Characterization of plasma cell subsets in the
human bone marrow
vorgelegt von
von
Dipl. Ing. Annika Glenzer
ORCID: 0000-0002-0013-6579
an der Fakultät III – Prozesswissenschaften
der Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften
– Dr.-Ing. –
genehmigte Dissertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. Hyun-Dong Chang
Gutachter: Prof. Dr. Roland Lauster
Gutachter: Prof. Dr. Thomas Dörner
Tag der wissenschaftlichen Aussprache: 29.01.2021
Berlin 2021
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I. Abstract
Human bone marrow plasma cells (BMPC) are continuously producing antibodies and thereby,
together with other mechanisms, protect the human body against foreign pathogens. While
BMPC are not proliferating and have specific requirements for their survival niche, e.g. contact
of adhesion molecules expressed on different cell types or secreted factors by surrounding
cells. PC are the producers of autoantibodies in autoantibody-mediated autoimmune diseases
and thus, a better understanding of their survival mechanisms can help to specifically target
PC.
During differentiation, PC consecutively lose typical B cell markers, e.g. CD20, and a subset
of human BMPC was described to lack the expression of CD19, whereas CD19 expression is
maintained in the majority of BMPC. Since CD19 is a co-stimulatory molecule of the B cell
receptor (BCR), we wondered whether expression of CD19 would affect cellular responses of
BMPC, e.g. to BCR stimulation, or expression of checkpoint molecules.
In total, 139 human BM samples from individuals undergoing total hip arthroplasty were
received and analyzed. BCR-associated molecules were investigated on mRNA and protein
level and were found to be expressed in both subsets with a higher expression of spleen
tyrosine kinase (SYK) in CD19+ BMPC. Short-term BCR stimulation resulted in kinase
phosphorylation with CD19+ BMPC being the subset to induce higher phosphorylation.
Interestingly, when taking the immunoglobulin (Ig) isotype into account, the BCR response was
restricted to IgA+ BMPC independently of their CD19 expression.
Analysis of checkpoint molecule expression revealed upregulation of programmed cell death
protein 1 (PD-1) on 30% of IgA-expressing BMPC after BCR stimulation in cell culture and
inhibition of SYK abrogated PD-1 upregulation.
Thus, BMPC do not only react to BCR stimulation by phosphorylation of kinases, but also by
upregulation of PD-1 surface expression. Enhanced PD-1 expression in a subset of IgA+ BMPC
upon antigenic stimulation could reflect a distinct functiotype that is independent of the
phenotypic heterogeneity of the subsets identified by CD19. These data suggest that specific
BMPC subsets underlie different regulatory principles and could be involved in regulatory
functions on surrounding cells in the human BM.
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II. Zusammenfassung
Plasmazellen (PZ) im humanen Knochenmark (KM) produzieren kontinuierlich Antikörper, die
zusammen mit anderen Mechanismen den menschlichen Körper gegen feindliche Pathogene
schützen. KMPZ proliferieren nicht und haben besondere Ansprüche an ihre
Überlebensnische, wie zum Beispiel Kontakt zu benachbarten Zellen über Adhäsionsmoleküle
oder von umgebenden Zellen sekretierte Faktoren. PZ sind die Produzenten von
Autoantikörpern in Autoantikörper-vermittelten Autoimmunerkrankungen, weshalb ein
besseres Verständnis ihrer Generierung sowie ihrer Überlebensmechanismen von Vorteil
wäre um diese Zellen spezifisch zu eliminieren.
Während der Differenzierung regulieren PZ die typischen B-Zell-Marker, wie z.B. CD20, auf
ihrer Oberfläche herunter und es wurde beschrieben, dass eine Subpopulation von KMPZ
keine Expression von CD19 mehr zeigt, während die Mehrheit die CD19-Expression beibehält.
Da CD19 ein ko-stimulatorisches Molekül des B-Zell-Rezeptors (BZR) ist, haben wir uns
gefragt ob die Expression von CD19 auf KMPZ Auswirkungen auf zelluläre Antworten wie z.B.
auf BZR-Stimulation oder die Expression von Checkpoint-Molekülen hat.
Insgesamt wurden in dieser Arbeit 139 KM-Proben von Individuen, die eine Hüft-
Totalendoprothese erhielten, erhalten und analysiert. Die Expression von mit dem BZR
assoziierten Molekülen wurde auf mRNA- und Protein-Ebene untersucht, wobei meist eine
vergleichbare Expression gefunden wurde. Nur die Kinase spleen tyrosine kinase (SYK) zeigte
eine höhere Expression in CD19+ KMPZ. Kurzzeitige BZR-Stimulation resultierte in der
Phosphorylierung von Kinasen, wobei CD19+ KMPZ durchweg einen höheren Grad der
Phosphorylierung zeigten. Bei gleichzeitiger Auswertung des Immunglobulin-Isotyps
reagierten hauptsächlich IgA+ KMPZ auf die Stimulation über den BZR und diese Antwort war
unabhängig von der CD19-Expression.
Bei der Analyse von Checkpoint-Molekülen in KMPZ zeigte sich eine erhöhte Expression von
programmed cell death protein 1 (PD-1) in 30% der IgA-exprimierenden KMPZ nach BZR-
Stimulation in Zellkultur. Die Inhibition der Kinase SYK konnte diese Hochregulation von PD-1
verhindern.
