TLR innate immune

receptors. Members of this family are thought to be either of activating (DAP12/ITAM)) or

inhibitory (ITIM) nature and participate in various cell processes (10-12). We were interested in

putative inhibitory regulators of inflammation. In 2003 one inhibitory ITIM carrying receptor

was predicted within the family and named treml1 (TLT-1), a second unnamed receptor (RIKEN

cDNA B430306N03 gene) was also predicted to be of inhibitory nature (10). In 2009, it was

named treml6 and its cytoplasmic ITIM motif was described (12, 19).

4.1.1 Treml6 protein product TLT-6 is a predicted ITIM-carrying receptor

Until today, treml6 protein (TLT-6) existence is only evidenced by mRNA expression. In order to

obtain deeper insights into the putative structure of TLT-6, we first performed in silico analyses

(Fig. 7A). Different programs and databases predicted a) a signal peptide at amino acid (aa)-

position 1-23 (Program Signal P), b) an extracellular region at aa-position 1-154, c)

transmembrane helices at aa-position 155-177, d) an intracellular tail at aa-position 178-289 (b

to d TMHMM v2.0 software), and e) an Immunoglobulin domain (position 26-122) (SMART

domain database) or f) a V-set domain at aa-position 20-122 (Pfam 24.0). ScanProsite software

was used to scan the C-terminal intracellular domain for canonical (…[ILV]-x-Y-x-x-[LV]…) and

non-canonical/permissive (…[ILVST]-x-Y-x-x-[VLI]…) ITIM motifs (13) and identified one canonical

at aa-position 222-227 and one non-canonical at aa-position 257-262 (Fig. 7A). We next

performed a protein blast on the C-terminal part to identify potential orthologs in other species

and analyzed whether the previously predicted ITIM motifs are conserved. Proteins from Equus

cabalus (XP_001496610), Canis lupus familiaris (XP_851243 similar to triggering receptor

expressed on myeloid cells-like 4), and Gallus gallus (NP_001074339 triggering receptor

expressed on myeloid cells-like 2 and NP_001074337 triggering receptor expressed on myeloid

cells) showed high sequence similarity. Amino acid sequences of these proteins and TREML6

were aligned using the CLUSTAL multiple sequence alignment program and the boxshade

program. Both ITIM motifs were conserved in all analyzed proteins (Fig. 7B).

FIGURE 7. In silico analysis of putative TLT6 protein structure. A, Predicted amino acid sequence of

treml6. Italics, predicted signal peptide sequence; grey-boxes, predicted V-set Ig domain; underlines,

predicted transmembrane helix; yellow boxes and bold, ITIM motifs. B, Blast and alignment of C-terminal

parts revealed conserved ITIM motifs (boxed). C, RT-PCR (gapdh, β-actin, and treml6 EEJ5 primers) on WT

(WT) and treml6

-/-

(KO) BM cells.

In silico analysis suggested TLT6 to be a functional receptor (13, 157), with a signal peptide, an

extracellular V-set immunoglobulin-domain, a transmembrane region and an intracellular tail,

potentially transmitting inhibitory signals via two conserved ITIM motifs. In order to investigate

the biological function of TLT6, treml6 gene deficient mice were generated by Dr. Mischo Kursar

as described in 3.3.1.2. The gene targeting strategy is depicted in Fig. 6A. RT-PCR on WT and

treml6

-/-

BM cells showed no presence of treml6 mRNA in treml6

-/-

mice (Fig. 7C) and confirmed

successful deletion of treml6.

4.1.2 Treml6 mRNA expression pattern and initial immunological characterization of

treml6 deficient mice

Treml6

-/-

mice were viable and fertile and showed no abnormalities or growth retardation. As

initial step, we studied consequences of treml6 deficiency on immune cell composition in

different organs.

TABLE 1: Absolute cell numbers

Absolute numbers of different immune cell

populations in various organs of WT and

treml6

-/-

mice. Leukocytes were isolated from

depicted organs and cell subsets phenotyped

by flow cytometry. Cell numbers were

calculated from frequencies among total

leukocytes. Results are means of 2

independent experiments with 3 mice per

group and experiment. Plus-minus indicates

the standard errors of the mean. Significance

was calculated with Student’s t test (p>0.05

non-significant).

We isolated leukocytes from thymi, MLN, spleens, BM and peritoneal cavities and stained for T

cell (CD4 and CD8), B cell (B220), DCs (CD11c) and macrophage (CD11b) surface markers. Flow

cytometric analysis revealed no significant differences in frequencies (data not shown) or

numbers of CD4

T cells, CD8

T cells, DP (CD4

CD8

) T cells, B cells, DCs and macrophages

between the analyzed tissues of WT and treml6

-/-

mice (Tab. 1).

In parallel to flow cytometric analysis of treml6

-/-

mice, we measured the expression pattern of

treml6 mRNA in various tissues and cell subsets of C57BL/6. To this end, we performed RT-PCR

on housekeeping-genes (gapdh and β-actin) and treml6. Treml6 mRNA expression in organs or

cells was normalized to expression of gapdh and β-actin and fold differences were calculated

relative to a virtual tissue/cell (gapdh and β-actin c

: 12; treml6 c

: 30). Treml6 mRNA expression

was found preferentially in immune-related organs such as fetal liver, BM, spleen, and MLN, but

also in peritoneal cavities (Fig. 8A). We further analyzed FACS-sorted cell subsets from

peritoneal cavities, spleen and BM and found broad treml6 mRNA expression in all analyzed

leukocytes (Fig. 8B). Only in leukocytes from BM, (B cells (CD19

), NK cells (NK1.1

), and myeloid

cells (CD11b

)), treml6 mRNA expression was higher (~2

(12-14)

) than general treml6 mRNA

expression in the corresponding organ (BM (~2

(10)

)) (Fig. 8A and 8B).

4.1.3 Lack of treml6 reduces numbers of B-1 B cell precursors in BM and fetal liver

In adult mice, the BM is the origin of all circulating blood cells. Moreover, it is the site of B cell

maturation (158). Since our initial broad immunological analysis did not reveal any differences

in composition and numbers of BM leukocyte populations (Tab. 1), we decided to study B cell

development in more detail.

We did not detect differences in frequencies (data not shown) or absolute numbers of pro-B

(B220

CD19

c-Kit

CD25

), pre-B1 (B220

CD19

c-Kit

dim

CD25

), pre-B2 (B220

CD19

c-Kit

CD25

immature (CD19

IgM(µ-chain)

kappa-light-chain

IgD

) and mature B cells (CD19

IgM(µ

chain)

kappa-light-chain

IgD

) between BM of WT and treml6

-/-

mice according to Melchers-

Rolink definition of B cell developmental stages (87) (Fig. 8C), indicating normal B cell

maturation in treml6

-/-

mice. In contrast, frequencies and numbers of a newly described B-1P

(B220

CD19

) (118) were reduced by half in treml6

-/-

mice (Fig. 8D and 8E). B-1 B cells represent

a B cell subset that differs from conventional B cells (namely B-2 B cells) by phenotype,

anatomic location, and immunological function. During embryonic development, B-1 B cells are

the main B cell population produced in the fetal liver (116).

FIGURE 8. Treml6 mRNA expression in WT mice and B cell development in treml6

-/-

mice.

A, Relative levels of treml6 mRNA expression in indicated tissues B, relative treml6 mRNA expression in B

cells, myeloid cells and NK cells in BM and T and B cells from spleens and peritoneal cavities. WT cells were

sorted by FACS (B cells (BM): CD19

; myeloid cells (CD11b

); NK cells (NK1.1

); T cells: CD5

B220

; B-2 B

cells: CD5

B220

; B-1a B cells: CD5

dim

B220

-/dim

). Relative mRNA quantification was performed by real-time

RT-PCR using primers for gapdh and β-actin as internal controls and intron-spanning treml6 specific primers

(EEJ5) for quantification. Treml6 mRNA levels in C57BL/6 mice are represented as fold differences (log2)

relative to virtual values (gapdh/β-actin c

: 12; treml6 c

: 30). Error-bars represent standard error of

triplicates. One representative experiment of at least two is shown. C, Numbers of B cell precursor cells in BM

of WT and treml6

-/-

(KO) mice and D, Frequencies and numbers of B-1 B cell progenitors in BM and fetal liver

of WT and treml6

-/-

(KO) mice. E, Dot blots show FSC/SSC gated lymphocytes and demonstrate gating of B-1 B

cell progenitors in BM and fetal liver of WT and treml6

-/-

(KO) mice. E, Numbers of B cell precursor cells in

fetal livers of WT and treml6

-/-

(KO) mice. Leukocytes were isolated from depicted organs and cell subsets

phenotyped by flow cytometry. Cell numbers were calculated from frequencies among total leukocytes.

Results are means of at least 2 independent experiments with 5 mice or more per group and experiment.

Error-bars represent the standard errors of the mean. Significant differences between 2 groups are indicated

by asterisks (Student’s t test: *, p < 0.05, **, p < 0.01 and ***, p < 0.001).

Therefore, we extended our studies on B cell development to fetal livers (day 18 of gestation).

As in BM, we found B-1P to be reduced in fetal livers of treml6

-/-

mice (Fig. 8D and 8E).

Moreover, in fetal livers of treml6

-/-

mice, numbers of pro-B cells were reduced by half, while

numbers of pre-B1, pre-B2, or total B220

B cells were similar to WT numbers (Fig. 8F).

4.1.4 Treml6 deficiency results in B cell subset changes in peritoneal cavities

Since our data pointed to a defect in B-1 B cell development in treml6

-/-

mice, we next analyzed

whether B-1 B cells occur at normal rate in peripheral organs of adult mice. The B-1 B cell

population is mainly present in pleural and peritoneal cavities, but can also be found at lower

frequencies in the spleen (94). Conventional B-2 B cells express CD19 and B220, while B-1 B cells

are CD19

B220

dim/-

, and can be sub-divided further into B-1a B cells expressing CD5 and CD43

and into B-1b B cells which are CD5 negative, but CD43

((94) and Fig. 9A). Frequencies of B-1a B

cells - among peritoneal lymphocytes - were reduced by 50% in 9-11 week old treml6

-/-

mice

compared to WT controls and frequencies of B-2 B cells were increased by a factor of 1.3.

Similar differences were observed in numbers of peritoneal B-1a and B-2 B cells. In contrast, no

differences in frequencies and numbers of peritoneal B-1b, splenic B-2, and splenic B-1a B cell

populations could be detected between WT and treml6

-/-

mice (Fig. 9B). We also analyzed other

splenic B cell populations and found no differences in immature or mature, namely FO and MZ B

cell populations (data not shown).

B-1 B cells are the prominent B cell subset in neonatal mice, and remain as a self-renewing

population in peritoneal cavities of adult mice (100). To address the issue of whether the defect

in peritoneal B-1a B cells appears already early in life or whether the population is lost over age,

we performed a kinetic analysis on B-1a and B-2 B cells in peritoneal cavities and spleens (data

not shown) of WT and treml6

-/-

mice. Treml6

-/-

mice showed reduced frequencies and numbers

of peritoneal B-1a B cells at the age of 4-5 weeks, B-2 B cell frequencies and numbers were

increased from the age of 7-8 weeks. At the age of 17-23 weeks these differences in B-1a and B-

2 B cells almost normalized to WT levels, at least in terms of numbers (Fig. 9C). In spleens no

differences were detected (data not shown).

FIGURE 9. Age dependent impact of treml6 deficiency on B-2 and B-1a B cell populations.

A, Dot blots represent B cell staining and gating of isolated leukocytes from spleens and peritoneal cavities.

Left dot blots show FSC/SSC gated lymphocytes. B, Frequencies and numbers of B-2 and B-1a B cells in spleen

and B-2, B-1a and B-1b B cells in peritoneal cavities of 9 to 11-weeks old WT and treml6

-/-

(KO) mice. C,

Frequencies and numbers of B-2 and B-1a B cells in peritoneal cavities of 4-5 weeks, 7-8 weeks, 9-11 weeks,

and 17-23 weeks old mice. Leukocytes were isolated from depicted organs and cell subsets phenotyped by

flow cytometry. Cell numbers were calculated from frequencies among total leukocytes. Results are means of

at least 2 independent experiments with a minimum of 5 mice per group and experiment. Error-bars

represent the standard errors of the mean. Significant differences between 2 groups are indicated by asterisks

(Student’s t test: *, p < 0.05,**, p < 0.01 and ***, p < 0.001).

4.1.5 Functional analysis of B-2 and B-1a B cells from treml6 deficient mice

To characterize the functional properties of peripheral treml6

-/-

B-2 and B-1a B cells and to find

further indications explaining the observed paucity in peritoneal B-1a B cells, we analyzed their

proliferation capacity, their ability to mount BCR-triggered Ca

responses, their viability status,

and their homing capacities.

4.1.5.1 IL-4 evoked in vitro proliferation is enhanced in treml6 deficient mice

In contrast to conventional B-2 B cells which are generated throughout life in the BM, B-1 B cells

are thought to be self-renewing once they reside in pleural or peritoneal cavities (100). To

analyze their turnover, WT and treml6

-/-

mice were fed BrdU via the drinking water for a period

of 12 days. At days 4, 8 and 12 the amount of incorporated BrdU was measured in B-2 and B-1a

B cell populations and served as indicator of proliferation. Both, splenic B-1a and B-2 B cells

from treml6

-/-

mice showed higher turnover rates compared to their WT counterparts after 12

days of BrdU treatment. In peritoneal cavities of treml6

-/-

mice B-2 B cells showed significantly

reduced turnover at day 8 compared to WT B-2 B cells. In contrast, more BrdU was incorporated

by peritoneal B-1a B cells of treml6

-/-

mice than by WT B-1a B cells. Notably, peritoneal B cells

showed strong differences in BrdU incorporation between single mice of one group, impeding

the interpretation of results (Fig. 10A).

Next, we sorted WT and treml6

-/-

peritoneal B-2 and B-1a B cells by FACS to investigate their

ability to proliferate in vitro upon various stimuli. We observed no major differences in

proliferation of peritoneal B-2 B cells of WT or treml6

-/-

mice. Although differences in basal

proliferation and proliferation upon stimulation with 15µg/ml F(ab’)

αIgM and CpG were

statistically significant, they were rather minor in actual numbers (e.g. WT B-2 B cells + CpG:

93%; treml6

-/-

B-2 B cells + CpG: 95%).

Generally, stimulated B-2 B cells from WT or treml6

-/-

mice proliferated stronger than B-1a B

cells, except when stimulated with IL-4. Addition of IL-4 did not increase the proliferation of WT

or treml6

-/-

B-2 B cells; however it did for B-1a B cells. The effect of IL-4 on B-1a B cells differed

in strength between WT and treml6

-/-

cells. Compared to WT, treml6

-/-

peritoneal B-1a B cells

proliferated significantly stronger in the presence of IL-4 (WT B-1a: 19%; treml6

-/-

B-1a: 31%).

Moreover, increased proliferative stimulation by IL-4 on treml6

-/-

B-1a B cells remained in the

presence of αCD40 and F(ab’)

αIgM stimulation, while LPS or CpG treatment outweighed the IL-

4-dependent effect on treml6

-/-

B-1a B cells. Notably, treml6

-/-

B-1a like treml6

-/-

B-2 B cells

showed stronger basal proliferation than their corresponding WT cells (Fig. 10B).

FIGURE 10. Treml6

-/-

B-1a B cells show enhanced homeostatic and IL-4 driven proliferation.

