HIV-1 infection on Follicular Helper T cells

Ana Raquel Lourenço de Sousa Licenciatura em Bioquímica HIV-1 infection on Follicular Helper T cells Dissertação para obtenção do Grau de Mestre em Bioquímica para a Saúde Orientador: Doutora Helena Soares, Principal Investigator, CEDOC/FCM-UNL Setembro de 2017 Ana Raquel Lourenço de Sousa Licenciatura em Bioquímica HIV-1 infection on Follicular Helper T cells Dissertação para obtenção do Grau de Mestre em Bioquímica para a Saúde Orientador: Doutora Helena Soares, Principal Investigator, CEDOC/FCM-UNL Setembro de 2017 HIV-1 infection on Follicular Helper T cells Ana Sousa 2017 ii iii HIV-1 infection on Follicular Helper T cells Copyright, Ana Sousa, FCT/UNL, UNL A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa têm o direito, perpétuo e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, e de a divulgar através de repositórios científicos e de admitir a sua cópia e distribuição com objectivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor. iv v Acknowledgements Em primeiro lugar, gostaria de agradecer à Doutora Helena Soares não só por me receber no seu laboratório como também pela paciência e orientação que me prestou. Consegui cumprir o meu objectivo de crescer tanto academicamente como pessoalmente e, por isso, estou-lhe muito grata. Quero agradecer também às minhas colegas que foram incansáveis. À Rita, por ser a nossa mãe no laboratório, à Sofia por me ensinar tanto e ouvir os meus desabafos, à Daniela e à Rute pela ajuda e pelas gargalhadas e à Juliana pela amabilidade e boa energia. Não podia pedir um grupo de trabalho mais unido e animado. Muito obrigada. Devo também um agradecimento ao Professor Luís Graça e à Doutora Ana ÁguaDoce, do Instituto de Medicina Molecular, bem como à Doutora Cláudia Andrade, da Flow Cytometry Facility do CEDOC, por toda a ajuda prestada. Por fim, mas não menos importante, quero agradecer à minha família e amigos. Aos meus pais e mano por me ouvirem, apoiarem e, acima de tudo, me animarem. Aos meus amigos por partilharem este momento comigo e me ajudarem a vivê-lo da melhor maneira. Em especial à Marisa, por ser a minha terapeuta e me manter equilibrada e à Joana, por todas as horas ao telemóvel a apoiarmo-nos mutuamente. Muito obrigada a todos os que contribuíram para a finalização desta etapa na minha vida! xii 2.2.3.2 CD4+ T cell isolation .......................................................................................... 16 2.2.4 CD4+ T cells HIV-1 infection ................................................................................ 16 2.2.5 TLR7 stimulation ................................................................................................... 16 2.2.6 Data acquisition and processing ............................................................................ 17 2.2.6.1 Flow cytometry ................................................................................................... 17 2.2.6.2 Statistical analysis ............................................................................................... 17 3. Results ..................................................................................................................... 19 3.1 GC TFH population identification ....................................................................... 19 3.2 Tonsils CD4+ T cells populations susceptibility to HIV-1 ................................ 19 3.3 HIV-1 effects on GC TFH cells ............................................................................. 21 3.3.1 HIV-1 expands GC TFH population ....................................................................... 21 3.3.2 HIV potentially augments GC TFH cell survival .................................................... 22 3.3.3 HIV-1 modulates GC TFH cell signaling ................................................................ 25 3.3.4 TLR7 modulated signaling is HIV-1 specific ........................................................ 26 3.3.5 GC TFH cytokine profile may be shaped by HIV-1 ............................................... 28 4. Discussion ................................................................................................................ 31 5. Conclusions and future perspectives .................................................................... 35 6. References ............................................................................................................... 37 xiii Index of figures Figure 1.1 - Worldwide Human Immunodeficiency Virus (HIV) prevalence in adults aged 15 to 49, in 2016 ....................................................................................................... 2 Figure 1.2 - Structure of HIV ........................................................................................... 