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Linköping University Medical Dissertations No.1256 Immunomodulatory Effects of Human Immunodeficiency Virus (HIV-1) on Dendritic Cell and T cell Responses Studies of HIV-1 effects on Dendritic cell functionality reflected in primed T cells Karlhans Fru Che Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University SE-58185 Linköping 2011 Copyright © Karlhans Fru Che, 2011 Division of Molecular Virology Department of Clinical and experimental medicine Linköping University SE-58185 Linköping Cover: An HIV-1 exposed Dendritic cell priming naïve T cells The cover picture and illustrations in this thesis were performed by Rada Ellegård. Published articles have been reprinted with permission of the copyright holder. Printed in Sweden by Liu-Tryck, Linköping, Sweden, 2011 ISBN: 978-91-7393-093-2 ISSN 0345-0082 “A determined hunter is not afraid of the jungle” Nelson Mandela “Our deepest fear is not that we are inadequate. Our deepest fear is that we are powerful beyond measure. It is our light not our darkness that most frightens us. We ask ourselves, “Who am I to be brilliant, gorgeous, talented and fabulous?” Actually, who are you not to be? You are a child of God. Your playing small does not serve the world. There is nothing enlightened about shrinking so that other people won’t feel insecure around you. We are born to make manifest the glory of God that is within us. It is not just in some of us. It’s in everyone. And as we let our light shine we give other people permission to do the same. As we are liberated from our own fear, our presence automatically liberates others”. Marianne Williamson “With the rapid developments in today’s life, the sky is now my spring board and no longer my limit”. Karlhans Fru Che Supervisor Committee Board Professor Marie Larsson Professor Sven Hammarström Division of Molecular Virology Division of Cell Biology Department of Clinical and Experimental Medicine Department of Clinical and Experimental Medicine Linköping University Linköping University Linköping, Sweden Linköping, Sweden Co supervisors Professor Jorma Hinkula Professor Markus Maeurer Division of Molecular Virology Department of Microbiology Tumor and Cell Biology (MTC) Department of Clinical and Experimental Medicine Karolinska Institute Linköping University Stockholm Linköping, Sweden Sweden Professor Karl-Eric Magnusson Associate professor Maria Jenmalm Division of Medical Microbiology Division of Pediatrics Department of Clinical and Experimental Medicine Department of Clinical and Experimental Medicine Linköping University Linköping University Linköping, Sweden Linköping, Sweden Faculty opponent Professor Douglas Nixon Professor Jan-Ingvar Jönsson Division of Experimental Medicine Division of Experimental Hematology Department of Medicine University of California San Francisco Department of Clinical and Experimental Medicine, San Francisco, California, USA Linköping University, Linköping, Sweden This work is dedicated to my entire Family CONTENTS LIST OF PAPERS ........................................................................................................................ 1 ABSTRACT ................................................................................................................................ 3 INTRODUCTION ....................................................................................................................... 5 Human Immunodeficiency Virus (HIV) ........................................................................................ 5 Discovery and Epidemiology ................................................................................................... 5 Etiology .................................................................................................................................... 5 Virology.................................................................................................................................... 5 Pathogenesis ........................................................................................................................... 7 HIV-1 Disease Progression ....................................................................................................... 9 Treatment .............................................................................................................................. 10 Dendritic Cell and Functions...................................................................................................... 11 DC subsets ............................................................................................................................. 11 In vitro generated DCs ........................................................................................................... 13 T cells ......................................................................................................................................... 13 T cell subsets.......................................................................................................................... 13 DC T cell priming .................................................................................................................... 15 DC-T cell-HIV-1 Interaction .................................................................................................... 17 HIV-1 Specific responses ........................................................................................................... 18 Immunomodulatory Potentials of HIV-1 ................................................................................... 19 Immunostimulatory and Inhibitory Molecules at a DC-T cell Encounter .................................. 20 Conclusions ................................................................................................................................ 23 AIMS AND OBJECTIVES .......................................................................................................... 25 Specific Aims .............................................................................................................................. 25 METHODS .............................................................................................................................. 27 Dendritic cell preparation.......................................................................................................... 27 Naïve T cell Separation .............................................................................................................. 28 Virus preparation....................................................................................................................... 28 DC T cell cocultures ................................................................................................................... 28 ELISPOT: ..................................................................................................................................... 29 Measurement of secretory factors............................................................................................ 29 ELISA: ..................................................................................................................................... 29 Intracellular cytokine staining ............................................................................................... 30 T cell proliferation analysis ........................................................................................................ 30 Fluorescence activated cell sorter (FACS) ................................................................................. 31 Gene expression by real time PCR (SYBR® Green) .................................................................... 32 Statistical analysis ...................................................................................................................... 32 FINDINGS AND DISCUSSION ................................................................................................... 33 Paper 1....................................................................................................................................... 33 Paper II....................................................................................................................................... 35 Paper III...................................................................................................................................... 37 Paper IV ..................................................................................................................................... 38 OVERALL FINDINGS/CONCLUSIONS ........................................................................................ 