A, Homeostatic proliferation of B-2 and B-1a B cells in spleens and peritoneal cavities of naïve WT and treml6

(KO) mice measured by BrdU incorporation. Mice received BrdU drinking water for 12 days. Results are

means of two independent experiments with 4 mice per group and experiment. Error-bars represent the

standard errors of the mean. B, In vitro proliferation of peritoneal B-2 and B-1a B cells from WT and treml6

-/-

(KO) mice. WT and treml6

-/-

(KO) B-2 (B220

CD5

) and B-1a B cells (B220

dim

CD5

dim

) from peritoneal cavities

(pool of 7-10 mice per group) were sorted by FACS, stained with CFSE and incubated with different stimuli for

2.5 days. CFSE dilution was measured by flow cytometry; dead cells were excluded by 7-AAD staining. Results

show mean values of two independent experiments each performed in triplicate. Error bars represent the

standard error of mean. Significant differences between 2 groups are indicated by asterisks (Student’s t test: *,

p < 0.05, **, p < 0.01 and ***, p < 0.001).

4.1.5.2 BCR-dependent Calcium-signaling is not affected by the lack of treml6

Since F(ab’)

αIgM (15µg/ml) induced proliferation was marginally stronger in both peritoneal B-

2 and B-1a B cells (w/o IL-4), we hypothesized that BCR-dependent calcium-signaling could be

enhanced in treml6

-/-

mice. Therefore, we isolated leukocytes from spleens and peritoneal

cavities, stained them with B-2 and B-1a B cell specific markers, and loaded them with indo-1.

We measured Calcium-signaling as increases in free intracellular calcium in gated B cell

populations as changes in ratio of Indo-violet (bound Ca

) to Indo-blue (unbound Ca

) signals

in real time. However, calcium responses to stimulation with high (10µg/ml) or low dose

(1µg/ml) F(ab’)

αIgM by splenic B-2 B cells, peritoneal B-2 or B-1a B cells were identical for WT

and treml6

-/-

B cells (Fig. 11A). Thus, treml6 is dispensable for BCR-dependent Ca

signaling.

4.1.5.3 Impact of treml6 deficiency on B cell maintenance and homing capacities

Reduced peritoneal B-1a B cells in treml6

-/-

mice might as well be a consequence of increased

apoptosis or of impaired homing to peritoneal cavities.

Therefore, we measured frequencies of early apoptotic cells (AnnexinV

7-AAD

) within splenic

and peritoneal B-2 and B-1a B cell populations and did not detect differences between WT and

treml6

-/-

cell populations, indicating similar fitness of cells (Fig. 11B).

To investigate the homing capacities of B-2 and B-1a B cells in treml6

-/-

mice, we isolated RNA

from BM, spleen, and peritoneal cavity and from FACS-sorted splenic and peritoneal T (B220

CD5

), B-2 B (B220

CD5

) and B-1a B (B220

dim/-

CD5

dim

) cells, as well as from in vitro generated

pre-B I cells. We then performed RT-PCRs specific for cxcl13, cxcr5 and ccr7 on transcribed

cDNAs from these tissues and cells.The homeostatic chemokine, chemokine (C-X-C motif) ligand

13 (CXCL13) and its receptor CXCR5, as well as chemokine (C-C motif) receptor 7 (CCR7) are

important for B-1 B cell homing to peritoneal cavities (159, 160). Moreover, CCR7 drives re-

circulation of B-2 B cells after peritoneal passage (160). We detected no considerable

differences in cxcl13 mRNA expression between WT and treml6

-/-

tissues (Fig. 11C). Cxcr5 mRNA

expression did also not differ between WT and treml6

-/-

T, B-2 and B-1a B cells from spleen and

peritoneal cavities, indicating that mature cells are able to home to the peritoneum. In contrast,

in vitro generated treml6

-/-

pre-B I cells showed a 4-fold reduction in cxcr5 mRNA compared to

WT (Fig. 11D).

FIGURE 11. BCR-dependent Ca

-signaling in B-2 and B-1a B cells from WT and treml6

-/-

mice.

A, Leukocytes from spleen and peritoneal cavities were isolated, surface stained for B220 and CD5 and, loaded

with indo-1. Increases in free intracellular calcium in gated B cell populations were measured as changes in

ratio of Indo-violet to Indo-blue signals in real time. Cells were stimulated with 10 µg/ml or 1 µg/ml F(ab’)

αIgM. Results are representative for two independent experiments and show pooled graphs of 3 independent

mice per group and experiment. B, Leukocytes from spleen and peritoneal cavities were isolated, surface

stained for B220, CD19, CD43 and CD5, followed by AnnexinV and 7-AAD staining. Early apoptotic cells were

defined as AnnexinV

7-AAD

. C, Fold differences of cxcl13 mRNA expression in indicated tissues of treml6

-/-

(KO) mice relative to WT and D, Fold differences of cxcr5 mRNA expression and E, Fold differences of ccr7

mRNA expression in T and B cells from spleens and peritoneal cavities of treml6

-/-

mice relative to WT. D/E,

WT and treml6

-/-

cells were sorted by FACS. C/D/E, Relative mRNA quantification was performed by real-time

RT-PCR. Real-time PCR was performed using primers for gapdh and β-actin simultaneously as controls and

intron spanning gene (cxcl13, cxcr5, and ccr7) specific primers for quantification. Dotted lines mark borders of

2-fold changes. Means of at least two experiments (each mean of triplicates) are shown. Error-bars represent

standard error of means.

Ccr7 mRNA expression did not differ between WT and treml6

-/-

T cells and B-2 B cells. However,

treml6

-/-

B-1a B cells showed a 4-fold up-regulation of ccr7 mRNA compared to WT cells (Fig.

11E). In order to determine if the reduction of ccr7 mRNA expression in treml6

-/-

B-1a B cells

translates into higher protein expression of CCR7 receptor, we stained B-1a B cells with αCCR7

mAbs and analyzed them by means of flow cytometry. In contrast to mRNA expression, we

observed no significant differences in surface CCR7 expression by WT and treml6

-/-

B-1a B cells.

Conclusively, the upregulation of ccr7 mRNA did not translate into measurable higher levels of

receptor expression (data not shown).

4.1.6 Immune responses of WT and treml6

-/-

mice to TI-2 antigens

A hallmark of B cells is their ability to secrete antibodies; B-1 and MZ B cells are the mayor

producers of natural antibodies, such as IgM and IgG3 (100). It is possible that changes in B-2:B-

1a B cell ratios observed in treml6

-/-

mice, lead to differences in serum immunoglobulin levels.

Therefore, we measured the amount of serum immunoglobulins in naïve WT and treml6

-/-

mice

over age by multiplex analysis. At 9 to 10 weeks of age no differences in IgG1, IgG2b, IgG3, and

IgM serum antibodies were detected between WT and treml6

-/-

mice. In between 12 to 20

weeks of age, treml6

-/-

mice showed a small, but significant reduction in IgM serum antibodies

(Fig. 12A) compared to WT mice. IgG1, IgG2b, and IgG3 serum levels were identical between

older (12-20 weeks) WT and treml6

-/-

mice (data not shown).

FIGURE 12. Lower IgM titers in aged treml6

-/-

mice and TI-2 immune responses. V

A, Multiplex analysis of serum titers of immunoglobulins (Serum Ig) in 9-10 weeks old WT and treml6

-/-

(KO)

mice and IgM titers in 12-20 weeks old WT and treml6

-/-

(KO) mice. Each symbol represents one mouse.

Significant differences between 2 groups are indicated by asterisks (Mann-Whitney U test: *, p < 0.05). B,

Immune responses of WT and treml6

-/-

(KO) mice after TI-2 immunization. Mice were immunized with 10µg

TNP-Ficoll, and at days 0, 5, 8 and 14 post immunization serum samples were taken and TNP-specific IgM and

IgG3 antibody responses were measured by ELISA. Results are representative for two independent

experiments with 3-5 mice per group and experiment. AU, arbitrary units.

Since B-1 B cells are involved in TI-2 immune responses (100, 161), we measured the ability of

treml6

-/-

mice to mount a normal antibody response. We immunized mice with trinitrophenol

(TNP)-Ficoll as TI-2 antigen and followed TNP-specific IgM and IgG3 in sera. Both IgM and IgG3

responses were similar to WT responses (Fig. 12B). In sum, lack of treml6 had no detectable

influence of TI-2 antibody responses against TNP-Ficoll, despite reduced levels of serum IgM in

older treml6

-/-

mice.

4.1.7 Unimpaired reconstitution of Rag1

-/-

mice with treml6

-/-

The observed contraction of the peritoneal B-1a B cell population in treml6

-/-

mice can be of

either fetal or adult origin. Moreover, it can be either caused extrinsically by other cells

expressing treml6 and influencing the development and/or maintenance of the B-1a B cell

population or it can be due to B cell intrinsic defects. Fetal pro-B cells as well as BM B-1 B cell

progenitors have been described to give rise to B-1 B cells in the periphery (86, 118) and both

were reduced in treml6

-/-

mice (Fig. 8). To investigate a putative connection with the peritoneal

B-1a B cell paucity, we studied the ability of treml6

-/-

fetal liver-derived and treml6

-/-

BM cells to

reconstitute the B-1 B cell compartment in spleens and peritoneal cavities in a WT environment,

namely irradiated Rag1 deficient mice (Fig. 13).

In vitro generated pre-B I cells are known to preferentially fill the B-1 B cell compartment ((162)

and (personal communication Szandor Simmons and Fritz Melchers)). Therefore, we generated

pre-B I cells from WT and treml6

-/-

fetal livers (day 18 of gestation) in vitro and transplanted

these cells into irradiated Rag1

-/-

hosts (Fig. 13A) to compare their capacity to generate B-1 B

cells in vivo. Notably, this experiment has only been performed once and therefore conclusions

should be drawn carefully. Yet, we observed reduced B-1a B cell numbers in peritoneal cavities

(4 weeks, WT: 20% and KO: 11%; 12 weeks, WT: 42% and KO: 20%) and spleens after transfer of

treml6

-/-

pre-B I cells compared to transfer of WT pre-B I cells 4 and 12 weeks post transfer,

indicating that treml6 is necessary for fetal liver-dependent B-1a B cell generation (Fig. 13B).

Transfer of treml6

-/-

pre-B I cells resulted also in reduced B-2 B cell engraftment in spleens, but

not in peritoneal cavities of recipient mice. To date we cannot explain why treml6

-/-

pre-B I cells

were not as efficient as WT pre-B I cells in filling the B-2 B cell compartment in spleens. Notably,

the differences were minor. Frequencies of B-2 B cells reached 5-4% (4 and 12 weeks) after

transfer of WT cells and 2 and 3% (4 and 12 weeks) after transfer of treml6

-/-

pre-B I cells, and

were thus far below normal B-2 B cell frequencies (60% of lymphocytes) found in spleen (Fig.

9B). Moreover, different numbers or fitness of transferred cells cannot have caused the

observed differences, since B-2 B cell populations in peritoneal cavity reached the same size

after either transfer (Fig. 13B).

FIGURE 13. Transplantation of fetal liver derived pre-B I cells or adult BM into Rag1

-/-

mice.

A, Scheme of pre-B I cell generation and cell transfer into Rag1

-/-

hosts. B, Frequencies and numbers of B-2

and B-1a B cells, at 4 and 12 weeks after reconstitution of Rag1

-/-

mice with in vitro generated WT or treml6

-/-

(KO) preB I B cells. Left dot blots show FSC/SSC gated lymphocytes. The respective B cell staining and gating

strategy is depicted in dot blots of C. Results are from 1 experiment with 5 mice per time-point, group and,

experiment. Error-bars represent the standard errors of the mean. D, Scheme of BM transfer, 1x 10

WT or

treml6

-/-

(KO) BM cells were adoptively transferred upon Rag1

-/-

mice. E, Frequencies and numbers of B-2 and

B-1a B cells after 4 and 8 weeks of re-constitution with BM cells in spleens and peritoneal cavities. Left dot

blots show FSC/SSC gated lymphocytes. The respective B cell staining and gating strategy is depicted in dot

blots of F. Results are means of 2 independent experiments with 5 mice per time-point, group and,

experiment. Error-bars represent the standard errors of the mean. Significant differences between 2 groups

are indicated by asterisks (Student’s t test: *, p < 0.05, **, p < 0.01 and ***, p < 0.001).

We also transplanted WT and treml6

-/-

BM cells into Rag1

-/-

mice (Fig. 13D). We found that

treml6-deficient B-1a B cells, like WT B-1a B cells, can be generated from progenitors found in

adult BM. Notably, B-1a B cell frequencies among lymphocytes were around 15% at 4 and 8

weeks post WT or treml6

-/-

BM transfer (Fig. 13E) and therefore lower than normal B-1a B cell

frequencies in WT animals (Fig. 9B). Eight weeks post transfer of either WT or treml6

-/-

frequencies of B-2 B cells in recipient spleens reached about 60% of lymphocytes, reflecting

normal WT levels (Fig. 13E). Peritoneal cavities of reconstituted mice showed higher B-2 B cell

frequencies than normally found in WT animals (Fig. 13E and 9B). Thus, BM from adult WT and

treml6

-/-

mice possessed the same capacity to reconstitute all analyzed B cell subsets when

transplanted in a WT environment (Rag1

-/-

4.1.8 Concluding remarks on the role of treml6 in immunity

In sum, we identified treml6 as an ITIM-containing receptor with preferential mRNA expression

in peritoneal cavities and lymphoid tissues. Treml6

-/-

mice revealed reduced B-1 B cell

precursors in fetal liver (B-1Ps and pro-B cells) and bone marrow (B-1Ps), as well as reduced

peritoneal B-1a B cells compared to WT mice. Treml6 deficient peritoneal B-1a B cells showed a

propensity for increased turnover and IL-4 driven proliferation. We detected no differences in

BCR-triggered calcium responses or apoptosis rate between B-2 and B-1a B cell populations

from treml6

-/-

or WT mice. However, we observed a) reduced expression of cxcr5 mRNA – a

chemokine receptor involved in homing to peritoneal cavities, and b) impaired B-1 B cell

engraftment in Rag1

-/-

mice by in vitro generated treml6

-/-

pre-B I cells when compared to

respective WT cells. In contrast, after transfer of treml6

-/-

BM or WT BM B cell engraftment of

Rag1

-/-

mice was equal. TI-2 antibody responses to TNP-Ficoll were identical between treml6

-/-

and WT mice.

4.2 Impact of ICOS on T cell responses and protection against Mtb infection

In an attempt to compile the kinetics of multiple co-stimulatory molecules during Mtb infection,

we observed that CD4

Th1 cells, notably IFN-γ-secretors, expressed ICOS during murine TB (Fig.

14B). Meanwhile, Quiroga et al. reported that the amount of IFN-γ secretion by T cells from

patients with active TB disease correlates with their ICOS surface expression, and that ICOS

signaling can increase IFN-γ secretion (155). Moreover, Urdahl and colleagues described that

pulmonary Tregs express elevated level of ICOS during murine Mtb infection (66). These data

point to ICOS as an important player during TB. Therefore, we investigated the role of ICOS co-

stimulation in TB using ICOS

-/-

mice. Here we describe the expression pattern of ICOS during

murine TB and its impact on T cell responses and disease outcome. We show that ICOS is

strongly expressed on CD4

T cells during Mtb infection and that ICOS deficiency differentially

affects CD4

and CD8

T cell responses, ultimately resulting in improved protection in the spleen

during later stages of Mtb infection.

4.2.1 ICOS expression by CD4

and CD8

T cells during Mtb infection

Both CD4

and CD8

T cells are crucial for control of TB (124, 125, 127). In order to investigate

the role of ICOS signaling in generation of Mtb-specific T cell responses, we first aimed at

characterizing ICOS expression by T cells during Mtb infection (Fig. 14A). C57BL/6 wild type (WT)

mice were infected via aerosol with a low dose of Mtb (approx. 200 CFU of strain H37Rv).