3 Figure 1.3 - HIV life cycle ............................................................................................... 4 Figure 1.4 - HIV course of infection ................................................................................ 5 Figure 1.5 - TFH cell differentiation ................................................................................. 9 Figure 1.6 - HIV-1 persistence in GCs, during ART ..................................................... 11 Figure 3.1 – Gating strategy used for GC TFH identification (CD4+PD-1highCXCR5high T cells) ................................................................................................................................ 19 Figure 3.2 – Tonsils CD4+ T cells populations susceptibility to HIV-1 ........................ 20 Figure 3.3 – HIV-1 increases the expression of the lineage specific transcription factor Bcl6 in GC TFH cells ....................................................................................................... 21 Figure 3.4 – HIV-1 seems to increase GC TFH chemotactic receptor CXCR5 .............. 22 Figure 3.5 – HIV-1 increases GC TFH cell proliferation ................................................ 23 Figure 3.6 – HIV-1 imposes an increased GC TFH metabolism ..................................... 24 Figure 3.7 – HIV-1 potentiates an activation status of GC TFH cells ............................. 24 Figure 3.8 – HIV-1 apparently modulates GC TFH signaling ........................................ 26 Figure 3.9 – HIV-1 increases TLR7 expression in GC TFH cells .................................. 26 Figure 3.10 – IMQ decreases TLR7 expression in NI GC TFH cells ............................. 27 Figure 3.11 – IMQ decreases proliferation capacity of NI GC TFH cells ...................... 28 Figure 3.12 – HIV-1 interferes with GC TFH cytokine production ................................ 29 xiv xv Index of tables Table 2.1 - Primary surface and intracellular antibodies used in FACS. ....................... 13 Table 2.2 - Dyes used in FACS ...................................................................................... 14 xvi xvii Abbreviations AIDS Acquired Immunodeficiency Syndrome APCs Antigen-presenting cells ART Antiretroviral therapy Bcl6 B-cell lymphoma 6 protein BSA Bovine serum albumin CCL Chemokine (motif C-C) ligand CCR C-C chemokine receptor CD Cluster of differentiation CXCL Chemokine (C-X-C motif) ligand CXCR C-X-C chemokine receptor DCs Dendritic cells DMEM Dulbecco’s modified Eagle’s medium DNA Deoxyribonucleic acid EDTA Ethylenediamine tetraacetic acid EIs Entry inhibitors FACS Fluorescence-activated cell sorting FACS-SAP Fluorescence-activated cell sorting-Saponin FBS Fetal bovine serum FDCs Follicular dendritic cells FIs Fusion inhibitors FSC Forward Scatter GCs Germinal centers GFP Green fluorescent protein HBS Hepes-buffered saline HIV Human Immunodeficiency Virus ICOS Inducible T-cell co-stimulator IFN Interferon IgG Immunoglobulin G IL Interleukin iMM Instituto de Medicina Molecular xviii IMQ Imiquimod INs Integrase inhibitors L/D Live/Dead cells LB Luria-Bertani MFI Mean fluorescence intensity MHC Major Histocompatibility MNCs Mononuclear cells mRNA Messenger RNA MT Mitotracker NF-ƙB Nuclear factor-kappa B nGC Non-Germinal center NKs Natural killer cells NNRTIs Non-nucleoside reverse transcriptase inhibitors NRTIs Nucleoside/Nucleotide reverse transcriptase inhibitors nTFH Non-TFH O/N Overnight P/S Penicillin-Spreptomycin PAMPs Pathogen-associated molecular patterns PBS Phosphate-buffered saline PD-1 Programmed cell death protein 1 PFA Paraformaldehyde PIs Protease inhibitors PLL Polylysine PRRs Pattern recognition receptors RNA Ribonucleic acid rpm revolutions per minute RPMI Roswell Park Memorial Institute RT Room temperature SIV Simian Immunodeficiency Virus SSC Side Scatter ssRNA Single-stranded RNA TCR T-cell receptor xix TFH Follicular helper T cells Th T helper cells TLR Toll-like receptor TNF-α Tumor necrosis factor α WHO World Health Organization xx 1 1. Introduction 1.1. Human Immunodeficiency Virus (HIV) 1.1.1 History AIDS (Acquired Immunodeficiency Syndrome) is characterized by a progressive failure of immune system leading to opportunistic infections and it is transmitted by bodily fluids as semen or blood (Fanales-Belasio, E. [et al.], 2010). It was first clinically described in the USA, in 1981, in a restrict group of homosexual men, intravenous drug users and hemophiliacs (Sharp, P.M. and Hahn, B.H., 2011). However, the number of cases continued to grow and it was not confined to the previously described community (Greene, W.C., 2007). In 1983-84, two separate research groups, led by Luc Montagnier and Françoise Barré-Sinoussi (Barresinoussi, F. [et