41 RECOMMENDATIONS AND FUTURE CHALLENGES .................................................................. 43 LAY LANGUAGE SUMMARY .................................................................................................... 45 ACKWOLEDGEMENTS............................................................................................................. 47 REFERENCES .......................................................................................................................... 51 LIST OF ABBREVIATIONS Ab Antibody AIDS Acquired immunodeficiency syndrome ANOVA Analysis of variance APC Antigen presenting cell ART Antiretroviral therapy AT-2 Aldrithiol-2 BLIMP-1 B-lymphocyte-induced maturation protein-1 BTLA B- and T-lymphocyte attenuator DCs Dendritic cells CCR Chemokine receptor CD Cluster of differentiation CFSE Carboxyfluorescein succinimidyl ester CTLA-4 Cytotoxic T lymphocyte antigen-4 CXCR4 C-X-C chemokine receptor type DNA Deoxyribonucleic acid DTX1 Deltex-1 EC Elite controllers EPG Epithelial growth factor FOXP3 Forkhead box P3 GM-CFS Granulocyte-macrophage colony stimulating factor gp Glycoprotein HAART Highly active antiretroviral therapy HIV-1 Human immunodeficiency virus-1 HVEM Herpes virus entry mediator IDCs Immature dendritic cells IDO Indoleamine 2, 3 dioxygenase IFN Interferon IL Interleukin JAK Janus kinase LAG-3 Lymphocyte-activation gene-3 LCs Langerhans cells LTNPs Long term non-progressors MAPK Mitogen activated protein kinases mDCs Myeloid dendritic cells MDC Mature dendritic cells MDDC Monocyte derived dendritic cells MHC Major Histocompatibility complex MLR Mixed lymphocyte reaction NFAT Nuclear factor of activated T cells NFkB Nuclear factor kappa-light-chain enhancer of activated B cells OIs Opportunistic infections PBMCs Peripheral blood mononuclear cells PCR Polymerase chain reaction pDCs Plasmacytoid dendritic cells PDGF Platelet derived growth factor PD-1 Programmed death-1 PD-1L Programmed death-1 ligand PHS Pooled human serum PI protease inhibitor PRR Pattern recognition receptors PAMP Pathogen associated molecular patterns PVL Plasma viral load RANTES Regulated upon activation, normal T cell expressed and secreted RNA Ribonucleic acid RT Reverse transcriptase SEB Staphylococcal enterotoxin B SIV Simian immunodeficiency virus STAT Signal transducer and activator of transcription SIV Semian Immunodeficiency Virus TCR T cell receptor TCM Central memory T cells TEM Effector memory T cells TGF-β Transforming growth factor-β TH1 T helper type 1 TH2 T helper type 2 TIM-3 T cell immunoglobulin domain and mucin domain-3 TLR Toll like receptor TNF Tumor necrosis factor TNFR Tumor necrosis factor receptor TRAIL TNF-related apoptosis inducing ligand Tregs Regulatory T cells Tr1 Regulatory T cells type 1 Tr3 Regulatory T cells type 3 VEGF Vascular endothelial growth factor VC Viremic controllers List of Papers LIST OF PAPERS 1 In vitro priming recapitulates in vivo HIV-1 specific T cell responses, revealing rapid loss of virus reactive CD4 T cells in acute HIV-1 infection. Lubong Sabado R, Kavanagh DG, Kaufmann DE, Fru K, Babcock E, Rosenberg E, Walker B, Lifson J, Bhardwaj N, Larsson M. PLoS One. 2009; 4(1): e4256. 2 HIV-1 impairs in vitro priming of naïve T cells and gives rise to contactdependent suppressor T cells. Che KF, Sabado RL, Shankar EM, Tjomsland V, Messmer D, Bhardwaj N, Lifson JD, Larsson M. European Journal of Immunology. 2010; 40(8): 2248-58. 3 Expression of a broad array of negative costimulatory molecules and Blimp-1 in T cells following priming by HIV-1 pulsed dendritic cells. Shankar EM, Che KF, Messmer D, Lifson JD, Larsson M. Molecular Medicine. 2011;17(3-4): 229-40. 4 Cross-talk between P38MAPK and STAT3 regulates expression of negative costimulatory molecules and transcription factors in HIV-1 primed T cells. Che KF, Shankar EM, Sundaram M, Zandi S, Sigvardsson M, Hinkula J, Messmer D, Lifson JD, and Larsson M (Submitted) 1 List of Papers 2 Abstract ABSTRACT The human immunodeficiency virus (HIV)-1 is the causative agent of acquired immune deficiency syndrome (AIDS) worldwide. Till date there are no vaccines or cure for this infection as the virus has adapted myriad ways to remain persistent in the host where it causes severe damage to the immune system. Both humoral and cellular immune responses are mounted against HIV-1 during the initial phase of infection but fail to control viral replication as these responses are severely depleted during disease progression. Of great importance in HIV-1 research today is the in depth understanding of the types of immune responses elicited, the mechanisms behind their decline and how these responses can be maintained overtime. The focus of this thesis was to examine the possibility of priming HIV-1 specific T cell responses in vitro from whole viral particles and in detail, scrutinize the type of T cell responses and epitope specificities generated. Next was to investigate in vitro the factors responsible for impaired immune responses in HIV-1 infected individuals. We were also interested in understanding the underlying mechanisms through which HIV-1 initiate suppression of T cell functionality. Results showed that using HIV-1 pulsed monocyte derived dendritic cells (DCs), we were able to prime HIV-1 specific CD4+ and CD8+ T cells from naïve T cells in vitro. The epitopes primed in vitro were located within the HIV-1 envelope, gag, and pol proteins and were confirmed ex vivo to exist in acute and chronically infected individuals. We established that many of the novel CD4+ T cell epitopes primed in vitro also existed in vivo in HIV-1 infected individuals during acute infection. These responses declined/disappeared early on, which is in line with HIV-1 preferential infection of HIV1 specific CD4+ T cells. Besides declining HIV-1 specific T cell responses, many HIV-1 infected individuals also have impaired T cell functionality. We established that one reason behind the decline and impairment in immune responses was the increased expression of inhibitory molecules PD-1, CTLA-4, and TRAIL on HIV-1 primed T cells. These T cells had the capacity to suppress new responses in a cell-cell contact dependent manner. The ability of the HIV-1 primed T cells to proliferate was severely impaired and this condition was reversed after a combined blockade of PD-1, CTLA-4 and TRAIL. Furthermore, more inhibitory molecules TIM-3, LAG-3, CD160, BLIMP-1, and FOXP3 were also found increased at both gene and protein levels on HIV-1 primed T cells. Additionally, we showed decreased levels of functional cytokines IL-2, IFN-γ and TNF-α, and the cytolytic proteins perforin and granzyme in DC T cell priming cocultures containing HIV-1. This could be as a result of the decreased T cell activation or impaired production by T cells. The mechanisms responsible for the elevated levels of inhibitory molecules emanated 3 Abstract mainly from the P38MAPK/STAT3 pathways. Blockade of these pathways in both allogeneic and autologous DC-T cell assays significantly suppressed expression of inhibitory molecules and subsequently rescued T cell proliferation. In conclusion, HIV-1 pulsed DCs have the capacity to prime HIV-1 specific responses in vitro that do exist in HIV-1 infected individuals and we found evidence that many of these responses were eliminated rapidly in HIV-1 infected individuals. HIV-1 triggers through P38MAPK/STAT3 pathway the synthesis of inhibitory molecules, namely CTLA-4, PD-1, TRAIL, TIM-3, LAG-3, CD160, and suppression associated transcription factors FOXP3, BLIMP-1 and DTX1. This is followed by decreased T cell proliferation and functionality which are much needed to control viral replication. 