At different time points p.i., ICOS expression by T cells from spleen and lungs was determined

by flow-cytometry. During the entire course of Mtb infection, numbers and frequencies of CD8

T cells expressing ICOS remained constant with less than 5% of all splenic and pulmonary CD8

cells (< 3x 10

CD8

T cells per organ) being ICOS

(Fig. 14A). In contrast to CD8

T cells, the

fraction of CD4

T cells expressing ICOS constantly increased during the acute phase of infection.

It reached its maximum at day 30 p.i. when up to 45% of all CD4

T cells expressed ICOS in lungs

(~ 2x 10

ICOS

CD4

T cells), and up to 35% in spleen (~ 4x 10

ICOS

CD4

T cells). Between days

30 and 45 p.i., the ICOS

CD4

T cell population contracted to approximately half the size, and re-

expanded during the chronic phase of infection (days 45 to 120 p.i.) to reach levels similar to

day 30 p.i.

FIGURE 14. ICOS expression by CD4

and CD8

T cells during Mtb infection.

A, Kinetics of frequencies and numbers of ICOS surface-expressing CD4

and CD8

lung and spleen T

lymphocytes from Mtb infected C57BL/6 (WT) mice. At indicated time-points p.i. lung and spleen lymphocytes

were isolated, surface-stained with αCD4 mAb, αCD8 mAb, and αICOS mAb, and analyzed by flow cytometry.

Percentages refer to gated CD4

or CD8

lymphocytes. Results are representative for 3 independent

experiments with 3 pools of 1-2 mice per group and experiment. Error-bars represent the standard errors of

the mean. B, Kinetics of frequencies and numbers of ICOS

IFN-γ

CD4

spleen and lung lymphocytes from Mtb

infected WT mice. Isolated cells from 5-8 mice were pooled and re-stimulated in vitro with a mixture of Mtb

MHC-II peptides or a mixture of αCD3ε/αCD28 mAbs. Subsequently, cells were surface-stained with αCD4

mAb and intracellularly with αICOS mAb and αIFN-γ mAb and analyzed by flow cytometry. Frequencies refer

to gated CD4

T lymphocytes. Cells cultured in medium served as background controls. Results represent

means of 2 independent experiments with 2 pools of 5-8 mice per experiment. Error-bars represent standard

errors of the mean. C, Representative dot blots of IFN-γ and ICOS expressing CD4

lung lymphocytes from

naïve and Mtb infected (day 24 p.i.) WT mice. Black squares indicate gating of ICOS

IFN-γ

CD4

T cells.

To further define the nature and cytokine secretion patterns of these ICOS

CD4

T cells, we

performed intracellular cytokine staining after short term in vitro re-stimulation with either

Mtb-derived peptides or αCD3ε/αCD28 monoclonal antibodies (polyclonal stimulation) (Fig.

14B). In either setting, almost all IFN-γ secreting cells also co-expressed ICOS (Fig. 14C)

suggesting a mayor role of ICOS co-stimulation in the development of Th1 cells during Mtb

infection.

4.2.2 Impact of ICOS deficiency on Mtb burden and pathology

Since ICOS was expressed by almost all IFN-γ secreting CD4

T cells during Mtb infection, we

next studied consequences of ICOS deficiency on TB disease progression. We infected ICOS-

gene deficient mice and WT animals with Mtb and followed bacterial burdens and pathology in

lungs and spleens during the course of infection (Fig. 15 and 16). We observed no differences in

Mtb numbers in lungs between both groups at all time-points analyzed (days 15 to 120 p.i.). In

contrast, spleens of ICOS-deficient mice revealed a significantly reduced bacterial burden on

days 60 and 120 p.i. (Fig. 15).

FIGURE 15. Mtb burden in lungs and spleen during Mtb infection. ICOS

-/-

and WT mice were infected with

Mtb by aerosol. At indicated time-points p.i., mice were killed and bacterial burdens in lungs and spleens were

determined. Medians with range of log CFU are plotted versus time. Open or filled diamonds represent ICOS

-/-

or WT mice, respectively. Data are representative of 3 independent experiments with 5 mice per group per

time-point. Significant differences between 2 groups are indicated by asterisks (Mann-Whitney U test: *, p <

0.05 and **, p < 0.01).

During the course of Mtb infection we observed no apparent differences in pathology of lungs

between WT and ICOS

-/-

mice (Fig. 15B). At day 30 p.i. lung parenchyma presented infiltrates

with granulomatous appearance, but no great differences were revealed between the groups in

terms of frequency or cellular composition of the inflammatory foci. Alveolitis, perivascular and

peribronchiolar accumulations of macrophages and lymphocytes were as well comparable

between WT and ICOS

-/-

mice. During chronic Mtb infection (120 p.i.) lung lesions extended to

almost the entire respiratory tissue. They became consolidated with defined clusters of

mononuclear cells, mostly lymphocytes. Within the inflammatory infiltrates and particularly in

proximity to the remnant alveolar spaces large foamy macrophages were present in both animal

strains. Compensatory emphysema and relatively few intact alveoli were present at this stage of

Mtb infection.

FIGURE 16. Pathology of lungs and spleen during Mtb infection. ICOS

-/-

and WT mice were infected with

Mtb by aerosol. Hematoxylin and eosin staining of lung and spleen sections from naïve and Mtb-infected

C57BL/6 and ICOS

-/-

mice; Upper panels of each time point, scale bar 500µm; lower panels of each time point,

scale bar 200µm.

The pathology of the spleens differed only slightly during the chronic phase of Mtb infection

(Fig. 16). During the acute phase of Mtb infection (day 30 p.i.) spleen architecture was not

dramatically changed. In both, WT and ICOS

-/-

mice spleen follicles were intact and sparse

mononuclear cell infiltrates were observed within the red pulp. At day 120 p.i. partial disruption

of the spleen follicles was recorded for both WT and ICOS

-/-

mice and slightly more infiltration of

the perifollicular space was noticed in ICOS

-/-

mice compared to WT mice.

4.2.3 Impact of ICOS deficiency on generation of CD4

Th1 responses during Mtb

infection

The CD4

Th1 response and its major effector cytokine IFN-γ are crucial for defense against Mtb

(125, 127). To gain detailed information about the kinetics and activation of the Th1 response in

absence of ICOS signaling, we infected WT and ICOS

-/-

mice with Mtb. At various time points

after infection, we isolated spleen and lung lymphocytes and restimulated them in vitro either

polyclonally (αCD3ε/αCD28) or with a cocktail of Mtb peptides (PepA, Ag85A

241-260

, Ag85B

240-260

and ESAT6

1-20

). We subsequently stained and analyzed these cells for co-expression of CD4 (and

CD8; Fig. 19A), IFN-γ and TNF-α (Fig. 17).

Kinetics of initial and late phase Mtb (ESAT6, Ag85A and Ag85B)-specific Th1 responses were

similar between WT and ICOS

-/-

mice, only the contraction of the response on day 40 p.i. was

less pronounced in ICOS

-/-

mice than in WT mice. Accordingly more than twice as many Mtb-

peptide-specific CD4

T cells were identified in lungs and spleens of ICOS

-/-

mice (~3.2x 10

ICOS

-/-

vs. ~1.5x 10

in WT lungs, and ~5x 10

in ICOS

-/-

and ~2x 10

in WT spleens). The total IFN-

γ secreting CD4

T cell response (after αCD3ε/αCD28 mAbs restimulation) differed significantly

between the two groups of mice in the chronic stage of infection. In ICOS

-/-

mice the Th1

response did not contract as profoundly as in WT mice, and remained elevated throughout the

later stage of infection, evidenced by significantly increased frequencies and numbers of IFN-

CD4

T cells in lungs and spleen of ICOS-deficient mice.

FIGURE 17. Increased frequencies of total IFN-γ

CD4

T cells in ICOS

-/-

mice during late stage of Mtb

infection. A, Kinetics of frequencies and numbers of IFN-γ

CD4

spleen and lung lymphocytes from Mtb

infected WT and ICOS

-/-

mice. WT and ICOS

-/-

mice were infected with Mtb and cells were isolated, re-

stimulated, stained, and analyzed, as in Fig. 14B. Percentages refer to gated CD4

T lymphocytes. Results are

mean values of 2 to 3 independent experiments with 3 pools of 1-2 mice per group and experiment. Error-

bars represent the standard errors of the mean. Significant differences between 2 groups are indicated by

asterisks (Student’s t test: *, p < 0.05, **, p < 0.01 and ***, p < 0.001). B, Representative dot blots of IFN-γ and

TNF-α expression by CD4

lung and spleen lymphocytes from Mtb infected (day 120 p.i.) WT and ICOS

-/-

mice.

Black squares indicate gating of IFN-γ

CD4

T cells.

From day 30 to 40 p.i., numbers of total IFN-γ producing CD4

T cells contracted approximately

by half in lungs of WT mice and only by a quarter in lungs of ICOS-deficient mice. After the

contraction phase, the amount of total IFN-γ producing CD4

T cells remained at about the same

level in both groups of mice until day 120 p.i. (~0.8x 10

in WT and 1.6x 10

in ICOS

-/-

lungs). In

spleens differences in contraction strength between the two groups of mice were even more

pronounced. From day 30 to 40 p.i. numbers of total IFN-γ producing CD4

T cells contracted

approximately by two-third in spleens of WT mice and only by a quarter in spleens of ICOS-

deficient mice. After the contraction of the CD4

Th1 response, numbers of total IFN-γ

producing CD4

T cells doubled in both groups from day 40 p.i. to 120 p.i. (~1.3x 10

to ~2.7x 10

in WT mice and ~3.6x 10

to ~7.5x 10

in ICOS

-/-

mice). While, ICOS-deficient mice had the

highest levels of total IFN-γ producing CD4

T cells on day 120 p.i. (~7.5x 10

), WT mice showed

highest numbers on day 30 p.i. at the peak of the acute immune response (~4.2x 10

) (Fig. 17A).

Thus, we assume that the contraction of the acute CD4

T cell response is likely governed by

ICOS signaling and that at later stages of infection, CD4

Th1 responses are restricted in an ICOS-

dependent manner.

4.2.4 Impaired maintenance of Mtb-specific effector CD8

T cells in ICOS

-/-

mice

In addition to CD4

Th1 cells, CD8

T cells contribute to protection against Mtb (124, 163, 164).

They secrete the mayor Th1 effector cytokine IFN-γ, and also lyse infected cells directly (140).

FIGURE 18. Reduced numbers of PepA-specific CD8

T cells in chronically Mtb infected ICOS

-/-

mice. A,

Kinetics of frequencies and numbers of PepA-Tetramer

CD8

spleen and lung lymphocytes from Mtb infected

ICOS

-/-

and WT mice. WT and ICOS

-/-

mice were infected with Mtb and cells were isolated as in Fig. 17. Isolated

cells were surface-stained with αCD8 mAb, PepA-Tetramer and αCD62L mAb and analyzed by flow cytometry.

Percentages refer to gated CD8

T lymphocytes. Results are mean values of 2 to 3 independent experiments

with 3 pools of 1-2 mice per group and experiment. Error-bars represent standard errors of the mean.

Significant differences between 2 groups are indicated by asterisks (Student’s t test: *, p < 0.05, **, p < 0.01

and ***, p < 0.001). B, Representative dot blots of PepA-Tetramer binding and CD62L expression by CD8

lung

and spleen lymphocytes from Mtb infected (days 30 and 120 p.i.) ICOS

-/-

and WT mice. Black circles indicate

gating of PepA-Tetramer

CD62L

low

CD8

T cells.

We stained PepA-specific CD8

T cells using fluorochrome-labeled MHCI-peptide-tetramers (Fig.

18). The initial acute CD8

T cell response did not differ between WT and ICOS-deficient mice,

whilst the late phase response did. At day 60 p.i., 4% and at day 120 p.i., about 5% of all CD8

cells were specific for PepA in lungs of Mtb-infected WT mice. In contrast, at the same time

points only about 2% of all CD8

T cells were PepA-specific in lungs of Mtb-infected ICOS

-/-

mice

resulting in significantly reduced numbers of these cells at day 120 p.i. (0.25x 10

in ICOS

-/-

and

0.7x 10

in WT mice). We also observed reduced PepA-specific CD8

T cell responses in spleens

of ICOS

-/-

mice. At day 120 p.i., 0.4% of all ICOS

-/-

CD8

T cells and about 1% of all CD8

T cells in

WT mice were PepA-specific. Not only frequencies, but also numbers of PepA-specific CD8

cells were reduced in spleens of ICOS

-/-

mice; however they did not differ significantly from

numbers in WT animals (Fig. 18A).

FIGURE 19. Reduced numbers of IFN-γ secreting PepA-specific CD8

T cells in chronically Mtb infected

ICOS

-/-

mice. A, Kinetics of frequencies and numbers of IFN-γ

CD8

spleen and lung lymphocytes from Mtb

infected ICOS

-/-

and WT mice. WT and ICOS

-/-

mice were infected with Mtb and cells were isolated as in Fig. 17.

Isolated cells were in vitro restimulated with an Mtb MHC-I peptide or a mixture of αCD3ε/αCD28 mAbs.

Subsequently, cells were surface-stained with αCD8 mAb and intracellularly with αTNF-α and αIFN-γ mAbs,

and analyzed by flow cytometry. Percentages refer to gated CD8

T lymphocytes. Cells cultured in medium

served as a background controls. Results are mean values of 2 to 3 independent experiments with 3 pools of

1-2 mice per group and experiment. Error-bars represent the standard errors of the mean. Significant

differences between 2 groups are indicated by asterisks (Student’s t test: *, p < 0.05, **, p < 0.01 and ***, p <

0.001). B, Representative dot blots of IFN-γ and TNF-α expression by CD8

lung and spleen cells from Mtb

infected (day 120 p.i.) ICOS

-/-

and WT mice. Black squares indicate gating of IFN-γ

CD4

T cells.

In order to assess effector functions of the CD8

T cell response, we first analyzed their ability to

produce IFN-γ and TNF-α after short term in vitro restimulation with PepA peptide (Fig. 19).

CD8

T cells from ICOS

-/-

mice were not affected in their ability to produce effector cytokines.

Frequencies and numbers of CD8

T cells secreting IFN-γ upon restimulation with Mtb-peptide

PepA were comparable to those of PepA-TCR expressing CD8

T cells as assessed by MHC-I-

tetramer staining. At day 120 p.i. we measured 2% PepA-TCR-specific CD8

T cells (Fig. 18A), as

well as 2% IFN-γ secreting PepA-specific CD8

T cells in ICOS

-/-

mice (Fig 19A). Although during

the late stage of infection lack of ICOS co-stimulation led to reduced PepA-specific CTL response

in magnitude, it did not affect their capacity to secrete IFN-γ (Fig. 18A and 19A).

Compared to WT mice, ICOS

-/-

mice had less PepA-TCR-specific CD8

T cells as well as PepA-

specific IFN-γ secreting CD8

T cells. However, pulmonary total IFN-γ-secreting CD8

T cells –

assessed through in vitro restimulation with αCD3ε/αCD28 mAbs – were only reduced in

frequencies, but not numbers, in ICOS

-/-

mice. Of note, in spleens of ICOS

-/-

mice the total IFN-γ

secreting CD8

T cell response was even elevated in the late stage of Mtb infection (Fig. 19A).

4.2.4.1 Reduced killing by PepA-specific CD8

T cell in ICOS

-/-

mice

A hallmark of CD8

T cell function is their ability to lyse target cells (165). To further address the

effector functions of the weakened PepA-specific CD8

T cell response in ICOS

-/-

mice, we

examined whether PepA-specific

CD8

T cells in Mtb infected mice were capable of lysing

target

cells presenting this epitope in vivo (Fig. 20).