KMPZ sind folglich über den BZR aktivierbar, was sich nicht nur in der Phosphorylierung von
Kinasen des BZR-Signalweges zeigt, sondern auch durch Expression von PD-1 auf der
Zelloberfläche. Die erhöhte Expression von PD-1 in einer Subpopulation von IgA+ KMPZ
könnte einen spezifischen Funktiotyp bedeuten, der unabhängig von der durch CD19-
Expression definierten phänotyischen Heterogenität ist. Die Daten deuten darauf hin, dass
verschiedene KMPZ-Subpopulationen besonderen regulatorischen Prinzipien unterliegen und
in regulatorische Funktionen auf umgebende Zellen im humanen KM involviert sein könnten.
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III. Table of contents
I. Abstract .......................................................................................................................... 1
II. Zusammenfassung ......................................................................................................... 2
III. Table of contents ........................................................................................................ 3
1. Introduction .................................................................................................................... 6
1.1 The human immune system .................................................................................... 6
1.2 The adaptive immune system .................................................................................. 7
1.3 The humoral immune response of the adaptive immune system ............................. 7
1.3.1 Development of B cells ..................................................................................... 7
1.3.2 The germinal center reaction ............................................................................ 9
1.3.3 The BCR signaling cascade ............................................................................11
1.4 The plasma cell – the terminally differentiated B cell ..............................................13
1.4.1 Differentiation of plasma cells ..........................................................................13
1.4.2 Long-lived plasma cells ...................................................................................15
1.4.3 The BM niche – cellular and soluble factors ....................................................15
1.4.4 CD19 as BCR co-receptor ...............................................................................17
1.4.5 A subset of BMPC lacks the expression of CD19 ............................................18
1.4.6 PC in diseases ................................................................................................19
1.5 Checkpoint molecules ............................................................................................20
2. Research Aim ................................................................................................................22
3. Materials and Methods ..................................................................................................23
3.1 Materials ................................................................................................................23
3.2 Methods .................................................................................................................29
3.2.1 Donors ............................................................................................................29
3.2.2 Isolation of mononuclear cells .........................................................................29
3.2.3 Enrichment of CD138+ cells by MACS .............................................................30
3.2.4 Depletion of CD3+CD14+CD235a+ cells by MACS ...........................................31
3.2.5 Whole bone marrow stainings .........................................................................34
3.2.6 Analysis by flow cytometry and fluorescence-activated cell sorting .................34
3.2.6.1 Gating strategy ............................................................................................34
3.2.6.2 Surface staining ...........................................................................................36
3.2.6.3 Absolute cell counts .....................................................................................36
3.2.6.4 Intracellular staining for BCR-associated kinases ........................................36
3.2.6.5 Sorting of BMPC ..........................................................................................36
3.2.7 Microarray analysis .........................................................................................38
3.2.8 Transcriptomics ...............................................................................................38
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3.2.8.1 RNA Sequencing .........................................................................................38
3.2.8.2 mRNA Seq Data processing ........................................................................39
3.2.9 Stimulation ......................................................................................................39
3.2.9.1 Short-term stimulation of PC ........................................................................39
3.2.9.2 Stimulation of BMPC in cell culture ..............................................................40
3.2.10 Data analysis and statistical evaluation ...........................................................40
4. Results ..........................................................................................................................41
4.1 BCR signaling in human BMPC ..............................................................................41
4.1.1 BCR-associated molecules are expressed in CD19+ and CD19- BMPC ..........41
4.1.1.1 Transcriptome analysis reveals expression of BCR components .................41
4.1.1.2 Whole BM stainings confirm expression of BCR-associated kinases ...........42
4.1.2 BCR activation induces phosphorylation of downstream kinases ....................44
4.1.2.1 CD19+ BMPC show higher response to BCR stimulation .............................46
4.1.2.2 BCR signaling can be inhibited by kinase inhibitors .....................................48
4.1.3 IgA and IgM are expressed on the surface of BMPC .......................................49
4.1.4 Expression of isotypes and reaction to stimulation is comparable in the same
donor six months apart ..................................................................................................51
4.1.5 IgA+ BMPC express BCR-associated kinases .................................................55
4.1.6 IgA-expressing BMPC respond to BCR stimulation .........................................57
4.1.7 Whole BM staining of IgA-expressing cells ......................................................63
4.2 Expression of checkpoint molecules on BMPC .......................................................65
4.2.1 BCR stimulation in cell culture leads to upregulation of the inhibitory checkpoint
molecule PD-1 ...............................................................................................................68
4.2.2 CD19 and Ig isotype expression are stable upon BCR stimulation ..................75
4.2.3 BTLA activation does not alter the response to BCR stimulation .....................77
4.2.4 Inhibition of SYK prevents upregulation of PD-1 ..............................................79
5. Discussion .....................................................................................................................80
5.1 BMPC are capable of BCR signaling ......................................................................81
5.2 CD19 and Ig expression do not change during in vitro stimulation ..........................81
5.3 IgA-expressing BMPC are the main contributors to increased phosphorylation
whereas CD19 is dispensable for BCR signaling ..............................................................82
5.4 PD-1 expression on BMPC – a regulatory function for BMPC? ..............................84
5.5 Conclusion and outlook ..........................................................................................86
6. References ....................................................................................................................88
7. Abbreviations ................................................................................................................97
8. List of Figures .............................................................................................................. 100
9. List of Tables ............................................................................................................... 101
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