4 Introduction INTRODUCTION Human Immunodeficiency Virus (HIV) Discovery and Epidemiology HIV-1, the primary cause of acquired immunodeficiency syndrome (AIDS) was initially discovered by Luc Montagnier at the Pasteur Institute in 1983 (1) and was later characterized in 1984 by Robert Gallo in Washington and Jay Levy in San Francisco. HIV-2, a milder form of HIV, was later isolated from a West African patient in 1986 (2). Current data suggests that HIV-1 comprises four groups, namely M, N, O, and P. The M (major) group is predominant (90%) and constitutes 11 subtypes/clades (denoted by AK), and is cosmopolitan in distribution. The O (outliers) group is restricted to western and Central Africa, whereas group N (Non-M and O), reported from Cameroon in 1998, is extremely rare (3). The presumable fourth (P group) of HIV-1 was discovered in 2009 in a Cameroonian woman and was closely associated with the gorilla simian immunodeficiency virus (SIV) (4). HIV-2 subtypes are mainly restricted to West Africa and comprise seven distinct phylogenetic lineages ranging from A-G. HIV-2 may be categorized as epidemic subtypes (A and B) and non-epidemic subtypes (C-G) (5, 6). As of 2009, over 33 million people are reportedly living with HIV-1 and among these, over 22 million were from sub-Saharan Africa. In 2009, 2.6 million new cases (~7000 new cases/day) and 1.8 million deaths were registered. Therefore HIV-1 ranks highest amongst the leading causes of death worldwide (www.uniaids.org). Etiology HIV-1 is a lentivirus similar to those found in primates called SIV believed to have existed for more than 32,000 years (7). Available literature suggests that human infections occurred through multiple zoonotic events (8). The human encounter with SIV through primates led to crossing, and via a series of reinfections and mutations, HIV-1 originated (9, 10). Phylogenetic investigations have traced the origin of HIV-1 in humans from chimpanzees, and HIV-2 from sooty mangabeys (10). Because these primate species are indigenous to Central and West Africa, it is believed that HIV originated from these African areas (9, 11). Virology HIV-1 is a positive, single-stranded (ss) RNA virus with a genome of 9.7 kilobases and is classified under family Retroviridae, sub-family Lentiviridae, and genus Lentivirus. The two ssRNA strands are surrounded by a capsid that encompasses ~2000 copies of the core protein p24 (12, 13). The capsid is enclosed in a lipid envelope formed from host cell membrane during viral budding (13)(see figure 1 for HIV-1 structure). 5 Introduction Figure 1: HIV-1 structure. HIV-1 life cycle involves a series of events within the host cell. Briefly, HIV-1 infects a susceptible CD4+ target cell via binding its gp120 receptors to corresponding host CD4 receptors and coreceptors; chemokine receptors 5 (CCR5) or 4 (CXCR4) depending on the viral tropism (i.e. R5 or X4 virus). This is followed by a conformational change in the gp120 trimer leading to membrane fusion and delivery of the viral RNA into the cytoplasm. Within the cytoplasm, viral RNA is transcribed by HIV-1 viral reverse transcriptase (RT) into ssDNA, and subsequently into double stranded (ds) DNA. With the help of viral integrase, the dsDNA is integrated into the host genome, and this integrated DNA is called a provirus. Proviral DNA is transcribed alongside host DNA during normal cell division (13) or remains latent in cells, which are called viral reservoirs ensuring bust of infections especially after the interruption of antiretroviral therapy (ART) (14-21). The RNA transcripts are spliced and translated into viral proteins or remain unspliced and exported to the cytoplasm, where they are assembled as new virions. Before viral assembly, immature viral polypeptides are processed into their functional forms by viral protease and packaged with two full-length ssRNA transcripts to make a mature viral particle. HIV-1 particles bud off from the cell membrane with an envelope of host cell plasma membrane ready to infect other cells (13, 22). (Figure 2) 6 Introduction Figure 2: HIV-1 replication cycle. HIV-1 binds and fuses to target cells by the help of CD4 receptor and CCR5/CXCR4 coreceptors. The virus uncoats and the viral ssRNA are released in the cytoplasm. Viral ssRNA is transcribed to dsDNA by the help of viral enzyme reverse transcriptase. The DNA is incorporated in the human genome by help of integrase. New viral transcripts (proviruses) are synthesized during cell division and assembled with translated protease-modified proteins at the cell membrane. New virions are formed and bud off from the host cell plasma membrane Pathogenesis The hallmark of HIV-1 infection is destruction of CD4+ T cells and eventual collapse of the host immune system. HIV-1 infects CD4+ immune cells including CD4+ T cells, dendritic cells (DCs), macrophages, monocytes, thymocytes, and microglial cells (13, 22). New receptors against HIV-1 albeit the main receptors CD4, CCR5, and CXCR4 are constantly emerging (23-25). HIV-1 transmission requires direct exposure of the body to infected blood or secretions via needles, sharps, or abrasions in the mucosa during sexual intercourse (26). The evolution of coreceptor use defines different phases of viral pathogenesis involving a change from CCR5 use (R5 phenotype) to CXCR4 use (X4) or in combination (R5X4) (27-31). The use of CXCR4 is characterized by rapid CD4+ T cell loss and onset of opportunistic infections (OIs) (32). Studies by Grivel et al (1999) 7 Introduction revealed that R5 HIV-1 is also highly cytopathic, but only for CCR5+/CD4+ T cells and because these cells constitute only a small fraction of CD4+ T cells, their depletion does not substantially change the total CD4+ T cell count. On the other hand, the effects of X4 HIV-1 is readily appreciated due to the extensive depletion of more CXCR4+CD4+ T cells within the T cell pool (33). Within 3 weeks of infection (acute phase) the viral load increases rapidly followed by increase in both cell-mediated and humoral responses (34, 35). The appearance of HIV-1 specific antibodies occurs approximately 3 weeks after the onset of infection and during this time the subject may potentially transmit HIV-1 (window period) as infection is not tested without detectable antibody levels (36, 37). Thereafter, the generated immune responses drastically curtail HIV-1 spread and keep viral replication under control for a considerable duration (5-10 years) strictly depending on both viral and host factors (38). During acute infection, subjects present flu-like symptoms like fever, rashes, oral ulcers, lymphadenopathy, arthralgia, pharyngitis, malaise, and weight loss (39). HIV-1 invades the gut, deplete CD4+ T cells and cause severe damage in body organs, especially the lymphoid tissues (13, 40-42). HIV-1 continues to replicate evading host anti-viral responses, directly or indirectly targeting HIV-1 specific T cells for elimination. This results to an eventual decline in immune responses, a steady establishment of viral dominance and the onset of OIs. Subjects with CD4+ T cell count ≤200cells/mm3 progress rapidly towards terminal AIDS and develop OIs owing to viral, bacterial, fungal, parasitic infections, and neoplasms (43). (Figure 3) Figure 3: HIV-1 pathogenesis. During acute infection, viral load increases rapidly with a corresponding drop in CD4+ T cell counts for both typical and long term non progressors. CD4+ T cells are significantly reestablished in LTNPs and maintained for over 10 years and viral replication is kept low or below the limit of detection as in elite controllers. In typical progressors, the CD4+ T cell counts slightly increase after the initial drop but then steadily declines as viral replication steadily rises. 8 Introduction Following onset of infection, HIV-1 establishes a latent infection in certain cell types namely resting memory CD4+ T cells (19, 44), dendritic cells (DCs) (20, 45, 46), monocytes (21, 47-50), and macrophages (51-53). Naïve CD4+ T cells, pluripotent progenitors in the bone marrow, and CD4+ T cells and macrophages in seminal fluid have also been reported reservoirs for latent infection in subjects undergoing ART (17, 54-56). Latent infection in cells located in the central nervous system (CNS), such as microglial cells and macrophages migrating into the CNS, are critical in protecting HIV-1 from antiretroviral drugs (18). Interruption of ART in patients with undetectable viremia results in the reappearance of viral plasma, a phenomenon brought about by viral release from the latently infected cells (44). HIV-1 Disease Progression The progression from HIV-1 infection to AIDS varies from person to person and depends on both host and viral factors. For the host dependent factors, individuals with mutation in the HIV-1 coreceptor CCR5Δ32 allele express a non-functional and truncated protein that is unfit for viral binding and fusion and this offers a strong, but not complete protection from infection in homozygous individuals (57), and a delayed disease progression in heterozygous individuals (57, 58). About 10-15% of Caucasians are heterozygous and 1% homozygous for the Δ32mutation (57). Another key host factor that determines disease progression is HLA haplotypes (59, 60). HLA molecules are amongst the most polymorphic gene products and therefore, depending on the composition of the HLA haplotypes, individuals respond differently in immune strength to the same pathogen (61). In humans, HLA B is the most diverse and polymorphic, rapidly evolving and has been connected to a majority of HLA class I related diseases and disease progression (62-64). In Chimpanzees as in humans, expression of the HLA B*27/B*57 haplotypes target conserved areas of SIV/HIV and minimize the chances of viral evasion (65). In a study by Carrignton et al (66), Caucasians with HLA B*35 and HLA Cw*04 alleles were consistently associated with rapid progression to AIDS, whereas the haplotypes HLA B*27, HLA B*57, and HLA B*5801 are associated with lower viral loads and slower disease progression (67, 68). HLA class II haplotypes have also been linked to disease progression. For instance, one study reported the association of HLA DRB1*1303 alleles with decreased viral loads and slower disease progression (69). HLA DRB1*01 was more common in HIV-1 negative as compared to positive individuals and the expression of HLA DRB1*08 corresponded to lower median viral loads (70). Interestingly, the age when acquiring the HIV-1 infection has also been shown to influence disease progression as rapid disease progression seems to be more common in older as compared to younger subjects (71, 72). Host habits and factors like smoking(73), malnutrition(74) and depression (75) have also been shown to affect HIV-1 disease progression, albeit not all studies confirm these findings. 