We used an in

vivo cytotoxicity assay, in which equal numbers of differentially CFSE-labeled

splenocytes from naïve

mice, pulsed either with control peptide LLO

91-99

or pulsed with Mtb-

derived peptide PepA, were adoptively transferred

upon Mtb infected mice. Peptide-specific

lysis of transferred

cells was assessed by flow cytometric analysis of spleens of recipient mice

and quantification of residual CFSE high (PepA pulsed) and CFSE low (LLO

91-99

pulsed) donor

cells. WT and ICOS

-/-

mice showed killing upon transfer of

target cells. The CTL activity of PepA-

specific CD8

T cell was significantly stronger in WT mice (~40%-specific killing of PepA loaded

cells) as compared to ICOS

-/-

mice (~25%-specific killing) (Fig. 20A). The observed reduction in

killing of donor cells can be due to either reduced killing capacity of PepA-specific CD8

T cells or

be a consequence of reduced numbers and frequencies of PepA-specific CD8

T cells in ICOS-

deficient mice.

FIGURE 20. Reduced PepA-specific in vivo lysis in

chronically Mtb infected ICOS

-/-

mice. A, CTL activity of

PepA-specific CD8

T cells in vivo. Listeriolysin O (control

peptide) loaded splenocytes (CFSE

low

) and PepA-loaded

splenocytes (CFSE

high

) from naive mice were transferred upon

Mtb infected mice (day 60 p.i.). Reduction in CFSE

high

populations reflects the amount of splenocytes killed in vivo

by PepA-specific CD8

T cells. Significant differences between

2 groups are indicated by asterisks (Student’s t test: *, p <

0.05, **, p < 0.01 and ***, p < 0.001). B, Representative

histograms show frequencies of CFSE

high

and CFSE

low

populations. Specific lysis was calculated as indicated.

Calculation: % specific lysis = 1-[AVG r

naive

/ r

inf

] x100. AVG

naive

: average of CFSE

low

/CFSE

high

ratios from individual naïve

mice; AVG r

naive

has been calculated for each experiment

independently. r

inf

: CFSE

low

/CFSE

high

ratios of individual

infected mice. Scatter plots show data from 3 independent

experiments with 3 mice per group and experiment. Results

were pooled with each diamond representing one mouse.

4.2.5 Impaired CD8

effector memory maintenance during chronic Mtb infection in

ICOS

-/-

mice

ICOS signaling has been suggested to serve as survival factor for effector and effector memory T

cells (85). We were interested in contribution of ICOS signaling on the generation or stimulation

of memory T cells in the context of a chronic TB disease (Fig. 21). Memory phenotype of PepA-

specific CD8

T cells was assessed by staining for CD127, a surface marker expressed only on

naïve and on memory T cells, and for CD62L, to differentiate between central and effector

memory cells (166).

No differences in numbers of effector and central memory CD8

T cells were detected in spleens

at days 60 and 120 p.i. between WT and ICOS

-/-

mice. Moreover, central memory T cells were

not affected by the lack of ICOS signals in lungs of Mtb infected mice as numbers did not differ

from those in WT mice. However, we observed differences in numbers of effector memory T

cells. PepA-specific effector memory CD8

T cells in WT mice increased from 1.8x 10

cells at day

60 p.i. to about 3x 10

cells at day 120 p.i., whilst effector memory CD8

T cells in ICOS

-/-

mice

remained at a stable level of 1.8x 10

cells (Fig. 21A). In sum, lack of ICOS had no impact on the

development of central memory T cells, but impaired formation and/or maintenance of PepA-

specific effector memory CD8

T cells in lungs of Mtb infected mice.

FIGURE 21. Reduced PepA-specific CD8

effector memory T cells at day 120 p.i. in ICOS

-/-

mice.

A, Numbers of CD127

and CD62L

/CD62L

PepA-Tetramer

CD8

spleen and lung lymphocytes from Mtb

infected ICOS

-/-

and WT mice on days 60 and 120 p.i. WT and ICOS

-/-

mice were infected with Mtb and cells

were isolated as in Fig. 17. Isolated cells were surface-stained with αCD8 mAb, PepA-Tetramer, αCD127 mAb

and αCD62L mAb and analyzed by flow cytometry. Results are mean values of 2 to 3 independent experiments

with 3 pools of 1-2 mice per group and experiment. Error-bars represent standard errors of the mean.

Significant differences between 2 groups are indicated by asterisks (Student’s t test: *, p < 0.05, **, p < 0.01

and ***, p < 0.001). B, Representative dot blots demonstrating PepA-Tetramer-binding and CD62L expression

by CD8

gated lung lymphocytes from Mtb infected (day 120 p.i.) WT and ICOS

-/-

mice. Black circles indicate

gating of PepA-tetramer-binding

CD8

T cells. Arrows point to dot blots which demonstrate further analysis of

CD127 and CD62L expression of the PepA-tetramer binding CD8

T cells.

4.2.6 Reduced frequencies and numbers of Treg in ICOS

-/-

mice during Mtb infection

Treg control the immune response against Mtb (64-66), and Treg upregulate ICOS surface

expression during Mtb infection (66), suggesting ICOS as a key regulator for these cells in TB. We

found that throughout the course of Mtb infection Treg frequencies were significantly reduced

in ICOS-deficient mice as compared to WT mice (Fig. 22). This also held true for Treg numbers in

the later stage of Mtb infection.

FIGURE 22. Reduced frequencies and numbers of Treg in ICOS

-/-

mice throughout Mtb infection.

A, Kinetics of frequencies and numbers of FoxP3

CD4

spleen and lung lymphocytes from Mtb infected WT

and ICOS

-/-

mice. WT and ICOS

-/-

mice were infected with Mtb and cells were isolated as in Fig. 17. Cells were

surface-stained with αCD4 mAb and αCD25 mAb and intranuclearly with αFoxP3 mAb, and analyzed by flow

cytometry. Percentages refer to gated CD4

T lymphocytes. Results are mean values of 2 to 3 independent

experiments with 3 pools of 1-2 mice per group and experiment. Error-bars represent standard errors of the

mean. Significant differences between 2 groups at analyzed time-points are indicated by asterisks (Student’s t

test: *, p < 0.05, **, p < 0.01 and ***, p < 0.001). B, Representative dot blots of FoxP3 and CD25 expression by

CD4

lung and spleen cells from Mtb infected (day 30 p.i.) ICOS

-/-

and WT mice. Black circles indicate gating of

CD4

FoxP3

cells.

Frequencies of Treg in spleen and lung of Mtb infected WT and ICOS-deficient mice were altered

reciprocally to the Th1 response. The Th1 response peaked around day 30 p.i. whereas the Treg

response contracted at this time point. WT Treg decreased from 8% at day 15 p.i. to about 4% of

CD4

T cells at day 30 p.i. Similarly, ICOS-deficient Treg decreased from 6% at day 15 p.i. to

about 2-3% of all CD4

T cells at day 30 p.i., indicating similar kinetics despite lower frequencies

during the acute response to Mtb in ICOS

-/-

mice. However, while the Treg response in WT mice

recovered and increased after day 30 p.i. (up to 7-8% of all CD4

T cells at days 60 and 120 p.i.),

Treg frequencies in lungs of ICOS

-/-

mice remained at a constant low level of 2-3% of all CD4

cells (Fig. 22A).

4.2.7 Concluding remarks on the role of ICOS during murine Mtb infection

In sum, all major T cell populations were altered in the absence of ICOS signaling during murine

Mtb infection. ICOS deficiency resulted in an increased polyclonal CD4

Th1 response against

Mtb, most likely caused by robustly reduced numbers and frequencies of Tregs in these mice. In

contrast to the CD4

Th1 response, the Mtb-specific CD8

T cell response during the later stage

of infection was reduced. In addition, not only Mtb-specific effector CD8

T cells, but also

effector memory CD8

T cells were reduced in Mtb infected ICOS

-/-

mice. As a result, Mtb

infected ICOS

-/-

mice were less susceptible during the late chronic stage of infection as

compared to wild type (WT) controls evidenced by decreased bacterial burden in the spleen.

5 DISCUSSION

5.1 Treml6 is a positive regulator of B-1a B cell development

This thesis is the first report describing treml6

-/-

mice and investigating the function of murine

treml6, an ITIM-containing receptor of the TREM protein family (12, 13). TREM receptors are of

particular interest in infection immunology since many members regulate inflammatory

responses by either amplifying or dampening TLR signals (12). In this context, we got interested

in the TREM family and in particular in treml6 as a putative inhibitor of inflammation. The basis

for understanding the role of a molecule in infection is a careful immunological characterization

of its function in the steady state.

As a result of this analysis, we provide data that expand the function of TREM receptors from

regulators of inflammation to regulators of lymphocyte development. In fact, we identified

treml6 as a positive regulator of B-1a B cell development. Its role in regulation of B-1a B cell

development was manifested in reduced B-1P in fetal liver and adult BM, and finally resulted in

a strong reduction of peritoneal B-1a B cells in young and adult treml6

-/-

mice. Moreover, treml6

deficient peritoneal B-1a B cells showed a propensity for increased turnover and IL-4 driven

proliferation that possibly led to re-normalized B-1a B cell levels in aged treml6

-/-

mice (older

than 17 weeks). We found no indications linking treml6-dependent regulation to BCR signaling

or increased apoptosis.

Treml6

-/-

mice were fertile and showed normal growth. Moreover, the initial immunological

analysis of treml6

-/-

mice revealed regular immune cell composition. In addition, treml6 gene

expression showed preferential expression in peritoneal cavities and lymphoid tissues, such as

bone marrow and spleen. Though, it was not focused to a specific cell population. In latter

regard, treml6 is not an exception in the TREM family, since other TREM members are also

widely expressed on various innate immune cells of the myeloid lineage, and on T and B

lymphocytes (12). Our assessment of treml6 expression was limited to mRNA expression

analysis by the unavailability of commercial αTLT-6 antibodies. Future plans comprise the

generation of a TLT-6-specific antibody to define the protein expression profile.

Although we observed high treml6 mRNA expression in bone marrow leukocyte populations, we

initially detected no differences in size of these populations in treml6

-/-

mice. Moreover, B-2 B

cell development was normal in BM of treml6

-/-

mice, evidenced in normal-sized pro-B, pre-B1,

pre-B2, and immature as well mature B cell subsets (87, 167). However, numbers of the newly

described B-1Ps were reduced in the BM of adult treml6

-/-

mice. This was particularly

interesting, since B-1 B cell ontogeny is still poorly understood and controversial, with two

general models proposed. The ‘lineage model’ claims that B-1 and B-2 B cells are derived from

two developmentally distinct cell lineages, and the alternative ‘induced-differentiation’ model

proposes that B-1a B cells derive from B-2 B cells exposed to strong BCR signals (86, 97, 100,

101). However, both models acknowledge the importance of the fetal microenvironment in B-1

B cell generation (95, 100). In line with these reports, we showed that treml6 was not only

necessary for B-1Ps generation in BM, but also in fetal livers (day 18 of gestation). In addition,

pro-B cells were reduced in fetal livers of treml6

-/-

mice, but fetal pre-B1 and pre-B2 populations

were unaffected. The reduction in fetal liver pro-B, but not bone marrow pro-B cells was

consistent with a specific role of treml6 in B-1 B cell development. Early cell transfer studies by

Hardy and Hayakawa with committed D-J rearranged B cell precursor, isolated from fetal liver or

bone marrow and transferred into adult scid mice, showed that most fetal-derived cells become

B-1 B cells, whereas most bone marrow-derived cells become B-2 B cells (86).

We could further underline the importance of treml6 in B-1 B cell development, by showing that

the defects in B-1 precursor (B-1Ps and fetal liver pro-B cells) translated into reduced peripheral

B-1 B cell numbers as early as at the age of 4-5 weeks. Notably, only frequencies and numbers

of B-1a B cells, but not B-2 and B-1b B cells were reduced in peritoneal cavities of adult treml6

-/-

mice. We observed the treml6-dependent paucity in peritoneal B-1a B cells until the age of 11

weeks. Interestingly, in aged treml6

-/-

mice (17-23 weeks) earlier differences in B-1a and B-2 B

cell populations had normalized to WT levels. This reconstitution of WT levels was most likely

due to the proliferative and self-renewing capacity of B-1a B cells (168). Peritoneal B-1a B cells

showed a higher, while peritoneal B-2 B cells showed a lower turnover rate in adult treml6

-/-

mice compared to WT mice. Moreover, basal and IL-4 driven in vitro proliferation was increased

in treml6

-/-

B-1a B cells, indicating a higher activation state of these cells (169). Similar

observations were made in IL-5

-/-

mice that lack the B-1 B cell growth factor IL-5, reduction in B-

1a B cells at the age of 2 weeks normalized by the age of 6-8 weeks (168). Notably, peripheral B-

1a and B-2 B cell populations in peritoneal cavities and spleens of treml6

-/-

mice did not

comprise more apoptotic cells than WT B cell populations. Thus, different degrees in fitness

cannot explain the paucity in peritoneal B-1a B cells that we observed in treml6

-/-

mice.

In spleens of treml6

-/-

mice, no differences in any mature B cell subset (MZ, FC, B-2, B-1a B cells)

were detected. Referring to our previous observation of normal B-2 B cell development in adult

BM, we expected the B-2 B cell compartment to be unaffected. However, it was puzzling to us

why the reduction in B-1 B cell precursors in fetal liver and adult bone marrow of treml6

-/-

mice

resulted in reduced peritoneal B-1a B cells, but not in reduced peritoneal B-1b and splenic B-1a

B cells, in respective mice.

Coinciding with independent roles of B cell subsets – and in support of our data that treml6

exclusively regulates peritoneal B-1a B cells - are recent studies that strengthen the hypothesis

that B-1a, B-1b and B-2 cells derive from distinct lineages (118, 170-172). Supporters of the

‘lineage model’ and ‘unified model’ (Fig. 3) of B-1 B cell development claim that B-1a B cells are

generated from fetal precursors, while precursor cells for B-1b B cells are present during fetal

and adult B cell development. Moreover, they showed that transferred adult bone marrow-

derived B-1Ps preferentially give rise to B-1b B cells, while fetal B-1Ps (BM and liver)

preferentially generate B-1a B cells (118, 170). In the context of these models, the reduced

numbers of B-1a B cells and WT-like numbers of B-1b B cells further underline that solely fetal

B-1 B cell development depends on treml6. Notably, the observed reduction of B-1Ps in adult

bone marrow in treml6

-/-

mice did not translate into reduced peripheral B-1b B cells.

Conclusively, either treml6

-/-

B-1b B cells must possess the propensity to compensate reduced

precursor numbers through a yet unknown mechanism, or other B-1b B cell precursor apart

from B-1P exist in the BM, or - despite its high expression in BM, and more probable, - treml6

only operates in the fetal environment and reduced numbers of B-1Ps in BM could be a relic of

defective B-1Ps generation in fetal livers of treml6

-/-

mice.