al.], 1983) and Robert Gallo (Gallo, R.C. [et al.], 1983), identified HIV (Human Immunodeficiency Virus) as the cause of this pandemic disease (figure 1.1). Later on, it was discovered the existence of two closely related viruses: HIV-1 and HIV-2. Both of them are believed to be originated from non-human primates in southern Cameroon infected with Simian Immunodeficiency Viruses (SIVs), by hunting or handling bushmeat (zoonosis). Due to rubber and ivory exploitation, there were fluvial connections between Cameroon and Kinshasa (capital of Democratic Republic of Congo), allowing the virus to travel into this growing city, in 1920. Being the biggest city in the region with a rapid population growth, sex trade, railways and emigration, offered the virus conditions to begin spreading from human-to-human regionally, at first, and soon after around the world (Faria, N.R. [et al.], 2014). In fact, it is believed that HIV entered in the USA, via New York City, from Caribbean (Worobey, M. [et al.], 2016). 8 circulating memory CD4+ T cells have been described as the hotspot of HIV-1 reservoir, recent work identified another one as the major source of replication-competent HIV-1: follicular helper CD4+ T cells (TFH) (Banga, R. [et al.], 2016). 1.2 Immune system The innate immune system is the first line of host defense comprising multiple mechanisms and cells (macrophages, DCs and natural killer cells (NKs)) that provide immediate protection against infection. It is composed by physical and chemical barriers, such as skin and tears, and by complement system that helps in pathogens clearance (Turvey, S.E. and Broide, D.H., 2010). The main innate immune response is inflammation which is initiated by the recognition of pathogen structural motifs, known as pathogenassociated molecular patterns (PAMPs), by receptors present on some leukocytes (such as macrophages and DCs) – the pattern recognition receptors (PRRs), such as toll-like receptors (TLRs). This recognition allows cell activation and release of inflammatory cytokines, like TNF-α, IL-1 and interferon gamma (IFNγ) (Brubaker, S.W. [et al.], 2015). Althought innate immune system provides a rapid response, it is not a long-lasting one and, more importantly, it is not pathogen-specific. Hence the evolutionary importance in acquiring a specific yet adaptable response able to prepare the host for future challenges and that generates an immunological memory enhancing the host response to subsequent encounter with the same pathogen – the adaptive immune system (Bonilla, F.A. and Oettgen, H.C., 2010). The major players of adaptive immune system are B and T lymphocytes and their activation relies on antigen-presentation. This process is taken by all nucleated cells but there is a group of professional antigen-presenting cells (APCs) – DCs, macrophages and B cells – that provide a stronger immune response. While all nucleated cells are capable of presenting endogenous peptides (including the ones resulting from viral infections and/or expressed by cancer cells), APCs distinguish themselves by the ability of internalizing and processing exogenous antigens into peptides and displaying them complexed with MHC class II on the surface. While MHC class I is present in non-APCs and is recognized by T-cell receptor (TCR) expressed on the surface of cytotoxic T cells 9 (CD8+), MHC class II is present in APCs and is recognized via TCR expressed by helper T cells (CD4+) (Bonilla, F.A. and Oettgen, H.C., 2010). Consequently, cytotoxic T cells are responsible for inducing programmed cell death to target cells (potentially harmful cells) (Berg, R.E. and Forman, J., 2006) and helper T cells have the crucial role of assisting other leukocytes, as it is the case of antibody secretion by B cells (Luckheeram, R.V. [et al.], 2012). 1.3 Follicular helper T cells (TFH) Follicular helper T cells are a specialized subset of helper T cells that are mainly localized in germinal centers (GCs) within secondary lymphoid organs, as lymph nodes and tonsils. They play an important role in the formation and maintenance of GCs and generating lasting immune memory, which is fundamental for vaccination, considering that TFH cells provide differentiation signals to B cells in order to secrete antibodies (Crotty, S., 2011). 