9 Introduction Viral factors, especially evolutionary changes in HIV-1 have been directly associated with disease progression (30). Different HIV-1 strains have different replicating fitness, which in turn can affect disease progression (76-78). Individuals with slow disease progression are thought to harbor attenuated strains of HIV-1 or mutant forms of important viral genes, e.g. nef, vpr, vif, or rev, which makes the virus less virulent (79). The ability or inability to control disease progression has led to the categorization of various stages of HIV-1 infection. Individuals with stable CD4+ T cell counts, lack of disease progression, and symptoms of HIV-1 infection, and low or intermediate plasma viral loads (PVLs) for >10 years of infection are classified as long term non-progressors (LTNPs). LTNPs constitute 5-15% of HIV-1 infected individuals worldwide (80-85). Elite controllers (ECs) make up <1% of all infected subjects and are characterized by their ability to control their viral load (<50 copies/mL) below the limit of detection for longer periods of time (64, 86, 87), whereas viremic controllers (VCs) display a lesser degree of viremic control (low but detectable HIV-1 RNA levels (50-2000 copies/mL)) as compared to ECs (88, 89). The central characteristic of ECs and VCs is their ability to control viral load even if some of these subjects experience severe CD4+ T cell depletion and advancement to AIDS. Likewise, LTNPs may remain clinically healthy with stable CD4+ T cell counts, but consistently have uncontrolled PVL (90, 91). Treatment There is no effective vaccine available against HIV/AIDS, but ART regimens are widely available to improve the life span and quality of life for infected individuals. Following the advent of ART regimens, there has been a dramatic reduction in AIDS related morbidity and mortality (92-94). According to recent treatment guidelines, patients who show symptoms of AIDS regardless of CD4+ T cell counts or PVL, as well as asymptomatic patients with PVLs ≥100,000copies/mm3 and CD4+ T cell counts ≤350cells/mm3 are eligible to initiate ART (95, 96). There are four categories of antiretroviral drugs; (1) nucleoside reverse transcriptase inhibitors that interfere with the viral RT, e.g. Zidovudine, Didanosine, and Stavudine, (2) Nucleotide reverse transcriptase inhibitors that terminates chain elongation during replication, e.g. Tenofovir and Adefovir, (3) Non-nucleoside reverse transcriptase inhibitors that act via non-competitive binding to a hydrophobic pocket close to the RT enzyme, e.g. Nevirapine, Efavirenz, and Delavirdine, and (4) Protease inhibitors that block HIV-1 proteases preventing assembly of new viral particles, e.g. Nelfinavir and Saquinavir (97, 98). Recently, several alternative approaches have gained acceptance as treatment options and include fusion inhibitors that prevent viral entry into the cell e.g., Maraviroc and Enfuvirtide (99-102). In addition, integrase inhibitors that block the viral integrase are being tested as potential regimens (103), and Raltegravir has recently been approved by FDA (104). Viral maturation inhibitors are being widely investigated and 10 Introduction they work by inhibiting formation of infectious viral particles and interferon alpha (IFNα) is the currently available agent in this class (105, 106). The CCR5 receptor antagonists, e.g., Maraviroc, are the first drugs against HIV-1 infection that do not target the virus directly (104) seeing that they shield viral attachment to the cell by blocking CCR5 (102, 107, 108). Efficient adherence to treatment is important and maintains PVLs at a constantly low and manageable level (109, 110). This also help prevent the emergence of drug resistant strains (111, 112). Dendritic Cell and Functions DCs are defined as professional APCs that capture, process and present antigens to T cells and are unique in their capacity to activate primary T cell responses (113). DCs upon encountering pathogens capture and migrate to regional lymph nodes during which the DCs mature, process, and load antigenic peptides on MHC class I or II molecules. During DC migration, DC functionality is modulated by decreased phagocytosis, enhanced antigen presenting abilities, and increased expression of MHC I/II molecules, and costimulatory molecules (114-116). DC-T cell encounter in lymph nodes result either in immune activation or suppression (117), the later function being the maintenance of immune tolerance by DCs. Central tolerance occurs in the thymus for T cells and bone marrow for B cells and acts to prevent the immune system from responding to self antigens. The responsibilities of DCs in the thymus cannot be over emphasized as they educate newly produced T cells and negatively selects or eliminates T cells reacting to self MHC/peptide complexes (118, 119). DCs can also maintain peripheral tolerance by active suppression of T cell responses deleterious to the host either through induction of Tregs, or direct cell-to-cell contact mediated killing and induction of anergy (120). DC subsets There are multiple subsets of DCs depending on phenotype, functions, and location in the body (Figure 4). Enormous knowledge exist regarding DC subsets found in mice (121). The functional subsets of classical DCs in mice include CD8α+ DCs that induce T helper type I (TH1) responses and CD8α- DCs that induce TH2 responses (122, 123). These subsets have also been shown to have differential antigen processing outcomes whereby CD8α+ DCs prime for CD8+ specific T cells and CD8α- DCs for CD4+ specific T cells (124). Human DCs can be found either in blood or tissues in several distinct subsets (121). 11 Introduction Figure 4: Anatomical locations of human dendritic cells. Human myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) are bone marrow derived cells and are distinct from follicular DCs (FDC), which are of stromal origin having no role in T cell, NK cell, or NKT cell immunity (125). FDC will henceforth not be discussed further in this thesis. Present in the body internal organs such as gut, lungs, liver and kidney are interstitial tissue mDCs with the capacity to maintain surveillance, bind antigen, and stimulate T cell responses in a similar manner to blood and cutaneous DCs (126). Upon antigen encounter in these organs, the mDCs migrate to adjacent lymph nodes where they present processed antigens to T cells thereby generating adaptive responses against the pathogen of interest. Cutaneous DCs: Different layers of human skin play host to different DC subsets. The epidermis play host to Langerhans cells (LCs) and dermis to CD1a+ and CD14+ DCs, distinguished by their phenotypic expression of surface molecules (127). LCs express Langerin, a specific C-type lectin that makes up 2-3% of epidermal cells (128, 129). LCs distinctively secrete large amounts of IL-15, whereas CD14+ DCs are unique in producing a wide range of secretory factors, including IL-1β, IL-6, IL-8, IL-10, IL-12, GM-CSF, MCP, and TGF-β (130). Some functional differences between CD14+ DCs and LCs are the ability by CD14+ DCs to induce naïve CD4+ T cells to differentiate to T follicular helper cells (131, 132), and induce naive B cells to switch isotype and become plasma cells (133), whereas LCs effectively polarize naïve CD4+ T cells towards TH2 responses (133, 134). However comparative studies between LCs and CD14-CD1c+ DCs 12 Introduction showed that LCs are significantly more efficient than CD14-CD1c+ DCs at secreting both TH1 (IFN-γ and TNF-α) and TH2(IL-4 and IL-15) cytokines (135). LCs are also efficient at priming and crosspriming naive CD8+ T cells that are highly