B-1a and B-1b B cells differ not only in phenotype, but also in immunological function. While B-

1a B cells participate in innate immune responses, B-1b B cells mediate a type of T cell

independent adaptive immune response, including an IgM memory response as has been

reported by two studies with Streptococcus pneumoniae and Borrelia hermsii (96, 173). Notably,

despite reduced numbers of B-1a B cells in treml6

-/-

mice, we detected no differences in TI-2

IgM and IgG3 antibody responses against TNP-Ficoll between WT and treml6

-/-

mice. One

explanation could be that although B-1a B cells are involved in TI-2 responses (100), they do not

respond strongly to TNP-Ficoll (161). Instead, MZ B cells and B-1b B cell appear to be the main

population responding to this antigen (174, 175). Conclusively, WT-like anti-TNP antibody

responses mounted by treml6

-/-

mice reflect normal MZ and B-1b B cell populations, in

respective mice. A similar explanation could be envisioned for unaffected serum titers of IgM,

IgG1, IgG2b and IgG3 in naïve treml6

-/-

mice. Although B-1 B cells - together with MZ B cells - are

the major source of serum IgM (94), the reduction in B-1a B cells is most likely not sufficiently

pronounced to translate into detectable reduction of serum IgM levels in treml6

-/-

mice.

Currently, we cannot explain why IgM levels in older treml6

-/-

mice (17-23 weeks) were reduced,

despite normalized B-1a B cell numbers in these mice.

In contrast to the observed reduction in peritoneal B-1a B cells, the splenic B-1a B cell

population was normal in treml6

-/-

mice. Notably, it has been demonstrated that splenic B-1a B

cells rather resemble splenic B-2 B cells than peritoneal B-1a B cells in terms of transcription

factor and gene expression, and in signaling for cell cycle progression (176). Conclusively, our

data support the hypothesis that peritoneal B-1a B cells and splenic B-1a B cells might be

distinct lineages with distinct developmental pathways (176). Given all these observations

underlining the phenotypical, transcriptional, functional, and developmental differences

between peritoneal B-1a, splenic B-1a and peritoneal B-1b cells, we were no longer surprised

that treml6 exclusively influences peritoneal B-1a cells.

Treml6 mRNA expression was not restricted to B cells; all analyzed leukocyte populations

showed treml6 mRNA expression. Thus, the reduction of peritoneal B-1a B cells could originate

from two possible mechanisms. First, it could be caused by an extrinsic mechanism – such as

other treml6 expressing cells or tissues influencing the B cells. Second, it could also be a B-1a B

cell autonomous defect caused by B cell intrinsic treml6 deficiency. We designed cell transfer

experiments that would help elucidate, or at least provide evidence to, whether B cell

autonomous or extrinsic mechanisms are operative. Moreover, these experiments were

designed to discriminate whether the observed peripheral defect in B-1a B cells is rooted in fetal

or adult B cell development.

Our transfer of fetal liver-derived in vitro generated pre-B I cells confirmed previous studies,

showing that these cells indeed preferentially fill the B-1 compartment ((87) and (personal

communication Szandor Simmons and Fritz Melchers)). Notably, compared to WT pre-B I cells,

transfer of treml6-deficient pre-B I cells resulted in reduced engraftment of B-1a B cells, while

numbers of peritoneal B-2 B cells were equal in recipient mice. This observation demonstrated

that fetal-derived treml6

-/-

cells were defective in B-1a B cell engraftment of a WT environment.

It is tempting to speculate that the observed impairment in B-1a B cell generation by treml-6

-/-

pre-B I cells is a B cell autonomous defect. But given the fact that pre-B I cells were generated

with fetal liver cells from day 18 of gestation, we cannot fully exclude that by that time other

treml6 expressing cells could have imprinted the B-1 pathway on the precursor cells. Moreover,

the fetal liver pre-B I cell transfer experiment has only been performed once and we

acknowledge the weakness of drawn conclusions. Nevertheless, resulting findings match our

previous observations strikingly and support the existence of a treml6-dependent regulatory

mechanism that origins in fetal B-1a B cell development.

Transfer of BM from adult WT or treml6

-/-

mice resulted in similar reconstitution patterns of all B

cell compartments in recipient mice. In either transfer B-2 B cell populations dominated over

other B cell populations, supporting previous reports that BM transfer favors B-2 B cell

development (97, 100). Thus, as WT BM, BM from adult treml6-deficient mice can give rise to B-

1a and B-1b B cells, confirming our previous observation that reduced B-1Ps in adult treml6

-/-

BM did not translate into reduced peripheral B-1b B cells. These results allow the following

conclusion: peripheral reduction of B-1a B cells did not result from a B cell autonomous defect

in adult BM precursor cells. However, the possibility of ‘trans’ effects on BM precursors or

peripheral B cells by other treml6 expressing cells still exists, e.g. through cytokine or chemokine

secretion – thereby altering the development or homing of B-1a B cells.

Hence, we measured the expression of CXCL13, a homeostatic chemokine crucial for B-1 B cell

homing to peritoneal cavities (159, 160). We did not detect significant differences in cxcl13

mRNA expression between peritoneal cavities of WT or treml6-deficient mice, providing strong

evidence that regarding the homing capacity no extrinsic mechanism operates in the periphery.

Nevertheless, ultimately only a transfer of WT BM into a treml6-deficient environment would

help us clarify this issue.

On the B cell side, CXCR5 - the receptor for CXCL13 - as well as CCR7 are important for B-1 B cell

homing to peritoneal cavities (159, 160). Moreover, CCR7 drives re-circulation of B-2 B cells

after peritoneal passage (160). We found that treml6

-/-

T cells, B-1a cells, and B2 cells from

peritoneal cavity and spleen were fully functional in terms of cxcr5 mRNA expression. Moreover,

no alteration in CCR7 surface expressions were detected on any cell type, despite increased

mRNA levels of ccr7 in peritoneal treml6

-/-

B-1a B cells. Notably, in vitro generated treml6

-/-

pre-

B I cells had decreased levels of cxcr5 mRNA expression compared to WT cells, indicating that

impaired homing capacities might have led to reduced B-1a B cell engraftment after transfer of

treml6

-/-

pre-B I cells.

An important factor, involved in controlling the development and/or persistence, of both B-1a

and B-2 B cells, is BCR signaling strength as indicated by multiple studies of genetically

manipulated mice. Mutations that impair BCR signal strength (e.g., CD19 deletion, vav-1

deletion, or phospholipase PLCγ deletion) result in reduced B-1a cell numbers (100, 107-109),

whereas mutations that enhance BCR signaling (e.g., SH2 domain containing phosphatase-1

(SHP-1) deletion, CD22 deletion, CD72 deletion, Siglec-G deletion, or protein tyrosine kinase Lyn

deletion), lead to the expansion of the B-1a B cell population (100, 110-112, 114). Since treml6

affected B-1a B cell development, we speculated whether treml6 signaling influences BCR

signals. We found that treml6 only marginally affected BCR-triggered in vitro proliferation of

peritoneal B-2 and B-1a B cells. Moreover, BCR-induced calcium responses in treml6

-/-

splenic B-

2 B cells, and treml6

-/-

peritoneal B-2 and B-1a B cells were similar to those of respective WT

cells. Notably, as shown by a previous report B-1P as well as pro-B cells do not have rearranged

immunoglobulin heavy chain V gene, meaning they do not express BCR or pre-BCR (95, 170).

Conclusively, the observed treml6 dependent effects on B-1P and pro-B cell populations could

not have been caused by alterations of BCR signaling. We cannot exclude that BCR-signaling in

fetal precursors that express the pre-BCR is affected by treml6, but absence of regulation of

BCR-signaling on mature B cells strongly suggest that mechanism other than strength of BCR

signal lead to depletion of precursor and peritoneal B-1a B cells in treml6

-/-

mice.

Treml6 contains two intracellular ITIM motifs that can be bound by src homology 2 (SH2)

domain bearing proteins, such as SHP-1, SHP-2 and SHIP. Opposing to our results, mice that

carry a general mutation in Shp-1 (motheathen mice) or a specific Shp-1 deficiency in B cells

have increased numbers of B-1a B cells (114, 177). The signal-attenuating effects of SHP-1 are

believed to be mediated primarily via its binding to inhibitory receptors (178). B cells express

several of such ITIM-containing receptors, including CD72, CD22, Siglec-G, and paired Ig-like

receptor (PIR)-B (179). The phenotypes of mice lacking these inhibitory receptors show

deficiencies in B-1a B cell development (110, 111, 180-185). As discussed above, these receptors

as well as SHP-1 act by inhibiting BCR signals and we excluded this as the mode of action of

treml6. Conclusively, the current data contradict the possibility of SHP-1 binding to treml6.

A possible intracellular binding partner for treml6 is Bruton’s tyrosine kinase (btk), which

contains both SH2 and SH3 domains. Moreover, btk deficient mice, like treml6 deficient mice,

have reduced levels of B-1Ps in fetal livers (170). Thus, btk is involved in B cell development

prior to pre-BCR expression (170). On the other hand, opposing to treml6

-/-

mice, btk

-/-

mice

have increased numbers of B-1P in adult BM (171). In fact, the role of btk in BCR signaling is well

established and btk

-/-

mice show, like other mediators of BCR signaling, a defect in mature B-1 B

cells (186-188). Thus, treml6 activity through btk would have to be restricted to the fetal

microenvironment. However, unless binding experiments demonstrate interaction of Btk and

treml6, this interaction remains speculative.

In sum, this study ascribes a new function to the TREM receptor family, namely regulation of

lymphocyte development. We could show that TREM family member, treml6 is a positive

regulator of B-1a B cell development. The reduction in B-1 B cell specific progenitors in the fetal

liver, as well as the impairment in B-1a B cell engraftment of Rag1

-/-

mice by treml6

-/-

pre-B I

cells and normal engraftment after transfer of adult treml6

-/-

BM, point to developmental

defects during fetal hematopoiesis. The paucity of B-1a B cells in peritoneal cavities is possibly

caused due to impaired homing capacities of fetal-derived precursors, evidenced in reduced

expression of cxcr5 mRNA by in vitro generated treml6

-/-

pre-B I cells. Moreover, maintenance of

peripheral B-1a B cells is most likely not affected by the lack of treml6.

Due to increased B-1a- and decreased B-2-homeostatic turnover, peripheral treml6 deficient B-

1a and B-2 B cells were able to restore B-1:B-2 WT ratios in peritoneal cavities of treml6

-/-

mice

at the age of 17-23 weeks. The specific paucity in peritoneal B-1a B cells, but not other B-1 B cell

subsets, underlines the high specificity of treml6 in regulating solely the fetal development of B-

1a B cells and thereby supports the ‘lineage model’ and ‘unified model’ of B-1 B cell

development.

Nevertheless, it remains an open question whether B-1 precursors or the fetal

microenvironment or both depend on treml6. In addition, the modes of treml6 signal

transductions as well as extracellular ligands remain elusive. We excluded interference with BCR

signaling, but possible roles of btk and, in the context of TREM family function, TLR signaling

(discussed separately below), will have to be subjects of future studies. Since treml6 has no

known ortholog in humans, further studies on treml6 could provide new insights into

differences of murine and human B-1 B cell development.

Possible involvement of treml6 in TLR signaling

Most TREM family receptors were described to act as regulators of inflammation by either

amplifying or dampening TLR signaling. Even though we are missing direct evidence, it is likely

that this family characteristic, namely regulation of TLR signaling, also applies to treml6.

In line with this thinking, it is a very interesting fact that the role of TLR signaling in

hematopoiesis begins to be established. It has recently been described that HSC and

multipotent progenitors from humans (189-191), or mice (192) express TLRs and respond to

some of their ligands by secreting cytokines and producing preferentially a myeloid progeny,

namely DCs, macrophages, and granulocytes. This feedback to the site of hematopoiesis was

interpreted to be beneficial in fighting bacterial or viral infections as it ensures the rapid

production and thus replacement of the mainly short-lived myeloid cells (192, 193).

Interestingly, this does not only apply to HSC and common myeloid progenitors, but also to

CLPs. Mouse CLPs cultured in conditions that generate B lymphocytes, supplemented with TLR2,

TLR4 or TLR9 ligands, PAM3, LPS, and CpG, respectively, showed a greatly reduced B cell

production and yielded preferentially DC-like cells (192, 193). More relevant to our study is the

so far unpublished observation by Lalanne AI and Vieira P, that TLR9 ligand CpG controls the

pool size of pre-B 1 cells, through the induction of apoptosis of respective cells, in both fetal

liver and bone marrow (Lalanne AI and Vieira P, personal communication).

A link to potential regulation of TLR9 signaling by treml6 stems from in vitro proliferation

experiments (Fig. 10B), treml6

-/-

B-1a B cells proliferated marginally, but significantly stronger in

response to CpG. Moreover, another TREM family member PDC-TREM amplifies TLR9 signals

(CpG) through association with DAP12 and Plexin-A1 in pDCs (18) and in silico analysis showed

that treml6 shares striking homology (86.9% identity and 90% similarity) to the first 130aa of the

extracellular domain of PDC-TREM. In contrast to treml6, PDC-TREM signals by binding the

ITAM-containing adaptor protein DAP12. In most cases, ITAM and ITIM receptors control each

other by binding the same or similar ligands; in this regard treml6 was predicted to be the

counter-receptor of PDC-TREM (13). Conclusively, subjects of future studies would be to

investigate a) whether treml6 and PDC-TREM are in fact counter-receptors and b) whether

treml6 is linked to TLR9 signaling and involved in the CpG induced apoptosis of pre-B 1 cells

described by Lalanne AI and Vieira P and thereby regulates B-1a B cell development.

Independent of a putative connection of treml6 with TLR signaling future studies would also

have to comprise a) generation of a treml6 specific antibody to compare mRNA expression

profile with the protein expression profile, b) identification of the putative ligand of treml6, and

c) analysis of signaling pathways operating downstream of treml6.

5.2 ICOS co-stimulation shapes T cell responses against Mtb

Acquired T-cell immunity is crucial for efficient control of Mtb infection, and its failure results in

TB disease outbreak (124, 125, 127). We studied the contribution of ICOS co-stimulation on

generation of protective T-cell immunity against Mtb in mice. Analysis of Mtb burden and tissue

pathology in WT and ICOS

-/-

mice revealed no apparent differences at the primary site of

infection, the lungs. In contrast, Mtb burden in spleens was significantly reduced in ICOS

-/-

mice

in the late phase of infection, suggesting that ICOS signaling influenced control of Mtb during

chronic stages. This notion is consistent with exclusive ICOS expression on activated T cells

which formerly led to the conclusion that ICOS sustains functions of effector and/or memory T

cells rather than participating in priming of naïve T cells (68, 81, 194).

The initial acute CD4

Th1 response was comparable in WT and ICOS

-/-

mice. In contrast, in the

late stage of Mtb infection we observed significantly higher frequencies of total IFN-γ secreting

CD4

effector T cells in lungs and spleens of ICOS

-/-

mice. This was particularly interesting since

in the steady state in naïve ICOS

-/-

mice, CD4

effector and effector memory T cells are reduced

(85). Furthermore, the majority of studies addressing a role of ICOS in infection with

intracellular pathogens revealed that in the absence or blockage of ICOS signaling, CD4

T cell

responses remained either unaffected (e.g lymphocytic choriomeningitis virus (LCMV) (77)), or

were reduced (e.g. Leishmania mexicana, Nippostrongylus brasiliensis, Toxoplasma gondii and

vesicular stomatitis virus (77, 79, 81)). In murine listeriosis, inhibiting ICOS signaling impaired

Listeria monocytogenes-specific CD4

, as well as CD8

T cell responses, and resulted in higher

susceptibility of mice (76). In contrast to the studies described above, but similar to our

observations in murine TB, genital tract infection with Chlamydia trachomatis increased CD4

Th1 responses in ICOS

-/-

mice compared to WT controls (78). On the whole, depending on the

type of infection analyzed, absence of ICOS signaling can lead to reduced, equal, or as for C.

trachomatis and Mtb even increased CD4

T cell responses.