1.3.1 TFH differentiation TFH cell differentiation is a multifactorial process and it can be divided into three stages: APC-dependent, B cell interaction and GC TFH cell (figure 1.5) (Crotty, S., 2014). Figure 1.5 - TFH cell differentiation. From (Crotty, S., 2014) 10 In the first stage, there is interaction between a naïve CD4+ T cell and a dendritic cell in an antigen-presentation context, priming the CD4+ T cell, in the T cell zone. Upon activation, it can differentiate into TFH cell if it is Bcl6+ (B cell lymphoma 6) which is an antagonist of another transcriptor factor Blimp-1, responsible for CD4+ T cell differentiation into Th1, Th2, Th17 (T helper 1, 2 and 17 cells) (Crotty, S., 2011). Their differentiation also depends on IL-21, IL-6 and inducible co-stimulator (ICOS) (Jogdand, G.M. [et al.], 2016). In the second stage, early-TFH cell needs to migrate from T cell zone to T-B cell border. B cells express chemokines (C-X-C motif) ligand 13 (CXCL13) which are recognized by CXCR5 present on the early-TFH. In addition to over-expressing CXCR5, early-TFH cells downregulate CCR7 since it is the primary chemotactic receptor for the T-zone expressed chemokines (motif C-C) ligand 19 (CCL19) and 21 (CCL21) (Crotty, S., 2014). The final stage is the germinal center formation where GC TFH cells drive B cell differentiation into memory B cells and plasma cells and where antibody diversification (somatic hypermutation) occurs. This stage relies mainly on cytokine secretion by GC TFH, such as IL-21 and IL-4, and surface protein CD40L which binds to CD40 expressed by GC B cells (Vinuesa, C.G. [et al.], 2016). TFH cells can also be found in peripheral blood althought they are less abundant under normal circumstances; in autoimmune disease or in infection context, they migrate from lymphoid tissues to peripheral blood. However, there is still some controversy whether lymphoid tissue TFH cells play the same role than peripheral blood ones. Lymphoid tissue TFH cells are commonly identified as high expressors of CXCR5 and programmed cell death protein 1 (PD-1) - CXCR5highPD-1high (Yu, D. and Vinuesa, C.G., 2010). 1.3.2 TFH cells as HIV reservoir As stated before, TFH cells have been described as the major reservoir for HIV active replication (Banga, R. [et al.], 2016, Lorenzo-Redondo, R. [et al.], 2016, Perreau, M. [et al.], 2013b) since they are primarily localized in an immune privileged site, considering that it is shielded from cytotoxic T cells, responsible for controlling HIV infection, and it is not accessible by ART as it does not permeate lymphoid tissue (Barton, K. [et al.], 11 2016, Paiardini, M. and Lichterfeld, M., 2016). Furthermore, these cells exhibit an enhanced susceptibility to HIV infection (about 40 times more than a non-GC cell) (Connick, E. [et al.], 2007) and they constitute 60-75% of HIV-producing cells (Connick, E. [et al.], 2007, Folkvord, J.M. [et al.], 2005). Moreover their lifespan is not affected by HIV infection. Actually, it has been reported that there is an expansion of this CD4+ T cells subset (Lindqvist, M. [et al.], 2012). This GC permissitivity and expansion may be partially explained by the presence of highly infectious virions harbored by follicular dendritic cells (FDCs) that are chronically exposed to TFH cells (figure 1.6) and by alterations in cytokine profile (as IL-6 and IFNγ) (Miles, B. and Connick, E., 2016). However, there is still much to know about how HIV achieves this seemingly antagonic effects of increased viral replication and TFH cell survival in order to maintain its own replication. Figure 1.6 - HIV-1 persistence in GCs, during ART. From (Paiardini, M. and Lichterfeld, M., 2016) 12 1.4 Objectives The work presented on this thesis was inserted in the project «HIV-1 replication on follicular sanctuaries», funded by Gilead (#PGG/009/2016) which aims at determining how HIV-1 manipulates TFH microenvironment in order to improve its chances to thrive, without implicating TFH cells survival, becoming a major reservoir. Ultimately, this project intends to provide the necessary tools to the pursuit of a functional cure for HIV1 infection, by immunotherapy. In the present work, the main goal is to get an insight of the nature of TFH viral sanctuary by studying ex vivo HIV-1-infected TFH cells isolated from healthy human tonsils. This constitute an advantage comparing with the previous studies that investigate TFH compartment from HIV-1-infected patients as a whole, not allowing to discriminate between HIV-1 infection direct from bystander effects (Buranapraditkun, S. [et al.], 2017, Perreau, M. [et al.], 2013b). By taking these first steps on understanding TFH behavior, upon HIV-1 infection, it will arise many other questions that can lead to the identification of novel therapeutic targets. 