efficient in tumor cell killing as compared CD14+ DCs (130, 133, 135, 136) owing to their ability to secret IL15 (133, 137). The third group of cutaneous DC population CD1a+ DC is also good at activating CD8+ T cells better than CD14+ T cells but less efficiently than LCs (133, 135). Blood DCs: Based on their differential expression of surface molecules, three subsets of blood DCs have been categorized. CD1c+ mDCs express a wide range of toll like receptors (TLRs) including TLR 1-6, 8, and 10, and secrete proinflammatory cytokines upon stimulation (138, 139). Secondly is the CD141+ mDC subset representing a minute population that uniquely express CLEC9A, and is thought to serve as the human accomplice for mice CD8+ DCs with an efficient ability to induce CD8+ T cell responses through cross presentation and have also been report in non lymphoid tissues (140, 141). The third subset of blood DCs are the pDCs expressing high levels of IL-3Rα chain (CD123), BDCA2, and ILT7 (142). pDCs are unique in their ability to secret enormous type 1 IFNs upon recognition of viral components through TLR7 and 9 (143). pDCs can be functionally subdivided into CD2low and CD2high with the CD2high pDCs being more potent than CD2 low pDCs in their ability to induce allogeneic T cell proliferation (144) In vitro generated DCs The low percentage of DCs in the body (1% of PBMC) has made DC studies cumbersome and has prompted the establishment of ex vivo generated DCs (145, 146). Several studies have established cytokine-driven methods for expanding and differentiating DCs ex vivo creating sufficient numbers of DCs suitable for large scale studies. According to a summary by Rossi et al (2005) (147), four categories of cytokine ex vivo generated DCs were highlighted. The interstitial or dermal mDCs (1) differentiated from the CD14+ precursor by GM-CSF, TNF-α, and IL-4; LCs (2) differentiated from the CD14+ precursor by GM-CSF, TNF-α, and TGF-β; monocyte derived DCs (MDDCs) (3) differentiated from CD14+ monocytes by GM-CSF, and IL4.The fourth group are the pDCs (4) which are sustained by IL-3 in cultures. In this thesis, we have consistently used MDDCs in view to understand HIV-1 interaction with DCs and T cells and their ability to prime T cell responses. T cells T cell subsets T cell development and editing occur in the thymus and are selected according to MHC class restrictions. Thymocytes responding to MHC II are positively selected as CD4+ T 13 Introduction cells destined to provide helper functions (T helper cells), whereas thymocytes responding to MHC I are selected as CD8+ T cells destined for cytotoxic activities (cytotoxic T cells). On the other hand, T cells with receptors that strongly react to selfantigens on DCs are negatively selected, i.e. depleted (148). Positively selected naïve T cells (TN) constantly circulate between lymph nodes and peripheral blood ready to be primed in to effector T cells by APCs in lymph nodes. Effector T cells migrate out to peripheral tissues to execute their functions as instructed by APCs (149). After clearance of antigens, a majority of the effector cells undergo apoptosis and are cleared by scavenger cells, i.e. macrophages and DCs. Sustained effector cells differentiate into memory cells. The memory T cell pool is the hallmark of the acquired immune system and persists for life (150). Memory T cells can be effector memory (TEM) or central memory (TCM). The TEM circulate in the peripheral tissue and will mount an immediate effector function, e.g. direct or cytokine-mediated cell killing after encountering a recall pathogen. In contrast, TCM home to the T cell areas of secondary lymphoid tissues and will expand to TEM upon antigenic stimulation to a recall pathogen (150, 151). Effector T cell functions are either helper (TH) or cytotoxic (Tc). Helper effector T cells are subdivided according to functionality. TH1 cells express transcription factor T-bet and produce mostly IL-2 and IFN-γ. On the other hand, TH2 cells confer humoral immune responses and possess transcription factor GATA-3 producing mostly IL-4, IL-5, and IL13 (152, 153). Regulatory T cells (Tregs) make up the third group of polarized TH cells and are unique in their coexpression of high CD25, CD4, and expression of transcription factor FOXP3. Induced Tregs have two functional subsets, type 1 (Tr1) and type 3 (Tr3), which depends on IL-10 and TGF-β, respectively (154, 155). Tregs function as key modulators of adaptive immunity and mediate tolerance to self. The fourth arm of polarization is the TH17 subset commonly associated with transcriptional factors retinoid acid receptor related orphan receptor (ROR) and signal transducer and activator of transcription 3 (STAT3). TH17 cells are key producers of proinflammatory cytokines IL17, IL-6, IL-1, and IL-23 (152, 156, 157) (Figure 5). 14 Introduction Figure 5: Dendritic cell polarized CD4+ T cells. Different pathogens condition dendritic cells through their pathogen associated molecular patterns (PAMP) to activate naïve CD4+ T cells and promote their development into TH1, TH2, TH17, or Tregs. This is strictly dependent on the type of cytokines generated during the initial interaction hardwiring the generation of the different polarized T cell subsets. Cytotoxic T cells are potent destructors of viral or intracellular pathogen infected cells via direct killing of target cells by apoptosis or necrosis mediated mechanisms. The common use of granzyme and perforin for cell killing is an important tool used by Tc to execute this function (158). Tc cells are further subdivided into two subsets depending on their cytokine profile with the Tc1 subset producing high amounts of IFN-γ and the Tc2 subset producing IL-4, IL-5, IL-10, IL-13 and low amounts of IFN-γ (159, 160). DC T cell priming The fate of DC T cell interaction is either activation or suppression of the immune responses and this is a highly regulated process. The initiation of an effective immune response involves 4 signals (Figure 6). 15 Introduction Figure 6: Dendritic cell stimulatory signals to T cells. Signal 1 is the antigen specific signal between the TCR and peptide/MHC molecule. Signal 2 is the costimulatory signal and it gives either a stimulatory or inhibitory programming of the T cells. Signal 3 is the polarizing signal responsible for the promotion of TH1, TH2, TH17, or Tregs subsets. Signal 4 (not indicated in the figure), also known as the homing signal, equips the polarized T cells with homing molecules so they can migrate to their specific destination in the body, e.g. peripheral blood, gut, or skin. Signal 1; an antigen specific signal that consists of the cognate interaction of T cell receptor (TCR) complex with the DC peptide/MHC complex (161). To achieve full activation, a costimulatory signal is required (signal 2) and without this signal the T cells become anergic, which is characterized by decreased proliferation and IL-2 production (162). Signal 3; also called the polarizing signal, is responsible for polarizing the T cells to elicit a TH1, TH2, Treg, or TH17 response. The polarization depend on the prevailing circumstances in the microenvironment and how the DCs have been activated i.e. the type of PRR engagement and the cytokine profile elicited after pathogen encounter and the quality of subsequent DC-T cell crosstalk (156, 157). For example, IL-12, IL-23, IL18, IL-27, type 1 IFNs, and the cell surface expressed intracellular adhesion molecule 1 (ICAM-1) polarizes T cells towards TH1 response, whereas the IL-4, CCL2, and OX40 ligand (OX40L) polarizes towards TH2. Tregs are polarized by IL-10 and transforming growth factor-β (TGF-β) into either type 1 Tregs (Tr1) or type Tregs 3 (Tr3), respectively (156, 157, 163). TH17 polarization is guided by the mixture of TGF-β, IL-6, and IL-23 differentiating from Treg (164) (refer to figure 5). After T cell stimulation, costimulation, and polarization, effector T cells will home to their various organ destinations, e.g. skin, mucosa, and brain, to exert their functions. Primed T cells are equipped with specific sets of chemokine and adhesion molecules to guide them towards their final destinations (165, 166). For instance, increased expression of α4β7 integrins and chemokine receptor 9 (CCR9) directs effector T cells to the gut (167, 168), expression of α4β1 integrins direct these cells to the brain (168), whereas T cells with high levels of cutaneous lymphocyte associated antigen (CLA+) preferentially home to cutaneous inflammatory sites (169, 16 Introduction 170). To sum it all, during a DC-T cell interaction, the DC activates (signal 1), costimulates (signal 2), polarizes (signal 3), and directs homing of primed cells (signal 4). DC-T cell-HIV-1 