It has been proposed that all effector T-cell populations express ICOS (85). We found that during

murine Mtb infection CD4

, but not CD8

effector T cells surface expressed ICOS. Moreover,

comparison of naive CD8

T cells with PepA(Mtb)-specific effector CD8

T cells, did not reveal

increased ICOS density (data not shown). Yet, the CD8

T cell response was affected by the lack

of ICOS, manifested in a weaker Mtb-specific CD8

T cell response during the late stage of Mtb

infection. Our data only allow speculations on whether this effect is caused by either CD8

T-cell

intrinsic or extrinsic mechanisms. Several studies have analyzed influence of ICOS on CD8

T cell

responses (76, 80, 195-197). In most infection models, (e.g. Nippostrongylus brasiliensis, LCMV

and vesicular stomatitis virus infection) the CD8

T cell response remained normal in the

absence of ICOS signaling (77). However, if CD8

T cell responses depended on ICOS signaling

(76, 80), it was difficult to discriminate between direct effects of ICOS signaling on CD8

T cells,

and indirect effects e.g. ICOS dependent CD4

T cell help (198, 199). To answer this question

Vidric et al. performed in vitro stimulation assays showing that αICOS mAb can provide co-

stimulation

to naïve CD8

T cells directly in the absence of CD4

T cell help (80). Yet, this

experiment does not fully reflect the in vivo situation during an infection, where CD4

T cells

strongly up-regulate ICOS, while CD8

T cells do not. Indeed, in the presence of inflammatory

stimuli CD8

T cell responses can be generated without CD4

T cell help. Yet, memory CD8

T cell

responses will be defective without CD4

T cell help during primary responses (166, 198, 199).

Further, CD4

T cells are important for CD8

memory T cell survival, as evidenced by the decline

of memory CD8

T cells in mice lacking CD4

T cells (200, 201). Moreover, during viral infections

with persisting antigen, chronically stimulated CD8

T cells require

CD4

T cells for survival (202-

204). It is tempting to speculate that ICOS signaling is involved in this dependence. In chronic

infections, when CD8

T cell survival depends on CD4

T cells (202-204), activated CD4

T cells

strongly express ICOS which promotes their interactions with APCs or epithelial cells expressing

ICOS-L. Such interactions in turn can stimulate CD40L surface expression on CD4

T cells (74,

205). CD40L can then interact directly with CD8

T cells expressing CD40 or indirectly with CD40

expressed on APCs, licensing them to stimulate CD8

T cell responses (41-43, 198, 199, 206,

207). Consequently, lack of ICOS would lead to reduced CD8

T cell responses during secondary

challenge or in the chronic phase of infection as observed by us in murine TB.

Generation of CD8

effector memory, rather than central memory T cells depends on CD40

stimulation (166). It is possible that the described feedback loop is already operative during the

priming phase, promoting the generation of effector memory CD8

T cells. Indeed, in the

absence of ICOS signaling we observed a reduction of effector memory, but not central memory

CD8

T cells on day 120 of Mtb infection in lungs. CD4

T cells can help CD8

T cells via IL-2 (41).

Diminished IL-2 secretion by CD4

T cells could therefore also impair CD8

T cell responses. We

consider this scenario of CD4

T cell help less likely in our study, since we observed as many IL-2

secreting CD4

T cells in ICOS

-/-

mice as in WT mice (data not shown). Studies analyzing the

biological role of CD8

T cells in TB revealed increased bacterial load from day 90 in CD8α-

deficient mice (163, 164). Analysis of these and other CD8

T cell defective mouse strains

stressed the importance of CD8

T cells in control of chronic pulmonary TB (163, 164). Although,

we observed impaired CD8

T cell responses during the chronic phase of Mtb infection, ICOS

-/-

mice showed improved control of Mtb at this stage. We conclude that the subtle reduction in

CD8

T cell responses in ICOS

-/-

mice did not suffice to increase susceptibility to Mtb, and was

perhaps compensated for by elevated CD4

Th1 responses.

The role of Treg during Mtb infection has already been addressed by several studies which

generally indicate that Treg suppress protective Th1 responses against Mtb (64-66). Since Treg

express high levels of ICOS during murine Mtb infection, we were interested in how the absence

of ICOS signaling would influence their response (66). Our studies revealed constant Treg

numbers during chronic murine TB in ICOS

-/-

mice, compared to WT mice, in which numbers of

Treg continuously increased throughout Mtb infection. This is consistent with a role of ICOS as

survival factor for Treg in the steady state in naïve mice and humans (85, 208). Moreover,

Umetsu and colleagues showed that antigen-specific Treg which protect from allergen-induced

airway hyperreactivity in the lung mucosa develop via the ICOS-ICOS-L pathway (209). Since

Treg have been shown to suppress Th1 responses against Mtb, we assume that their reduction

allowed a stronger Th1 response as evidenced by increased IFN-γ secretion by bulk CD4

T cells

in ICOS

-/-

mice. In genital tract infection with C. trachomatis ICOS drives Th2 immunity and anti-

inflammation through IL-10 production and promotion of Treg populations (78). In line with

these results is the finding that blockage of ICOS co-stimulation during priming in Experimental

Autoimmune Encephalomyelitis (EAE) leads to increased IFN-γ secretion and Th1 polarization

causing exacerbated immunopathology (84). It is tempting to speculate that during Mtb

infection ICOS deficiency not only reduced Treg but also Th2 responses leading to enhanced IFN-

γ secretion by CD4

T cells (210, 211).

Impact of ICOS deficiency on T cell responses differed in strength between the organs. Overall,

Mtb-peptide specific CD4

Th1 responses developed normal in the absence of ICOS co-

stimulation, whereas bulk IFN-γ

-secreting CD4

T cell responses were increased in the late

phase of Mtb infection. In addition, ICOS signaling was necessary for the maintenance of Mtb-

specific CD8

T cell - and Treg responses. In combination however, these effects at the level of

single T cell subpopulations influenced each other. Thus mutual interdependency could explain

minor effects on control of Mtb infection. Notably, ICOS deficiency did not affect Mtb load in

lungs. We assume that beneficial effects of increased CD4

Th1 cell responses were countered

by the detrimental reduction of pulmonary CD8

T cell responses. Reduced Mtb numbers in

spleens of ICOS

-/-

mice resulted most likely from increased protective IFN-γ secreting CD4

cells, in combination with marginally reduced Mtb-specific CD8

T cell responses. Thus different

outcomes in distinct organs were the result of differential balances between T cell populations

notably CD4

Th1, Treg, and CD8

T cells.

6 SUMMARY

6.1 Treml6 regulates B-1a B cell development

TREM receptors are of particular interest in infection immunology since they are able to

regulate inflammatory responses by either amplifying or dampening TLR signals (12). Here we

investigated the immunological function of TLT-6 (gene: treml6), a so far undescribed ITIM-

containing receptor of the TREM protein family (12, 13) using treml6-WT and in-house

generated treml6

-/-

mice.

Treml6 mRNA was broadly expressed among leukocytes of the peritoneal cavity and lymphoid

tissues. Treml6

-/-

mice were fertile and showed normal growth. Analysis of their immune cell

composition revealed reduced numbers of B-1 B cell specific precursors in fetal liver and adult

BM of treml6

-/-

mice. Likely as a consequence of this reduction, young and adult treml6

-/-

mice

bared reduced numbers of peritoneal B-1a B cells. Notably, solely peritoneal B-1a B cells, but no

other B-1 or B-2 B cell subsets were affected by the lack of treml6. No abnormalities in

maintenance of B-1a B cells or in their functionality, such as increased apoptosis rates of B cells

or altered antibody responses, respectively, were evident in treml6

-/-

mice. Moreover, in

contrast to most B-1 B cell defects described in the literature, the defect observed in treml6

-/-

mice was independent of BCR signaling. By means of transplantation experiments it became

apparent that the origin of the peritoneal B-1a B cell defect in treml6

-/-

mice was rooted in a

defect during the fetal B cell development. Transplantation of treml6

-/-

adult BM cells resulted in

normal - WT-like - B cell engraftment of Rag1

-/-

mice. In contrast, transplantation of treml6

-/-

fetal liver-derived pre-B I cells resulted in poorer B-1a B cell engraftment of recipient Rag1

-/-

mice when compared to WT pre-B I cells.

In sum, we propose that treml6 is a positive regulator of fetal B-1a B cell development. Hence,

our data provide new insights into B-1a B cell development; an area of research that is still

controversial. Moreover, our study expands the knowledge of the TREM receptor family by

ascribing it an additional function, namely regulation of lymphocyte development.

6.2 ICOS co-stimulation shapes T cell responses against Mtb

Mtb is still a global health threat. T cells, notably CD4

Th1 cells and their major effector

cytokine IFN-γ are crucial for protective immunity against Mtb. However, the immune responses

including the T cell response are not sufficient to eliminate the bacilli and to provide sterile

immunity. In an initial attempt to identify factors that shape the Th1 response, we observed

that CD4

Th1 cells, notably IFN-γ-secretors, co-expressed ICOS during murine TB. These data

pointed to ICOS as an important player in the formation of Th1 responses against Mtb.

To gain detailed information about the kinetics and nature of the T cell response in absence of

ICOS signaling, we infected WT and ICOS

-/-

mice with Mtb. ICOS deficiency resulted in an

increased polyclonal CD4

Th1 response against Mtb, most likely caused by robustly reduced

numbers and frequencies of Treg in these mice. In contrast to the CD4

Th1 response, the Mtb-

specific CD8

T cell response was reduced in the absence of ICOS, but only during the later stage

of infection. In addition, not only Mtb-specific effector CD8

T cells, but also effector memory

CD8

T cells were reduced in Mtb infected ICOS

-/-

mice. The reduction in CD8

T cells was most

likely not CD8

T cell intrinsic – since CD8

T cells revealed only marginal ICOS surface

expression, but caused by impaired CD4

T cell help.

In sum, we confirmed our initial assumption; ICOS indeed influenced the Th1 cell response

against Mtb, and moreover presence of ICOS was mandatory for normal Treg responses. The

extent to which ICOS influenced single T cell populations, notably CD4

Th1, Treg, and CD8

cells, and conclusively the interaction among themselves, differed between spleen and lung. As

a result, ICOS

-/-

mice showed improved control of Mtb in the spleens, but not in the lungs during

the late chronic phase of infection.

7 ZUSAMMENFASSUNG

7. 1 Treml6 reguliert die Entwicklung von B-1a B Zellen

TREM Rezeptoren sind aus infektionsimmunologischer Sicht von großem Interesse, da sie TLR

Signale inhibieren bzw. verstärken können und somit in der Lage sind, inflammatorische

Immunantworten zu regulieren (12). Diese Doktorarbeit untersucht die immunologische

Funktion von murinem TLT-6 (Gen: treml6), einem bislang unerforschten ITIM-tragenden

Rezeptor der TREM Rezeptor Familie (12, 13), mittels Analyse von treml6-WT und eigens

generierten treml6

-/-

Mäusen.

Treml6 mRNA wurde vornehmlich von Leukozyten der Peritonealhöhle und lymphoiden

Organen exprimiert. Treml6

-/-

Mäuse waren fortpflanzungsfähig und zeigten normales

Wachstum. Die Analyse ihrer Immunzellzusammensetzung zeigte, dass treml6

-/-

Mäuse eine

verringerte Anzahl an B-1 B Vorläuferzellen in der fetalen Leber und im adulten Knochenmark

aufwiesen. Möglicherweise bedingt durch diese Reduktion an Vorläuferzellen, hatten sowohl

junge als auch erwachsene treml6

-/-

Mäusen weniger peritoneale B-1a B Zellen als WT Mäuse.

Andere B-1 oder B-2 Zellpopulationen in treml6

-/-

Mäusen waren hingegen nicht beeinträchtigt.

Ferner wiesen treml6

-/-

Mäusen keine Defekte in der Aufrechterhaltung von B-1a B Zellen in der

Peripherie, wie zum Beispiel eine erhöhte Apoptosisrate der B Zellen, noch Unterschiede in der

Funktionalität der B Zellen, wie z.B. abnormale Antikörpersekretion, auf. Der B-1a B Zelldefekt in

treml6

-/-

Mäusen kann, im Gegensatz zum Großteil der in der Literatur bekannten B-1 B

Zelldefekte, ebenfalls nicht auf eine veränderte Signaltransduktion des BCR zurückgeführt

werden. Die Ursache für die Reduktion der peritonealen B-1a B Zellen vermuten wir vielmehr in

einem Defekt im fetalen Stadium der B-1a B Zellentwicklung in treml6

-/-

Mäusen. Hinweise

hierfür lieferten Zelltransferexperimente. Entsprechend resultierte die Transplantation von

adultem treml6

-/-

KM in Rag1

-/-

Mäuse in einer normalen Rekonstitution aller B Zellpopulationen

des Rezipienten. Transplantierten wir jedoch aus treml6

-/-

fetalen Leberzellen generierte pre-B I

Zellen, so zeigte sich, dass sie im Vergleich zu WT pre-B I B Zellen, eine beschränkte Fähigkeit

aufwiesen, das B-1a B Zellkompartiment im Rag1

-/-

Rezipienten zu füllen.

Zusammenfassend schlussfolgern wir, dass treml6 ein positiver Regulator der fetalen B-1a B

Zellentwicklung ist. Unsere Daten liefern somit neue Einblicke in die Regulation der B-1a B

Zellentwicklung, einem nach wie vor stark diskutierten Forschungsfeld. Ferner haben unsere

Untersuchungen die Erkenntnisse über die TREM Rezeptor Familie um eine weitere Funktion,

nämlich ihre Beteiligung an der Regulation der Lymphozytenentwicklung, erweitert.

7.2 Kostimulation durch ICOS prägt die T Zellantwort gegen Mtb

Mtb ist weiterhin als globale gesundheitliche Bedrohung anzusehen. T Zellen, insbesondere

CD4

Th1 Zellen und ihr wichtigstes Effektorzytokin IFN-γ, sind für eine schützende

Immunantwort gegen Mtb entscheidend. Dessen ungeachtet gelingt es der Immunantwort

inklusive der T Zellantwort nicht, die Bakterien vollständig zu eliminieren und Sterilität zu

erreichen. Im Zuge vorrausgehender Bemühungen, Faktoren zu identifizieren, die die Th1

Antwort gegen Mtb beeinflussen, stellten wir fest, dass in der murinen Mtb Infektion ein

Großteil der IFN-γ sekretierenden CD4

Th1 Zellen ICOS exprimierten. Dementsprechend lag die

Vermutung nahe, dass ICOS eine wichtige Rolle in der Ausbildung der Th1 Antwort gegen Mtb

spielt.

Wir infizierten WT und ICOS

-/-

Mäuse mit Mtb, um ein detailiertes Bild der Kinetik und der Art

der T Zellantwort in Abwesenheit von ICOS-Kostimulation zu erhalten. ICOS-Defizienz führte zu

einer verstärkten polyklonalen CD4

Th1 Antwort gegen Mtb. Die Ursache hierfür lässt sich in

der stark reduzierten Anzahl und Frequenzen der Treg in ICOS

-/-

Mäusen vermuten. Im

Gegensatz zur CD4

Th1 war die Mtb-spezifische CD8

T Zellantwort in ICOS

-/-

Mäusen in der

chronischen Phase der Infektion abgeschwächt. Die Reduktion betraf nicht nur Effektor CD8

Zellen, sondern auch Effektor-Gedächtnis CD8

T Zellen in Mtb infizierten ICOS

-/-

Mäusen. Da nur

eine marginale ICOS Expression auf CD8

T Zellen während der Mtb Infektion festzustellen war,

lässt sich die Reduktion der Mtb-spezifischen CD8

T Zellen nicht auf intrinsische Defekte

zurückführen, sondern wurde wahrscheinlich durch beeinträchtigte CD4

T Zell-Hilfe in ICOS

-/-

Mäusen verursacht.