13 2. Materials and Methods 2.1 Materials 2.1.1 General reagents and materials Luria-Bertani (LB) Broth, Miller medium was obtained from Fisher BioReagentsTM, NucleoBond® Xtra Column Filters was from Macherey-Nagel, Dulbecco’s modified Eagle’s (DMEM) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (P/S) was from ThermoFisher as well as Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% P/S. Fluorescenceactivated cell sorting (FACS) buffer is constituted by Phosphate-buffered saline 1X (PBS) from VWR and 2% of FBS from Biochrom. Fluorescence-activated cell sorting-Saponin (FACS-SAP) buffer is constituted by FACS buffer and saponin 0.1%. Biocoll (density 1,077 g/mL) used in density gradient cell separation was from Biochrom. For CD4+ T cell isolation, MojoSortTM Human CD4 T cell isolation Kit from Biolegend was used. The MojoSort buffer is constituted by PBS 1X, 100 mM ethylenediamine tetraacetic acid (EDTA) and 5 mg/mL bovine serum albumin (BSA). For HIV-1 infection, it was used a ThermoMixer C for 15 mL tubes from Eppendorf. 2.1.2 FACS antibodies Table 2.1 - Primary surface and intracellular antibodies used in FACS. Antibody Isotype Species Supplier Fluorochrome Concentration (μg/ml) Surface CD4 IgG1 Mouse Biolegend PerCP-Cy5.5 100 CXCR5 IgG1 Mouse Biolegend APC-Cy7 200 IgG1 Mouse Biolegend PE 100 PD-1 IgG1 Mouse Biolegend BV421 100 IgG1 Mouse Biolegend PE-Cy7 200 CD3 IgG2a Mouse Biolegend APC-Cy7 200 IgG1 Mouse Biolegend PerCP 100 ICOS IgG Hamster Biolegend APC 200 CD38 IgG1 Mouse Biolegend APC-Cy7 400 CD69 IgG Hamster Biolegend PE 200 Intracellular Ki67 IgG1 Mouse BD Pharmog PE-Cy7 100 Bcl6 IgG2 Rat Biolegend PE-Cy7 25 IL-17 IgG1 Mouse Biolegend APC-Cy7 100 14 IL-21 IgG1 Mouse Biolegend A647 80 IFNγ IgG1 Mouse Biolegend Pacific-Blue 100 IL-10 IgG1 Rat Biolegend A647 100 TLR7 IgG1 Mouse Novus PE 700 2.1.3 FACS dyes Table 2.2 - Dyes used in FACS Dye Supplier Fluorochrome LIVE/DEAD™ Fixable Aqua Invitrogen BV510 MitoTracker® Deep Red FM Invitrogen A647 2.2 Methods 2.2.1 Viral plasmid production 2.2.1.1 Bacterial transformation 1 µL of viral DNA (HIV-NL-4-3 (Silva, J.G. [et al.], 2016) or NLENG1-IRES (Trinite, B. [et al.], 2013), both containing a green fluorescent protein (GFP) reporter gene) was added to DH5α competent cells and incubated on ice for 20 min. The mix was placed in a water bath at 42ºC, for 45 sec and placed back in ice for 2 min. 1 mL LB medium was added to the mix, incubated in a shaking incubator at 37ºC, 0,45 x g for 1 h and centrifuged at 3200 x g for 5 min. The mix was incubated overnight (O/N), at 37ºC, in a 10 cm LBagar plate supplemented with 100 µg/mL ampicillin. 2.2.1.2 Bacterial inoculation A single bacterial colony was pre-inoculated at 37ºC, 0.45 x g for 8 h (logarithmic phase of bacterial growth) in 5 mL LB medium supplemented with 100 µg/mL ampicillin. Pre-inoculum was placed in 200 mL LB medium supplemented with 100 µg/mL ampicillin and incubated O/N at 37ºC, 0.45 x g (saturation phase of bacterial growth). 15 2.2.1.3 Viral DNA purification Bacterial culture was centrifuged at 800 x g for 30 min. DNA was recovered from bacterial culture’s pellet with NucleoBond® Xtra Column Filters by anion-exchange chromatography, according to manufacturer’s instructions (Macherey-Nagel). Viral DNA was resuspended in 100 µL milliQ water and stored at -20ºC. 2.2.2 Viral particles production 2.2.2.1 Transfection 293T packaging cells were seeded O/N at 37ºC, 5% CO2, at 4x106 cells in a 10 cm plate in 10 mL supplemented DMEM medium. They need to grow until they reach 7090% confluency before transfection. A mix with 20 µg of packaging plasmid (HIV-NL43) and 20 µg of envelope plasmid (ENV-VSV) or 40 µg of whole plasmid (NLENG1IRES), CaCl2 and milliQ water (to a final volume of 1 mL) was added dropwise to 1 mL Hepes-buffered saline (HBS) 2X, while bubbling it. The final mix was added dropwise to 293T cells (1 mL/10 cm plate). The packaging cells were incubated O/N at 37ºC, 5% CO2. Media was changed at 24 h. 2.2.2.2 Supernatant collection Viral particles (supernatant) were collected with a 10 mL syringe, filtered through a 0.45 µm filter and stored at -80 ºC, at 48 h post-transfection. 2.2.3 CD4+ T cells isolation from human tonsils 2.2.3.1 Human tonsils processing The human tonsils were gently provided by Professor Luís Graça, from Instituto de Medicina Molecular (iMM), in the scope of a protocol established between this laboratory and Hospital de Santa Maria, in Lisbon. The human tonsils were placed in FACS buffer (PBS 1X with 2% FBS) in a 10 cm plate and cauterized parts and inflamed tissue were 16 removed, the remaining tissue was mechanically disintegrated and was filtered with a 100 µm mesh. Then, it was diluted (1:2) with PBS 1X, added carefully on top of Biocoll (1:3) and centrifuged at 700 x g for 30 min (without brake) for density gradient cell separation. The mononuclear cells (MNCs) ring was carefully collected and washed two times with PBS 1X at 700 x g for 10 min. 2.2.3.2 CD4+ T cell isolation For CD4+ T cell isolation, it was followed the MojoSort™ Isolation Kits No Wash Protocol. They were culture with supplemented RPMI medium at 2x106 CD4+ T cells/mL, O/N at 37ºC, 5% CO2. 