Interaction Mucosal DCs are amongst the first targets of infections during sexual transmission of HIV-1 (129). At the site of infection, DCs pick up HIV-1, migrate to peripheral lymph nodes, and effectively present HIV-1 peptides to naïve T cells and prime specific T cell responses (171-175). Nonetheless, DCs play the role of a double-edged sword by transferring whole viral particles across the immunological synapses, and in this case it is called a virological synapse (129, 176-179). This leads to an efficient viral spread to CD4+ T cells. The DC-induced T cell activation and subsequent proliferation triggers a vicious cycle of viral dissemination (129, 179). (See figure 7). HIV-1 exposed DCs and their ability to prime T cell responses will be the basis of this thesis. Figure 7: HIV-1 dissemination from mucosal surfaces to lymph nodes. During sexual transmission, HIV-1can establish contact with the intraepithelial DCs/LCs in the luminal surfaces as they extend their dendrites to sample for luminal content or penetrate through lesions caused by venereal diseases, breaks in the mucosa caused by the sexual intercourse, or through direct capture and transfer of virus by epithelia cells to the DCs located underneath. Within the DCs, HIV-1 is trapped in specialized compartments that communicate with the external medium ready for transfer to T cells (178). DCs 17 Introduction migrate to the regional lymph nodes where they form clusters with T cells and transfer infectious viral particles through the infectious synapse to the T cells. So far, in vitro studies by Granelli-Piperno et al (2004) have demonstrated that supernatants derived from cocultures of HIV-1 pulsed MDDCs and autologous T cells from either uninfected or infected individuals suppressed T cell proliferation in an IL-10 dependent manner (180). Of note, the HIV-1 infection did not induce DC maturation nor were maturation stimuli able to induce maturation in already infected DCs (180). Kawamura et al (2003) found that HIV-1 infected DCs significantly downregulated CD4 expression on DCs, after which stimulation with CD40L induced increased levels of IL12p70 and decreased levels of IL-10. Allogeneic cocultures of CD4+ T cells with HIV-1 infected DCs impaired T cell proliferation and IL-2 production, which was reversible by addition of sCD4 (181). On the contrary, findings by Smed-Sorensen et al (2004) revealed that HIV-1 infected DCs failed to produce IL-12p70 in response to CD40L stimulation and had increased production of TNF-α (182). The formation of immunological synapses are important for adequate TCR stimulation and immune responses are impaired by HIV-1 as HIV-1 infected T cells poorly conjugate with APCs and the synapses are abnormal due to the accumulation of TCR and Lck in the recycling endosomal compartments. This was shown to be nef dependent as nef severely affected their intracellular trafficking and signal transduction. Therefore, alteration of endocytic and signaling networks at the immunological synapse likely impacts the function and fate of HIV-1 infected cells (183). This important research area is still lacking many facts and numerous studies are giving opposing findings. We have therefore conducted these studies to gain better understanding regarding the immunological responses generated from an HIV-1-DC-T cell interaction. HIV-1 Specific responses As discussed above, rapid or slow disease progression, exemplified in LTNPs, ECs, and VCs, is dependent on both the host and viral properties/factors (29-31, 57-85). The importance of HIV-1 specific immune responses in these individuals has been thoroughly investigated and found to play critical roles in controlling viral replication and disease progression (171-174, 179, 184, 185). In rapid progressors, HIV-1 specific CD4+ T cell responses are generally weak or absent at all stages of the disease, whereas HIV-1 specific CD8+ T cell responses are more noticed but also wane as the disease progresses (175, 185). However, increasing evidence suggest that early on in infection, HIV-1 specific CD4+ T cell responses are available but declines two - three weeks after infection (171, 186) probably due to lack of CD127 expression (187). Longitudinal studies revealed gag specific cytotoxic responses, very low numbers of infected CD4+ T cells, and higher CD4+ T cell numbers for over 8 years in LTNPs as compared to rapid progressors. Of note, gag specific responses were also noted during acute infection in rapid progressors, but eventually disappeared with the onset of AIDS (173). Findings by 18 Introduction Chinis et al (2003) (172) in pediatric patients showed highest HIV-specific CD8+ responses to gag, followed by gp120, gp41, and the V3 loop. Our in vitro studies confirmed that HIV-1 exposed DCs could prime gag, env, and pol-specific CD4+ and CD8+ T cell responses as well. We identified new CD4+ T cell specific epitopes and follow up studies in acutely infected individuals revealed that these epitopes were amongst the earliest recognized in vivo, but were lost presumably through activation induced CD4+ T cell depletion (171). Rosenberg et al (1997) (174) showed that individuals controlling viremia in the absence of ART, mount polyclonal, persistent, and vigorous HIV-1 specific CD4+ T cell proliferative responses with increased production of IFN-γ and antiviral chemokines. Therefore, the presence of protective cellular immunity in LTNPs offers hope for a possible control of HIV-1 infection, if effective vaccines can be created with the ability to elicit similar responses. However, the major hurdle to achieve this vaccine is the discovery that HIV-1 specifically infects HIV-1 specific T cells (184). The phenomenon that HIV-1 specifically infect the very cells that are supposed to respond to it (184) adds skepticism to the fact that vaccines generating HIVspecific T cells may simply create potential targets for efficient HIV-1 replication. Immunomodulatory Potentials of HIV-1 HIV-1 has adapted myriad ways to evade and hijack the immune system and to establish persistent infection in humans (188). The hallmark of HIV-1 infection is the progressive loss of immune cells ultimately leading to increased susceptibility to OIs and eventually AIDS (189). HIV-1 affects the immune system in several ways that range from general activation of the immune system, induction of general immune suppressive mechanisms, and to a greater extent initiates direct and indirect killing of infected and uninfected cells (189). The CD4+ T cells, known to regulate the adaptive arm of the immune system, are the same cells preferentially infected by HIV-1. CD4+ T cell depletion during HIV-1 infection occurs via accelerated destruction, relocation of cells to special compartments due to changes in homing receptors, chronic activation and T cell death, and impaired regeneration of new T cells (190). APCs including DCs, monocytes, and macrophages are also main targets of HIV-1 infection and contribute considerably to viral pathogenesis (15). The levels of macrophages are not significantly affected in HIV-1 infected individuals as oppose to monocytes and DCs. Their role as reservoirs for HIV-1 infection provides sources of virions, which can infect more cells (16, 51). DCs contribute even further to HIV-1 pathogenesis as they besides their ability to serve as reservoirs are reduced in number in peripheral blood (191, 192), exhibit changes in their phenotype and have impaired functionality (180, 193). HIV-1 proteins have been shown to alter the expression of costimulatory molecules as well as chemokine receptor expression on DCs (193-195). HIV-1 infected DCs in contact with T cells fail to give optimal feedback signaling to T cells, due partly to impaired IL-12 production, which in turn fail to provide optimal signals for DCs survival owing to decreased CD40L expression by the T cells 19 Introduction (182). Furthermore, sustenance of T cell proliferation is impaired and this is partly due to decreased IL-2 secretion (181, 196). Immunostimulatory and Inhibitory Molecules at a DC-T cell Encounter Stimulation via positive costimulatory molecules during a DC-T cell encounter enhances initial activation, provides additional signals for cell division, and favors survival or induction of effector functions, such as cytokine secretion and cytotoxicity (197). The most important costimulatory molecules belong to the B7-1 (CD80), B7-2 (CD86), CD28 or CTLA-4 superfamilies and upon ligation, depending on the prevailing circumstances, they will positively or negatively regulate immune responses. Positive costimulatory molecules are generally members of the immunoglobulin superfamily, e.g. CD28, and the inducible T cell costimulator (ICOS), or the tumor necrosis factor receptor (TNFR) family, e.g. CD27, OX40 (CD134), 4-IBB (CD137), and CD30 (198-200). In addition to inducing costimulatory signals to T cells, DCs can also be activated via the ligation of CD40 on its surface with CD40L on the T cells. Ligation of CD40 license DCs to increase expression of B7 molecules, secretion of IL-12 needed for TH1 responses, and also to drive effector Tc responses (201, 202) enhancing both positive costimulation and survival of the DCs and T cells. On the contrary, negative costimulatory molecules inhibit TCR mediated responses, impair T cell division, functional maturation, and induces T cell tolerance or anergy (197). Normal physiological immunosuppression makes sure to prevent responses to self antigens and will suppress excessive immune responses deleterious to the host and involve for instance Tregs. Tregs suppress myriad responses against autologous tumor cells (203), allergens (204), pathogenic or commensal microbes (205), allogeneic organ transplants (206), and fetus during pregnancy (207). Tregs may be naturally developed in the thymus and delivered to the periphery called natural Tregs (nTregs) or induced from circulating lymphocytes called adaptive Tregs (aTregs). aTregs have two functional subsets, type 1 Tregs (Tr1) and Tr3 Tregs depending on IL-10 and TGF-β, respectively (208, 209). Absence of Tregs leads to autoimmune disease as shown in both mice and humans (210-212). Some pathogens (HIV, HCV, and parasites) exploit the normal physiological immunosuppressive responses to subvert protective immune responses to the host enabling the pathogens to persist and establish chronic infections (205). The mechanisms of action of Tregs vary according to type. Once activated by a particular antigen, Tregs can suppress responder T cells irrespective of whether they share antigen specificity with the Tregs or not (213). Tregs function by secretion of immunosuppressive cytokines, e.g. IL-10 and TGF-β, establishing cell contact with the unwanted cell, or by functional modification or killing of APCs (214). Contact-dependent suppression by Tregs (Figure 8) may involve granzyme and perforin mediated killing, downregulation of costimulatory molecules on APCs, or stimulation of DCs to express indoleamine 2, 3 dioxygenase (IDO), an enzyme 20 Introduction that catabolizes the essential amino acid tryptophan to kynurenines that are toxic to T cells. Tregs suppressive mechanisms appear to also depend on expression of the negative costimulatory molecules, cytotoxic T lymphocyte antigen-4 (CTLA-4) and partially on lymphocyte-activation gene 3 (LAG-3) on their surfaces (214). Figure8: Contact dependent mechanisms of regulatory T cell functions Regulatory T cells use different mechanisms of action and they include out competing effector T cells for binding on DCs due to their increased affinity, modulation of DC functions by dampening the expression of costimulatory molecules and or direct cell killing of DCs or effector cells Thus far, several negative costimulatory molecules, viz., programmed death-1 (PD-1), T cell immunoglobulin and mucin domain 3 (TIM-3), TNF-related apoptosis inducing ligand (TRAIL), and CD160 together with suppression associated transcription factors like B-lymphocyte-induced maturation protein-1 (BLIMP-1), FOXP3 and deltex-1 (DTX1) have been reported on activated T cells exerting suppressive responses (215221). Inhibitory costimulatory molecules regulate cell activity through diverse mechanisms. For instance, the engagement of CTLA-4 to CD80/86 or PD-1 to PD-ligand 1/2 induce signaling cascades leading to impaired TCR mediated IL-2 production and T cell proliferation (221-223). HIV-1 has been shown to exploit the immune modulatory phenomena to their advantage by converting more naïve T cells to Tregs thereby suppressing HIV-1 specific CD4+ and CD8+ T cells (224, 225). Assessment of T cells ex vivo from HIV-1 infected patients and in vitro allogeneic mixed lymphocyte reactions (MLRs) showed increased expression of PD-1 and CTLA-4 in the presence of HIV-1 and blockade of these molecules significantly improved T cell functionalities (219, 221). The mechanisms behind impaired T cell activity in HIV-1 infection have been reported, and are largely attributed to PD-1 and CTLA-4. PD-1 expression on HIV-specific CD8+ T cells correlated with impaired HIV-1 specific CD8+ T cell functions as well as predictors of disease progression. Increased expression of PD-1 on CD4+ and CD8+ T cells correlated positively with viral load and inversely with CD4+ T cell counts (226). CTLA-4 expression was also upregulated on HIV-1 specific T cells and was positively correlated with disease progression and negatively with the capacity of CD4+ T cells to 21 Introduction produce IL-2 in response to viral antigens (221, 227). Blockade of these molecules significantly restored T cell functions and their ability to respond to viral antigens (221, 226, 227). We also demonstrated in vitro, the expression of PD-1 and CTLA-4 on HIV-1 primed T cells (219, 220). Recent studies suggest a role of TIM-3 in mediating T cell impairment and primarily acts following its ligation with galactin-9 and/or phosphatidylserine ligands, triggering dysfunctional T cells and eventual cell death (215, 228-230). The levels of TIM-3 expression on T cells from HIV-1 subjects have correlated positively with HIV-1 viral load and inversely with CD4+ T cell count. Progressively infected patients upregulated TIM-3 expression on HIV-1 specific CD8+ T cells resulting in failure to proliferate and secret cytokines in response to antigenic stimuli (230). CD160, another inhibitory molecule impairs T cell activation through its ligand HVEM by interfering with tyrosine phosphorylation proteins of CD3/CD28 T cell activation pathways (231). The expression of CD160 has been reported in both acute and chronic HIV-1 infections. Yamamoto et al (2011) (232) showed expression of multiple inhibitory molecules, e.g. PD-1, CD160, and 2B4, on HIV-1 specific CD8+ T cells, which correlated both with viral load and dysfunctional cytokine production (232, 233). In addition, LAG3 expression on T cells in HIV-1 subjects has been disputed seeing that comparative studies between CMV and HIV-1 showed increased expression PD-1, CD160, 2B4, and LAG-3 on CMV specific CD8+ T cells but not LAG-3 on the HIV-1 specific CD8+ T cells (232). However, other reports have shown enhancement of LAG-3 on T cells in HIV-1 infected subjects with unrestrained HIV-1 replication (234, 235). Our in vitro studies showed that HIV-1 exposed DCs primed naive T cells with elevated gene and protein expression levels of LAG-3 (220). LAG-3 binds with a higher affinity to MHC class II molecules compared to the CD4 receptor and facilitates immunosuppression, especially via Tregs (236), and has been linked to functional exhaustion of CD8+ T cells in persistent viral infections (PVI) (237). The role of TRAIL as a negative inhibitory molecule was brought to light when Lunemann et al (2001) (238) demonstrated that TRAIL could inhibit antigen specific T cell activation without necessarily inducing apoptosis. TRAIL also expanded CD4+CD25+ Tregs in mice to control experimental autoimmune thyroiditis, another possibility that TRAIL is not just an apoptosis inducing molecule, but could also serve as a regulator of a hyperactivated immune system (239). We confirmed this by showing that HIV-1 exposed DCs gave an increased expression of TRAIL on T cells, which suppressed immune activation without inducing apoptosis (219, 220). We further showed that only a synergistic effect of a combined blockade of TRAIL, CTLA-4, and PD-1 could significantly restore T cell proliferation, projecting TRAIL as a potential candidate in the ranks of inhibitory molecules (219). Notably, the transcription factor DTX1 has been linked to T cell anergy and suppression of T cell activation through NFATc by both ubiquitin E3-dependent and independent 22 Introduction mechanisms. Deletion of DTX1 promoted T cell activation, conferred resistance to tolerance and led to increased inflammation and autoimmunity (218). On the other hand, NFATc1 signaling has been shown to regulate PD-1 (240) and CTLA-4 (241) expression upon T cell activation. BLIMP-1 has been termed a transcriptional repressor and regulates T cell homeostasis and function (242) likewise regulating T cell exhaustion in chronic viral infections (217). Elevated expression of BLIMP-1 in virus specific CD8+ T cells repressed key aspects of normal memory CD8+ T cell differentiation and promoted high expression of inhibitory receptors, PD-1, LAG-3, CD160, and 2B4, during chronic LCMV infections (217). Both IL-2 and IL-2 activator