Insgesamt bestätigten wir unsere Anfangsvermutung, dass ICOS die Th1 T Zellantwort gegen

Mtb maßgeblich beeinflusst und konnten zusätzlich zeigen, dass die Stärke der Treg Antwort

ICOS-abhängig war. Das Ausmaß, in dem ICOS die einzelnen T Zellpopulationen wie CD4

Th1,

Treg und CD8

T Zellen und somit auch ihre Interaktion untereinander beeinflusste, unterschied

sich zwischen Milz und Lunge. So zeigten ICOS

-/-

Mäuse in der späten chronischen Phase der

Mtb Infektion eine bessere Kontrolle der Erreger in der Milz, nicht aber in der Lunge.

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9 ACKNOWLEDGEMENTS

Mein besonderer Dank gilt Prof. Stefan H. E. Kaufmann, dafür dass er mir ermöglichte diese

Doktorarbeit in seiner Abteilung durchzuführen. Ich möchte Ihm auch für die anregenden

Diskussion, seine Betreuung und vor allem seine Unterstützung danken, als das „treml“ Projekt

uns unerwarteterweise in infektionsfreie B-Zellgefilde führte.

Mein ausdrücklicher Dank gilt auch Herrn Prof. Roland Lauster für seine Tätigkeit als Betreuer

und Ratgeber. Ich danke auch Prof. Dr. Jens Kurreck für seine Bereitschaft Gutachter dieser

Doktorarbeit zu sein.

Dr. Mischo Kursar und Dr. Markus Koch möchte ich ebenfalls von ganzem Herzen danken, für

die Betreuung dieser Doktorarbeit, für die zahlreichen und lehrreichen Diskussion, für ihre

Unterstützung und für ihr Vertrauen. Diese Arbeit wäre nicht möglich gewesen ohne ihren

unermüdlichen Einsatz. Ich möchte auch Dr. Mischo Kursar dafür danken, dass er mich weiter

betreut hat und bei Sorgen und Problemen für mich da war, nachdem er das MPIIB bereits

verlassen hatte und dafür, dass er diese Arbeit korrekturgelesen hat.

Ich möchte für die Mitarbeit am ICOS Projekt Dr. Tracey Walker, Pia Gamradt, Steffi Kuhlmann

und Delia Loewe danken. Tausend Dank geht auch an Dr. Anca Dorhoi und Lydia Pradl für ihre

Unterstützung bei der histologischen Untersuchung. Mein Dank gilt auch Andraes Hutloff für die

ICOS Mäuse und Diskussionen der Daten. Im Rahmen des treml6 Projektes möchte ich

insbesondere Szandor Simmons danken, für die experimentelle Unterstützung und für

Nachhilfestunden in B Zellimmunologie. Mein Dank gilt ebenfalls Prof. Melchers und Marco

Knoll für ihre Unterstützung und Diskussionen. Vielen Dank auch an Steffi Kuhlmann für ihren

unermüdlichen Einsatz.

Nicht unerwähnt bleiben sollten auch January Weiner, vielen Dank für die Nachhilfestunde in in

silico Analyse, Christian Köberle für das FACS Analyser Programm, Julia Jellusova für die TI-2

Immunisierungsprotokolle und Robert Hurwitz für die PepA-Tetramere.

Danken möchte ich auch allen Kollegen und Doktoranden für eine herzliche, inspirierende und

motivierende Atmosphäre in und außerhalb des Instituts. Insbesondere danke ich Dr. Christian

Ganoza, der so freundlich war diese Arbeit ebenfalls Korrektur zu lesen. Auch meiner Freundin

Lisa Marit Otte möchte ich dafür danken, dass sie diese Arbeit korrekturgelesen hat.

Meine Liebsten zum Schluss, ein riesen Dank für ihre Unterstützung geht an meine Freunde,

Mischo, Tracey, Lisa, Katrin und Anke und an meine Familie. Julien, mein Bruderherz, Du bist

mein Fels in der Brandung. Papa, Dein naturwissenschaftliches Wissen, von Pflanzen und Tieren

bis zur Physik war mir viele Jahre ein unerschöpflich scheinendes lebendiges Lehrbuch. Merci.

An meine Mama unendlichen Dank natürlich dafür, dass Du immer für mich da warst und bist,

aber auch dafür, dass Du mich immer darin bestärkt hast neugierig zu sein, die Welt zu

hinterfragen und mir meine eigene Meinung zu bilden. Tausend Dank.

10 ABBREVIATIONS

α anti

aa amino acid

Ag85A Antigen 85A

Ag85B Antigen 85B

AP alkaline phosphatase

APC antigen presenting cell

β-actin beta-actin

B-1P B-1 B cell progenitor

BCG Bacille Calmette-Guérin

BCR b cell receptor

BM bone marrow

BrdU bromodeoxyuridine

BSA bovine serum albumin

btk Bruton’s tyrosine kinase

calcium

CCR chemokine (C-C motif) receptor

CD cluster of differentiation

CD4

Th1 cells CD4

T cells of Th1 type

cDNA complementary DNA

CFP10 culture filtrate protein 10

CFSE carboxy-fluorescein diacetate succinimidyl ester

CFU colony forming units

CLPs common lymphoid progenitors

CLRs C-type lectin receptors

carbon dioxide

CpG ODN oligodinucleotides containing CpG motifs

Cre causes recombination

cRPMI complete RPMI

threshold cycle

CTL cytotoxic T lymphocytes

CXCL chemokine (C-X-C motif) ligand

CXCR chemokine (C-X-C motif) receptor

DAP-12 DNAX activating protein of 12kDa

DC dendritic cell

ddH

O double distilled H

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetic acid

EEJ exon-exon junctions

ELISA enzyme-linked immuno sorbent assay

ES cells embryonic stem cells

100

ESAT6 six kilodalton early secretory antigenic target

F(ab’)

αIgM F(ab’)

fragment goat anti-mouse IgM

FACS fluorescence activated cell sorting

FCS fetal calf serum

FMO fluorescence minus one

FO follicular

FoxP3 forkhead box transcription factor P3

γδ T cells gamma delta T cells

gapdh glyceraldehyde-3-phosphate dehydrogenase

H&E hematoxylin and eosin Y

HA homology arm

HIV human immunodeficiency virus

HSC hematopoietic stem cells

i.p. intraperitoneal

i.v. intravenous

ICOS inducible co-stimulatory molecule

ICOS-L ICOS-ligand

ICS intracellular cytokine staining

IFNγ interferon-gamma

Ig immunoglobulin

IL interleukin

IMDM Iscove‘s modified Dulbecco‘s medium

ITAM immunoreceptor tyrosine-based activating

ITIM immunoreceptor tyrosine-based inhibitory

ITIM-Rs ITIM-containing receptors

iTreg induced Treg

KO knock-out

lacZ beta-galactosidase

LCMV lymphocytic choriomeningitis virus

LLO listeriolysin

loxP locus of X-ing over

LPS lipopolysaccharide

mAb monoclonal antibody

MEM minimum essential medium

MHC major histocompatibility complex

MHCI MHC class I

MHCII MHC class II

MLN mesenteric lymph nodes

Mtb Mycobacterium tuberculosis

MZ marginal zone

NLRs

nucleotide-binding domain and leucine-rich repeat containing

molecules

NOD nucleotide oligomerization domain

101

nTreg naturally occurring Treg

OP9-PH puromycine-hygromycine-resistant OP9 cells

p.i. post infection

PBS phosphate buffered saline

PBST PBS/Tween solution

PCR poly chain reaction

pDC plasmacytoid dendritic cell

PepA Mtb32A-derived peptide

PFA paraformaldehyde

PIR paired Ig-like receptor

PMN polymorphonuclear leukocytes

PMT photo multiplier tubes

PRR pattern recognition receptors

Rag recombination-activating gene

rIL-7 recombinant IL-7

RLRs RIG-I-like receptors

RNA ribonucleic acid

RORγt retinoic acid-related orphan receptor γ expressed in T cells

RPMI Roswell Park Memorial Institute

RT room temperature

RT-PCR real-time polymerase chain reaction

SH2 src homology 2

SH3 src homology 3

SHP-1 SH2 domain containing phosphatase-1

STAT signal transducer and activator of transcription

tACE

testes-specific promoter from the angiotensin-converting enzyme

gene

TB tuberculosis

central memory T cells

TCR T cell receptor

effector memory T cells

TGF-ß transforming growth factor beta

Th1 cells helper T cells type 1

Th2 cells helper T cells type 2

Th17 cells helper T cells type 17

TI T-independent

TI-2 thymus independent-type-2

TK thymidine-kinase

TLT TREM-like transcript (alternative name for TREML, used for protein)

TNF-α tumor necrosis factor alpha

TNP-Ficoll trinitrophenol

Treg regulatory T cells

TREM triggering receptors expressed on myeloid cells

102

TREML TREM-like (alternative name for TLT, used for genes)

T-bet T box transcription factor

heavy chain variable region

WHO World Health Organization

WT wild-type

XDR extensively drug-resistant

7-AAD 7-amino-actinomycin D

103

APPENDIX 1: MATERIAL

APPENDIX 1.1: Buffers and solutions

Standard laboratory chemicals used to prepare buffers were purchased from Sigma, Merck or

Roth. Solutions were prepared with a Millipore purified H

O and sterilized by autoclaving for 25

min at 121°C or filter-sterilized through a 0.2 µm membrane.

1x phosphate buffered saline (1x PBS) 8 g NaCl

0.2 g KCl

1.44 g Na

*2H

0.24 g KH

add to 1000 ml with H

PBS, 0.2% bovine serum albumin 2 g BSA

(PBS/BSA) solution add to 1000 ml with 1x PBS

PBS, 0.1% bovine serum albumin 1 g BSA

add to 1000 ml with 1x PBS

4% Paraformaldehyde (4% PFA)

solution

40 g PFA

add to 1000 ml with 1x PBS

stir O/N at 50°C, keep dark

Saponin buffer 5 g saponin

500 ml PBS/BSA

add to 1000 ml with H

Red Blood Cell Lysis buffer 8.29 g NH

1 g KHCO

0.037 g EDTA

add to 1000 ml with H

Trypan blue solution 10 ml 10x trypan blue

add to 100 ml with 1x PBS

PBST (PBS/Tween solution) 5 ml Tween20

add to 1000 ml with 1x PBS

40% Percoll/RPMI 40 ml Percoll

add to 100 ml with RPMI solution

70% Percoll/RPMI 70 ml Percoll

add to 100 ml with RPMI solution

TAE 1x 40 mM TRIS (pH 8.0)

1 mM EDTA

Krebs-Ringer solution [+CaCl2] 140 mM NaCl

4 mM KCl

1 mM MgCl

104

10 mM D-Glucose

10 mM HEPES (pH 7.4)

with or without 1 mM CaCl

add to 1000 ml with ddH

sterile filtration, store at 4°C

Proteinase K buffer 50 mM TRIS (pH 8.0),

100 mM NaCl,

100 mM EDTA,

1% SDS

in dH2O

TE 10 mM TRIS (pH 8.0),

1 mM EDTA

in dH2O

6x Gel loading dye 30% Glycerol

0.25% Bomophenol blue

in dH20

ELISA blocking solution

1 % BSA

0.05% sodium azide

in 1x PBS

ELISA dilution buffer 0.1 % BSA

0.05 % sodium azide

FACS Clean BD Pharmingen

FACS Rinse BD Pharmingen

APPENDIX 1.2: Media and cell culture reagents

IMDM Gibco

MEM alpha medium Gibco

RPMI (L-Glutamine) Gibco

D-MEM Gibco

Opti-MEM Gibco

PBS Dulbeccos (low endotoxin) Biochrom AG

FCS Sigma-Aldrich

L-Glutamine Gibco

2-Mercapto-ethanol Fluka

Trypsin/EDTA 5% Gibco

Insulin Sigma-Aldrich

Penicillin/Streptomycin (100x) PAA

MEM non-essential amino acids Gibco

Primatone RL Quest

RPMI-10 complete medium RPMI media plus:

105

0.2 mM L-Glutamine

10 U/ml Penicillin/Streptomycin

10 mM HEPES buffer

0.05 mM β-mercaptoethanol

10% heat inactivated (1 h 65°C) FCS

Collagenase medium RPMI-10 complete medium plus:

0.3 mg/ml Collagenase D

0.7 mg/ml Collagenase VIII

Freezing medium 45 ml FCS

5 ml DMSO

5% RPMI RPMI media plus:

5% heat inactivated FCS

IMDM based serum-free medium 5 l dddH2O

1 can IMDM powder, dissolve

30.24 g NaHCO3

100 ml Kanamycin Sulphate (100x)

100 ml MEM non-essential amino acids solution

(100x)

10 ml insulin (5 mg/ml), sterile

10 ml 2-Mercapto-ethanol (50 mM)

30 ml Primatone RL 10% solution, sterile

Fill up with triple-distilled H2O to 10 l total volume

Set pH to 7 with NaOH 10 M

Filtrate with 1 l sterile filter-units (0.22 µm) into 1 l

sterile flasks

αMEM based serum-free medium 5 l dddH2O

1 can αMEM powder, dissolve

20.02 g NaHCO3

100 ml Kanamycin Sulphate (100x)

100 ml MEM non-essential amino acids solution

(100x)

10 ml insulin (5mg/ml), sterile

10 ml 2-Mercapto-ethanol (50mM)

30 ml Primatone RL 10% solution, sterile

Fill up with triple-distilled H2O to 10 l total volume

Set pH to 7 with NaOH 10 M

Filtrate with 1 l sterile filter-units (0.22 µm) into 1 l

sterile flasks

APPENDIX 1.3: Reagents

0.1 M DTT Invitrogen

1 kb Plus DNA Ladder Invitrogen

106

10 mM dNTP Mix Invitrogen

10% Pluronic F127 Invitrogen

10 mM dNTP set Invitrogen

2-Propanol Carl Roth

2x Fast SybrGreen PCR Master Mix Applied Biosystems

2x SybrGreen PCR Master Mix Applied Biosystems

5x First-Strand Buffer Invitrogen

7-AAD BD Pharmingen

αCD40 [4.49µg/µl] DRFZ

AffiniPure F(ab’)