2.2.4 CD4+ T cells HIV-1 infection On the next day, CD4+ T cells were centrifuged, the supernatant was removed and they were resuspended with HIV-1 (500 μL HIV-NL4-3/million cells or 75 μL NLENG1IRES/million cells) and 15 μg/mL of polybrene, in 15 mL tubes. They were placed in a Thermo Mixer C, for 4 h, at 37ºC with agitation cycles of 300 rpm for 1 min every 5 mins. Then, they were incubated for another 4 h at 37ºC, 5% CO2. After that time, cells were centrifuged at 300 x g for 5 min, resuspended in supplemented RPMI medium at 2x106 CD4+ T cells/mL and cultured for 3 days at 37ºC, 5% CO2. 2.2.5 TLR7 stimulation For TLR7 stimulation in non-infected CD4+ T cells, it was used its synthetic agonist Imiquimod (IMQ) (1 mg/mL, from InVivoGen) at 0, 0.5, 1, 2.5, 5 and 10 μg/mL. CD4+ T cells were culture at 2.5x106/mL, for 3 days at 37ºC, 5% CO2. 17 2.2.6 Data acquisition and processing 2.2.6.1 Flow cytometry Uninfected and infected CD4+ T cells were washed with PBS 1X (at 700 x g for 3 min) and they were stained with MitoTracker® Deep Red FM and posteriorly with LIVE/DEAD™ Fixable Aqua, following manufacturer’s instructions. For surface staining, cells were washed twice with FACS buffer and incubated for 20 min, at RT (room temperature), in the dark, with the surface antibodies (table 1). This staining was followed with two washes with FACS buffer in order to remove the unbound antibodies. Cells were fixed with paraformaldehyde (PFA) 1%, for 20 min, at RT, in the dark and then washed with FACS buffer. For membrane permeabilization, it was used FACS-SAP with the same incubation’s condition as cell fixation. For intracellular staining, cells were centrifuged at 700 x g for 3 min and incubated for 30 min, at RT, in the dark, with the intracellular antibodies (table 1). Finally, cells were washed twice with FACS-SAP and once with FACS buffer and the data was acquired in FACS Canto II from BD Biosciences. The data was analyzed with Flow Jo software (BD Biosciences). 2.2.6.2 Statistical analysis GraphPad Prism software was used for statistical analysis. Statistical significance (pvalues) was obtained using Wilcoxon matched-pairs test; p values of less than 0.05 were considered significant. 24 Figure 3.6 – HIV-1 imposes an increased GC TFH metabolism. (A) Percentage (left) and MFI (right) of MT+ (Mitotracker+) NI and HIV-1-infected GC TFH cells. (B) Fold change in the percentage and MFI of MT+ GC TFH cells upon HIV-1 infection. Grey bars represent the median. Each symbol represents an individual donor. p values were determined by Wilcoxon matched-pairs test; ns, not significant (p > 0.05), * p < 0.05. Figure 3.7 – HIV-1 potentiates an activation status of GC TFH cells. (A) Percentage (left) and MFI (right) of CD69+ NI and HIV-1-infected GC TFH cells. (B) Fold change of percentage and MFI of CD69+ GC TFH cells upon HIV-1 infection. Grey bars represent the median. Each symbol represents an individual donor. p values were determined by Wilcoxon matched-pairs test; ns, not significant (p > 0.05). 25 3.3.3 HIV-1 modulates GC TFH cell signaling Bearing in mind that HIV-1 seemingly enhances TFH metabolism and, consequently, proliferation, the virus is also likely to control TFH signaling. Accordingly, HIV-1 slightly upregulates TCR (CD3) in GC TFH cells (figures 3.8 A and B), possibly to increase TFH activation. Regarding ICOS expression, there is a decrease in the percentage of expressing-TFH cells (figure 3.8 A) which is corroborated by the fold decrease (figure 3.8 D) although there is a subtle upraise of their ICOS expression (figure 3.8 B and D). However, ICOS expression was highly related with donor-specific characteristics, producing inconsistent outcomes. 26 Figure 3.8 – HIV-1 apparently modulates GC TFH signaling. (A) Percentage of CD3+ (top) and ICOS+ (bottom) NI and HIV-1-infected GC TFH cells. (B) MFIs of CD3 (top) and ICOS (bottom) in NI and HIV-1-infected GC TFH cells. Fold change of percentage and MFI of (C) CD3+ and (D) ICOS+ GC TFH cells upon HIV-1 infection. Grey bars represent the median. Each symbol represents an individual donor. p values were determined by Wilcoxon matched-pairs test; ns, not significant (p > 0.05), * p < 0.05. 3.3.4 TLR7 modulated signaling is HIV-1 specific TLRs act as sensors of PAMPs expressed on infectious agents, providing a positive co-stimulatory signal that induces pro-inflammatory cytokines and cell proliferation. There are surface and endosomal TLRs, allowing a broad pathogen recognition and immune response. Among several receptors, there is an intracellular one capable of recognize ssRNA, such as HIV-1 – the TLR7. So far, this work demonstrated that HIV-1 modulates GC TFH cells signaling aiming at improving this cellular reservoir capacities. Therefore, it is imperative to understand the extent of HIV-1 influence on TFH cellular machinery. In fact, HIV-1-infected GC TFH cells present a higher expression of TLR7 (figure 3.9). Figure 3.9 – HIV-1 increases TLR7 expression in GC TFH cells. (A) Percentage (left) and MFI (right) of TLR7+ NI and HIV-1-infected GC TFH cells (B) Fold change of percentage and MFI of TLR7+ GC TFH cells upon HIV-1 infection. Grey bars represent the median. 27 Each symbol represents an individual donor. p values were determined by Wilcoxon matched-pairs test; ns, not significant (p > 0.05), ** p < 0.01. In order to determine if the upregulation of TLR7 was a simple response to ligand abundance, a TLR7-synthetic agonist Imiquimod (IMQ) was added to non-infected cells. Strikingly, IMQ induced different outcomes comparing with HIV-1-infected cells. Upon IMQ stimulation, there is a trend for TLR7 expression decrease in NI TFH cells (figure 3.10), more pronounced for the highest IMQ concentration (10 μg/mL), indicating cellular defensive mechanism (negative feedback) to avoid chronic stimulation. Additionally, IMQ decreases GC TFH cells proliferation (figure 3.11). Altogether, these data show that HIV-1 may induce an alternative signaling pathway of TLR7 in order to escape a cellular protective response and to allow its cellular reservoir expansion. Figure 3.10 – IMQ decreases TLR7 expression in NI GC TFH cells. Percentage (left) of TLR7+ TFH cells and MFI (right) of TLR7 from NI GC TFH, for each IMQ concentration used (0, 0.5, 1, 2.5, 5 and 10 μg/mL). 28 Figure 3.11 – IMQ decreases proliferation capacity of NI GC TFH cells. Percentage (left) and MFI (right) of Ki67+ NI GC TFH cells, for each IMQ concentration used (0, 0.5, 1, 2.5, 5 and 10 μg/mL). 3.3.5 GC TFH cytokine profile may be shaped by HIV-1 A recent work demonstrated that TLR7 engagement by HIV-1, in circulating CD4+ T cells, induces a state of anergy and an inability to secrete cytokines (Dominguez-Villar, M. [et al.], 2015). In the light of the previous results, it was important to check if this hold true to the present work. For that reason, the pro-inflammatory cytokines IL-21, IL-17 and IFNγ were measured in GC TFH, without any stimulation, and compared with HIV1-infected ones, knowing that they present an amplified TLR7 expression. The TFH cytokine profile seems to be affected by HIV-1 infection, since it is visible an expansion of IL-21+, IL-17+ and IFNγ+ TFH cells (figure 3.10 A and B) and in their expression levels (figure 3.10 C), even though it is more evident in IL-21 and IFNγ considering their major role in directing antibody production. Hence, contrary to circulating CD4+ T cells, HIV-1 does not induce anergy in GC TFH cells. All points out to an infection mechanism, orchestrated by HIV-1, aiming at improving its replication and reservoir maintenance in GC TFH cells, through metabolism and signaling modulation. 29 Figure 3.12 – HIV-1 interferes with GC TFH cytokine production. Percentage of IL-21+ (left), IL-17+ (middle) and IFNγ+ (right) (A) NI and (B) HIV-1-infected GC TFH cells. (C) MFIs of described cytokines in NI (grey) and HIV-1-infected (red) GC TFH cells. 30 31 4. Discussion The combination antiretroviral therapy, introduced in 1996, has been one of the most outstanding achievements of medical research for the treatment of HIV infection, preventing millions of AIDS-related deaths and improving HIV-infected individual’s life expectancy. However, it is unable to clear persistent HIV reservoirs which remain the major obstacle to a functional cure and enforces a life time of treatment (Chun, T.W. [et al.], 2015). Such therapy entails a number of serious side effects such as heart and liver diseases and drug interactions (Marzolini, C. [et al.], 2011). Moreover, a life time of ART leads to development of drug resistance, an evasion mechanism in which occurs mutations in ART-targeted viral proteins, resulting in a great genetic variation within HIV populations. This phenomenon poses a real concern since it generates a high-fitness virions population capable of evade treatment (Clavel, F. and Hance, A.J., 2004). In fact, it is estimated that, in the United States of America, 76% of ART-treated patients developed resistance to one or more antiretroviral drugs (Richman, D.D. [et al.], 2004). That is why a therapy targeting the host signaling instead of the virus offers, not only the advantage of minimize the described selective pressure on HIV, but also the opportunity to compile a library of signaling inhibitors that, unlike ART, are capable of permeate lymphoid tissue (the major HIV-persistent reservoir). Follicular helper T cells constitute the most important cellular reservoir for HIV-1 persistence and replication (Paiardini, M. and Lichterfeld, M., 2016). As described previously in HIV-1-infected patients (Perreau, M. [et al.], 2013b), they exhibited an enhanced susceptibility to HIV-1 infection compared to non-follicular helper T cells or even non-germinal center ones (figure 3.2 B). In addition to that, GC TFH cells display a longer lifespan upon infection (figure 3.2) and an increased differentiation and recruitment, by the upregulation of TFH transcription factor Bcl6 and the chemokine receptor CXCR5, respectively (figures 3.3 and 3.4). These data suggest that HIV-1 preferentially drives GC TFH population expansion. Furthermore, HIV-1 augments GC TFH cells proliferation and potentiate an activated state (figures 3.5 and 3.7) in order to favor viral replication. These events impose a higher demand for energy that is visible by the increased mitochondrial mass in infected cells (figure 3.6). The essential role of TFH cells in GCs requires a mixed metabolism. It is 32 known that TFH cells rely both on glycolysis and mitochondrial oxidative phosphorylation (Ray, J.P. [et al.], 2015, Zeng, H. [et al.], 2016), being the last one the most efficient pathway in energy generation but glycolysis enables “building blocks” formation which is crucial for viral particles production. In a previous study, it was demonstrated that HIV1-infected circulating CD4+ T cells display an upregulated glucose transporter Glut1 expression meaning that HIV-1 most likely shifts the metabolism towards glycolysis (Loisel-Meyer, S. [et al.], 2012). Nevertheless, whether HIV-1 reprograms GC TFH metabolism towards one or the other metabolic pathway and, if so, which metabolic factors are involved, is still unknown. Combining the data obtained in the present work with the data from Loisel-Meyer, S. [et al.], 2012, it rises the idea that HIV-1 shapes TFH metabolism by activating the oxidative phosphorylation metabolic pathway to fulfill the energetic needs along with upregulation of the glycolytic pathway to provide “building blocks” to sustain a highly active HIV-1 replication. Even though HIV-1 replication also rely on an activated cellular state (Stevenson, M. [et al.], 1990), there is still much to know about how this virus affects TCR signaling in TFH cells. It is described that the increased pathogenicity observed in HIV-1 results in a loss of capacity of Nef protein to downregulate TCR-CD3, as seen in less pathogenic HIV strains (Feldmann, J. [et al.], 2009, Schindler, M. [et al.], 2006) where it act as a host protective measure by ensuring a minimal cellular activation and, consequently, a reduced activation-induced T cell death (Foster, J.L. and Garcia, J.V., 2006). Knowing that GC TFH cells are in an activated and proliferative state, upon HIV-1 infection, it is not surprising that TCR-CD3 is upregulated (figure 3.8 A). ICOS co-stimulatory signal is crucial for TFH differentiation (Rolf, J. [et al.], 2010) and it may play a role in HIV-1 infection since it highly activates PI3K/Akt/mTOR signaling pathway, responsible for cell activation and proliferation (Wikenheiser, D.J. and Stumhofer, J.S., 2016). However, in this study, it was not possible to determine if this idea is correct since ICOS expression was donor-related. On the other hand, PD-1 suppresses cell signaling promoting apoptosis and it was previously described that HIV1 downmodulates this inhibitor in order to ensure its own replication on the host cells (Kohler, S.L. [et al.], 2016). Nevertheless, it was not possible to conclude the effect of HIV-1 infection in GC TFH cells, in the present study, since only half of the donors exhibited that downmodulation, exposing the need to test in more donors. 33 Toll-like receptors represent a critical role in activating an innate immune response, TLR7, in particular, is responsible for ssRNA recognition in intracellular compartments. This study showed that HIV-1 enhances TLR7 expression in GC TFH cells (figure 3.9) indicating a modulated TLR signaling in order to promote HIV-1 expansion. These data raise questions regarding the altered TFH signaling by HIV-1 that need to be answered in the near future. Although TLR engagement act as a positive co-stimulatory signal to increase proinflammatory cytokines secretion and cell proliferation, it was demonstrated that, in circulating HIV-1-infected CD4+ T cells, it induces an anergic state (Dominguez-Villar, M. [et al.], 2015). This does not appear to apply to GC TFH cells considering the increased pro-inflammatory cytokines expression in HIV-1-infected cells (figure 3.12). This data also corroborates the chronic state of inflammation as a HIV-1 infection hallmark. Altogether, these data indicate a metabolic control and signaling modulation, by HIV1 as a disease mechanism, in order to turn GC TFH cells into viral factories. 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