Fragment Goat Anti-Mouse IgM Jackson ImmunoResearch

Agarose (NEEO) Carl Roth

Ampicillin Sigma Aldrich

Bio-Rad Protein Assay Bio-Rad

BrefeldinA Sigma-Aldrich

CellTrace CFSE Invitrogen

Chloroform, 99% Sigma-Aldrich

CpG ODN 1826 invivoGen

Cyclohexamide Sigma-Aldrich

Dimethyl Sulfoxide Sigma-Aldrich

Distilled water, DNase, RNase free Gibco

EDTA Carl Roth

Ethanol Carl Roth

Ethanol absolut Merck

Ethidium bromide solution Carl Roth

Glycerol Sigma-Aldrich

GM-CSF (5x10

U/ml) Active Bioscience

HCL Sigma-Aldrich

HEPES Solution Sigma-Aldrich

IL-4 (mouse) Active Bioscience

Indo-1, AM, 1 mM solution in anhydrous DMSO Invitrogen/Molecular Probes

Ionomycin Invitrogen

Isopropanol Merck

Kanamycin Sulphate (100x) Sigma-Aldrich

KCl Sigma-Aldrich

Ketamin Bayer

Sigma-Aldrich, Seelze

LPS (Salmonella typhimurium SL1344) (1 mg/ml) invivoGen

mAb CD28 (1,15 mg/ml) ATCC

mAb CD3 (2,2 mg/ml) ATCC

HPO

Sigma-Aldrich

107

NaCl Sigma-Aldrich

NaHCO

Sigma-Aldrich

NaOH Sigma-Aldrich

PAM3CSK4 (1 mg/ml) invivoGen

Paraformaldehyd (PFA) Sigma

Percoll Biochrom

Poly (I:C) (10 µg/µl) invivoGen

p-Nitrophenyl Phosphate Tablets, SIGMA FAST

Sigma-Aldrich

Saponine Carl Roth

SDS Solution 20% Fluka

Streptavidine Jackson ImmunoResearch

TRIS Carl Roth

Triton X-100 Sigma-Aldrich C

TRIzol Reagent Invitrogen

Trypan blue Sigma-Aldrich

Tween20 Sigma-Aldrich

APPENDIX 1.4 Plastic ware

0.2-ml Thermo Strip Thermo Fischer

14-ml tube, round bottom BD Pharmingen

15-ml, 50-ml tube, conical bottom Sarstedt

24 well cell culture cluster Nunc

48 well cell culture plate, flat bottom Corning

70-µm Filters BD Pharmingen

96 MicroWell™ Plate Nunclon Delta - 96x0,36 cm² Nunc, Roskilde

96 well cell culture plate, U bottom Cellstar

BD Falcon

Polysterene Round-Bottom Tube 5ml BD Pharmingen

BD Microtrainer (serum seperator) BD Pharmingen

BD PlastipakTM 1-ml Sub-Q BD Pharmingen

Cap Lock 0.2-ml Thermo Strip Thermo Fischer

Cell strainer, nylon 40-µm BD Pharmingen

CFU-bags (Whirl Pak) Nasco

Costar®Stripette 50-ml, 25-ml, 10-ml, 5-ml Corning Life Scienes

Hypodermic needle (1.20x40 mm; 0.7x 30 mm) B. Braun

MACS Separation Columns (MS; LS Columns) Miltenyi

MACS® Pre-Separation Filters 30-µm Miltenyi

Maxisorp Plate for ELISA 96 well Nunc

MicorAmp

Optical 96-Well Reaction Plate Applied Biosystems

108

MicroAmp™ Fast Optical 96-Well Reaction Plate Applied Biosystems

Microtube 0.5-ml, 1.5-ml, 2.0-ml Sarstedt

Omnifix syringe (5-ml, 10-ml, 20-ml) B. Braun

Petri dish 92 x 16 mm Sarstedt

Pipet tips Fischer Scientific

Rotilabo® - cassettes for biopsies Carl Roth

Surgical disposable scalpel Carl Roth

Tissue culture flasks (75 cm

and 150 cm

) TPP

Vacuum Filter (0.22 µm GP Express Plus Membrane) Millipore

APPENDIX 1.5: Antibodies

Abbreaviations used in application section; IVR: in vitro restimulation, FC: flow cytometry

surface staining, FC-ICS: flow cytomertry intracellulary cytokine staining, FC-INS: flow cytometry

intranuclear staining, B: blocking antibody, ELISA: enzyme linked immuno sorbent assay.

Specificity Clone Fluorochrome

/Enzyme

Application Source

AA4.1 (CD93) AA4.1 APC FC ebioscience

B220 RA3-6B2 PE-Cy7 FC ebioscience

B220 RA3-6B2 PacificBlue FC ebioscience

B220 RA3-6B2 eFluor

450 FC ebioscience

B7RP-1 (ICOS-L) MIL-5733 AlexaFluor647 FC RKI

CCR7 4B12 APC FC ebioscience

CD103 2E7 FITC FC ebioscience

CD11b 5C6 Cy5 FC ATCC

CD11b M1/70 PE-Cy7 FC BD Pharmingen

CD11b M1/70 APC-Cy7 FC BD Pharmingen

CD11c N418 Cy5 FC ATCC

CD11c N418 FITC FC ATCC

CD11c N418 Pacific Blue FC eBioscience

CD127 A7R34 PE-Cy7 FC eBioscience

CD19 ID3 PerCP-Cy5.5 FC BD Pharmingen

CD19 1D3 PE FC BD Pharmingen

CD19 ID3 APC-Cy7 FC ebioscience

CD19 ID3 PE-Cy7 FC ebioscience

CD21/CD35 eBio8D9 FITC FC ebioscience

CD23 B3B4 PE FC ebioscience

CD25 PC61 PerCP-Cy5.5 FC eBioscience

109

CD25 PC61.5 PE-Cy7 FC eBioscience

CD27 LG.3A10 FITC FC eBioscience

CD27 LG.7F9 APC FC eBioscience

CD28 37.51 IVR ATCC

CD3 145-2C11 IVR ATCC

CD4 RM4-5 PerCP FC BD Pharmingen

CD4 RM4-5 PacificBlue FC BD Pharmingen

CD4 FITC RM4-5 FC ebioscience

CD4 RM4-5 FITC FC eBioscience

CD40L MR1 FITC FC-ICS ATCC

CD40L MR1 PE FC eBioscience

CD43 1B11 FITC FC BioLegend

CD43 eBioR2/60 FITC FC ebioscience

CD44 IM7 Pacific Blue FC eBioscience

CD5 53-7.3 APC FC ebioscience

CD62L MEL-14 APC FC ATCC

CD62L MEL-14 APC-

APCAlexa750

FC ATCC

CD69 H1.2F3 PE-Cy7 FC BD Pharmingen

CD8α 53-6.7 PerCP FC BD Pharmingen

c-Kit ACK4 biotin FC ATCC

c-Kit ACK4 Cy5 FC ATCC

F(ab’)

αIgM

IVR Jackson

Fas (CD95) Jo2 PE FC ebioscience

Fc Receptor* 24G2 B ATCC

FoxP3 FJK/16S PE FC-INS eBioscience

GL7 GL7 FITC FC ebioscience

GR1 RB6-8C5 PacificBlue FC ATCC

GR1 RB6-8C5 PE-Cy7 FC ebioscience

ICOS 7E.17G9 AlexaFluor647 FC eBioscience

ICOS MIC280 Cy5 FC RKI

IFN-γ XMG1.2 PE-Cy7 FC-ICS ATCC

IgD 11.26C PacificBlue FC ATCC

IgD 11-26C PE FC ebioscience

IgG3 LO-MG3 AP ELISA Southern Biotech

IgM M41 Cy5 FC ATCC

IgM M41 biotin FC ATCC

IgM 1B4B1 AP ELISA Southern Biotech

IL-10 JES5-16E3 PE FC-ICS BD Pharmingen

IL-17 TC11-18H10.1 PE FC-ICS BD Pharmingen

110

IL-2 JES6-5H4 APC FC-ICS BD Pharmingen

MHCII T1B120 FITC FC ATCC

MHCII M5/114.15.2 APC FC ebioscience

NK1.1 PK136 PE-Cy7 FC BD Pharmingen

NK1.1 PK136 FITC FC eBioscience

NKp46 29A1.4 PE FC eBioscience

Rat serum αRat.-Ktr. B ATCC

TNF-α XT-22 FITC FC-ICS ATCC

APPENDIX 1.6: Primers

Primer for qRT-PCR or tail DNA PCR were synthesized by Metabion and delivered at a

concentration of 100 pg/mol.

name sequence 5' to 3' application

Random Primer

(3µg/ml)

NNN NNN cDNA-Synthese

b-actin fwd TGG AAT CCT GTG GCA TCC ATG AAA C qRT-PCR

b-actin rev TAA AAC GCA GCT CAG TAA CAG TCC G qRT-PCR

GAPDH fwd GCA ACT CCC ACT CTT CCA CCT TC qRT-PCR

GAPDH rev CCT CTC TTG CTC AGT GTC CTT GCT qRT-PCR

treml6 EEJ5 L TGAGGAAAGCTCGAAGAAAG qRT-PCR

treml6 EEJ5 R TGC TGA TTG AAG CTA GTG GT qRT-PCR

cxcl13 F CTC TCC AGG CCA CGG TAT T qRT-PCR

cxcl13 R TAA CCA TTT GGC ACG AGG AT qRT-PCR

cxcr5 F TGC AGA ACC GTG AAG ACA CCT G qRT-PCR

cxcr5 R TTC CCA GCT GGT TGT TGG ATG C qRT-PCR

ccr7 F AGG CCA TCA AGG TGA TCA TTG C qRT-PCR

ccr7 R TGT AGG GCA GCT GGA AGA CTA TG qRT-PCR

il-5 F AGC ACA GTG GTG AAA GAG ACC TT qRT-PCR

il-5 R TCC AAT GCA TAG CTG GTG ATT T qRT-PCR

B4 Tail WT fw1 (P11) GTG CTA GGA TTT TGG CTC ATA TAC PCR

B4-Tail WT rev1 (P12) TTG CTA CTT TGT ATT TTC TGA CCT ATA PCR

TP_B4_KO-sHA_ fw (P9)

GAC TCT GTC ACC TGC CTT CTG T PCR

TP_B4_KO-lacZ-rv (P10) TGG GGT CTT CTA CCT TTC TCT TC PCR

APPENDIX 1.7: Enzymes

Collagenas Type VIII from Clostridium histolyticum Sigma-Aldrich

111

Collagenase D from Clostridium histolyticum Roche

SuperScript III Reverse Transcriptase (200 U/µl) Invitrogen

GenTherm DNA- Polymerase Rapidozym

APPENDIX 1.8: Kits

B Cell Isolation Kit Miltenyi Biotech

Bio-Plex Cytokine Assay Bio-Rad

BrdU Flow Kit (APC) BD Pharmingen

Mouse Immunoglobulin Isotyping Kit Millipore

Mouse Regulatory T cell Staining Kit eBioscience

APPENDIX 1.9: Machines

7900 HAT Fast Real-Time PCR System AB Applied Biosystems

Bio-Rad Bio-Plex HTF System Bio-Rad Laboratories GmbH

Centrifuge 5417 R eppendorf AG

-incubator Nuaire Autoflow Zapf, Sarstedt

FACS-Sorter, FACSAria Cell Sorting System Becton Dickinson GmbH

Haemocytometer LO Labor Optik GmbH

Homogenizer, ULTRA-TURRAX T8 IKA Labortechnik

Hood, LaminAir HB 2448 Heraeus-Instruments

Incubator, Modell 800 Memmert

Lab-Shaker, KS 250basic IKA Labortechnik

Light microscope, LEICA DMLB Leica Microsystems AG

LSR II Becton Dickinson

Megafuge 2.0R Heraeus Instruments

MS1 Minishaker IKA Labortechnik

NanoDrop ND-1000 Spectrophotometer peqLab Biotechnologie GmbH

Pipetboy acu comfort IBS Integra Bioscience GmbH

Pipettes, FINNPIPETTE (0-10 µl, 0.3-3 µl, 3-

300 µl, 100-1000 µl) Thermo Electron GmbH

SpectraMAX 190 Microplate Reader with

PatchCheck Molecular Devices

Thermomixer 5431 eppendorf AG

Vacuumfiltrationsystem, MultiScreen

vacuum manifold Millipore GmbH

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APPENDIX 1.10: Software

Tables, calculations, statistics GraphPad Prism (GraphPad Software)

Excel (Microsoft)

Graphics GraphPad Prism (GraphPad Software)

PowerPoint (Microsoft)

Flow cytometric analysis BD FACSDiva software (BD)

FACS Data Analyser (V0.9.9) (Köberle, MPIIB)

FlowJo (TreeStar)

DNA/RNA quantification ND-1000 (peqLab Biotechnologie)

RT-PCR analysis SDS 2.2.2 (AB)

Proteinanalysis Bio-Plex Manager (Bio-Rad)

ELISA SoftMax Pro v3 and v5

Text Word (Microsoft) and SciWriter

APPENDIX 1.11: Online programs and databases

Program Signal P http://www.cbs.dtu.dk/services/SignalP/

TMHMM v2.0 software http://www.cbs.dtu.dk/services/TMHMM/

SMART domain database http://smart.embl-heidelberg.de/

ScanProsite software http://www.expasy.ch/tools/scanprosite/

Pfam 24.0 http://pfam.sanger.ac.uk/

CLUSTAL multiple sequence

alignment program http://www.ebi.ac.uk/Tools/clustalw2/index.html

boxshade program http://www.ch.embnet.org/software/BOX_form.html

APPENDIX 1.12: Suppliers

Supplier Location web address

AB Applied Biosystems Darmstadt, Germany www.appliedbiosystems.com

Active Bioscience Hamburg, Germany www.activebioscience.com

ATCC Manassas, VA, USA www.atcc.org

B. Braun Melsungen, Germany www.bbraun.de

Bayer AG Leverkusen, Germany www.bayer.com

Becton Dickinson GmbH Heidelberg, Germany www.bdbiosciences.com

(BD Biosciences): comprising

products from Pharmingen

and Clontech

Biochrom Berlin, Germany www.biochrom.com

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BioLegend Uithoorn, The Netherlands www.biolegend.com

Bio-Rad Laboratories GmbH München, Germany www.bio-rad.com

Carl Roth Karlsruhe, Germany www.carl-roth.de

Cellstar/Greiner Bio-One Solingen, Germany www.greinerbioone.com

Charles River Laboratories Wilmington, MA, USA www.criver.com

Corning Life Scienes Amsterdam, The

Netherlands

www.corning.com/lifesciences/

ebioscience Kranenburg, Germany www.ebioscience.com

eppendorf AG Hamburg, Germany www.eppendorf.de

Fermentas St. Leon-Rot, Germany www.fermentas.de

Fischer Scientific Schwerte, Germany www.de.fishersci.com

Fluka Basel, Switzerland www.sigmaaldrich.com

Gibco Karlsruhe, Germany www.Invitrogen.com

GraphPad Software San Diego, CA, USA www.graphpad.com

Heraeus-Instruments Hanau, Germany www.heraeus-instruments.de

IBS Integra Bioscience GmbH Fernwald, Germany www.integra-biosciences.de

IKA Labortechnik Staufen, Germany www.ika.de

Invitrogen/Molecular Probes Karlsruhe, Germany www.Invitrogen.com

invivoGen Toulouse, France www.invivogen.com

Jackson ImmunoResearch Suffolk, UK www.jacksonimmuno.com

Leica Microsystems AG Bensheim, Germany www.leica-microstystems.com

LO Labor Optik GmbH Friedrichsdorf, Germany www.lo-laboroptik.de

Memmert Büchenbach, Germany www.memmert.com

Merck Darmstadt, Germany www.merck.de

Metabion Martinsried, Germany www.metabion.com

Microsoft Deutschland Berlin, Germany www.microsoft.de

Millipore GmbH Schwalbach, Germany www.millipore.de

Miltenyi Biotech Bergisch Gladbach, Germany

www.miltenyibiotec.com

Molecular Devices Ismaning, Germany www.moleculardevices.com

Nasco Fort Atkinson, WI, USA www.enasco.com

Nunc Wiesbaden, Germany www.nuncbrand.com

PAA Pasching, Austria www.paa.com

peqLab Biotechnologie GmbH

Erlangen, Germany www.peqlab.de

Quest Almere, The Netherlands www.sheffield-products.com

Rapidozym Berlin, Germany www.rapidozym.de

Roche Diagnostics Mannheim, Germany www.roche-applied-

science.com

Sarstedt Nuembrecht, Germany www.sarstedt.com

Sigma-Aldrich München, Germany www.sigmaaldrich.com

Southern Biotech Birmingham, AL, USA www.southernbiotech.com

Thermo Fischer/Electron

GmbH

Dreieich, Germany www.thermo.com

TPP Trasadingen, Switzerland www.tpp.ch

TreeStar Olten, Switzerland www.treestar.com

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