Fos were directly repressed by BLIMP-1, which attenuated T cell proliferation and survival (243). The mechanisms whereby HIV-1 induces the upregulation of inhibitory molecules remains unclear and therefore efforts to uncover the molecular mechanisms underlying their upregulation will open up newer avenues for preventing viral suppression of the host immune system. Conclusions In the acute phase of HIV-1 replication humoral and cellular immune responses are generated and control this chronic disease for a considerable time period. Due to several factors in the HIV-1 pathogenesis, viral immunity starts declining including loss of CD4+ T cells, DCs, and other immune cells, resulting in gradual increase of the PVL. The individual eventually becomes vulnerable to opportunistic infections and malignancies, which leads to death unless treated with ART. Humans can manage chronic viral infections, e.g. CMV, EBV, and HSV, by generating immune responses that control infection throughout life but has failed to do so against HIV-1. Therefore, knowledge about the type of responses generated by the HIV-1 infection and subsequently, the mechanisms whereby these immune responses gradually decline over time is of paramount importance. Furthermore, in depth studies are needed to unveil the underlying mechanisms contributing to HIV-immunomodulation, a critical aim in our strides which can help design an effective anti-HIV vaccine. 23 Introduction 24 Aims and Objectives AIMS AND OBJECTIVES The studies were aimed to investigate the HIV-1 specific T cell responses generated by DCs pulsed with HIV-1, and to understand the molecular mechanisms and underlying signaling pathways associated with the onset of immune impairment in HIV-1 infection in vitro. Specific Aims Paper 1: To decipher the type of HIV-1 specific T cell responses primed from naïve T cells by HIV-1 pulsed DCs in vitro and to compare these responses with those existing in HIV-1 infected individuals in vivo. Paper 2: To investigate the effects HIV-1 exert on DCs’ ability to prime naïve allogeneic T cell responses. Paper 3: To investigate the array of inhibitory molecules upregulated by the presence of HIV-1 during DC priming of T cell responses. Paper 4: To unveil the underlying mechanisms through which HIV-1 give rise to immunosuppressive T cells when exposed and primed with DCs. 25 Aims and Objectives 26 Methods METHODS Dendritic cell preparation DCs were differentiated from monocytes in peripheral blood mononuclear cells (PBMCs). Fresh blood samples from HIV-1 infected individuals or healthy donors were diluted (50%) with RPMI1640 supplemented with 2mM EDTA and layered on FicollHypaque. The Buffy coat was separated by density gradient centrifugation at 2200rpm for 20minutes with no brakes. Buffy coat was carefully collected and washed 4 times in RPMI1640 at 1800rpm, 1500rpm, 1100 rpm, and 900rpm every 10minutes. Thirty to forty million PBMCs were plated in tissue culture dishes in 1% plasma at 37°C in 5% CO2 for 1-2 hours for monocytes (CD14+) to adhere. The non-adherent cells (NAC) were washed off with RPMI1640. Adherent cells (DC progenitors) were differentiated in 1% plasma supplemented with 100IU/mL recombinant granulocyte macrophage–colony stimulating factor (rhGM-CSF) 300U/mL and recombinant human IL-4 (rhIL-4) (Figure 9). Figure 9: Propagation of monocyte derived dendrite cells from blood progenitor Cytokines were replenished every 2 days to facilitate the differentiation of progenitors into MDDCs. Immature MDDCs were harvested on day 6. DC maturation was induced by adding 30ng/mL of polyinosinic acid (Poly I:C). 27 Methods Naïve T cell Separation Naïve bulk T cells were prepared from NACs derived from PBMCs by magnetic bead separation following manufacturer’s instructions (Miltenyi Biotech). In brief, naive T cells were negatively selected using anti-CD19 microbeads for B cells, anti-CD56 for NK cells, anti-CD45RO for memory T cells and anti-CD14 for monocytes. NACs were incubated for 15 minutes with bead antibodies, washed once in MAC buffer and loaded on a magnetic column. Labeled cells were trapped in the column while the unlabelled (negatively selected naïve bulk T cells) cells were collected as the effluent. Virus preparation Virions of HIV-1MN (X4-tropic), clade B were produced by infection of CL.4/CEMX174 (T1) cells and purified by sucrose gradient ultracentrifugation as described previously (244). Samples were titrated for the presence of infectious virus using AA2CL.1 cells and HIV-1 p24 antigen capture kits (AVP, NCI). Aldrithiol-2 (AT-2)-inactivated HIV-1 (AT2 HIV) was prepared as described previously (245). In paper 1, infectious HIV-1MN (Lots 3807, 3966, and 3941: AVP, SAIC Frederick, Inc., NCI Frederick) and AT-2 HIV-1MN (Lots 3808, 3965, 3937, and 3934) were used. HIV-1 BaL/SUPT1-CCR5 CL.30 (Lot P4143) was produced using chronically infected cultures of ACVP/BCP Cell line (No. 204), originally derived by infecting SUPT1-CCR5 CL.30 cells (graciously provided by Dr. J. Hoxie, University of Pennsylvania) with an infectious stock of HIV-1 BaL (NIH AIDS Research and Reference Reagent Program, Cat. No. 416, Lot 59155). Virus was purified by continuous flow centrifugation using a Beckman CF32Ti rotor at 30,000 rpm (~90,000 x g) at a flow rate of 6 liters/hour followed by banding for 30 minutes after sample loading. Sucrose density-gradient fractions were collected and virus-containing fractions were pooled and diluted to less than 20% sucrose, and virus pelleted at ~100,000 x g for 1 hour. The virus pellet was resuspended at a concentration of 1,000 x g relative to the cell culture filtrate and aliquots frozen in liquid N2 vapor. 1500ng/106 p24 equivalents of HIV-1 were pulsed with mature or immature DCs for 24 hours before culturing with naïve T cells. DC T cell cocultures HIV-1-overnight-exposed immature(I) and mature(M) DCs were harvested, washed twice, and cocultured with CFSE labeled naïve T cells in 5% pooled human serum (PHS) in RPMI1640 at a ratio of 1:10 in 96-well plates. Autologous cocultures as in paper 1 were restimulated at day 7, 14, 21 and 28 by adding autologous HIV-pulsed DCs, whereas allogeneic cocultures as in papers II, III and IV were restimulated once on day 7 by adding the same groups of DCs utilized to initiate the priming event. Expanded T cells were analyzed for numerous parameters, e.g. antigen-specific epitopes on primed T cells by ELISPOT assays, cytokine analysis by ELISA, Luminex, and intracellular staining. T 28 Methods cell proliferation was measured by flow cytometry or 3H-Thymidine incorporation using a β-counter. Gene expressions in primed T cells was by microarray and qRT-PCR, whereas expression of cell surface markers was by flow cytometry ELISPOT: The ELISPOT assay is a widely used technique primarily developed to identify antibody secreting cells (246). Today ELISPOT has been used to measure specific cytokineproducing cells as well as measuring epitope responses to particular peptides. ELISPOT assays employ the sandwich enzyme-linked immunosorbent assay (ELISA) technique. In brief, a polyvinylidene difluoride (PVDF) microplate is pre-coated with a monoclonal or polyclonal antibody specific for the chosen analyte overnight at 4oC or 2-3 hours at 37oC. The cells under investigation are then pipetted into the wells together with the stimulant and the microplate incubated. During incubation (usually overnight), secreted antibodies or cytokines bind to the immobilized antibody, in the immediate surroundings. Unbound cells are washed and a biotinylated polyclonal antibody specific for the chosen analyte is added to the wells for about 2 hours, washed and further incubated with an alkalinephosphatase conjugated to streptavidin. The unbound enzyme is subsequently removed by washing and a substrate solution (usually BCIP/NBT) is added. Blue-black colored precipitates are formed and appear as spots at the sites of analyte localization, with each individual spot representing an individual analyte-secreting cell. The spots can be counted with an automated ELISPOT reader system or manually, using a stereomicroscope. In our system, expanded T cells were harvested and added to IFN-γ ELISPOT plates at 50,000 T cells/well and restimulated with DCs infected with Vaccinia-vectors (MOI = 2) (V-ctr, V-env, V-gag, V-pol, V-nef) to measure class 1 responses or pulsed with gp160, nef, p66, or p24 proteins (5 μg/ml; Protein Sciences Corporation, Meriden, CT) for class II responses. The ratio of DCs to T cells was 1:10 and the assays were carried out as described above. Epitope mapping was by ELISPOT using pools of overlapping peptides to initiate the responses. Positive wells were reconfirmed with the single peptides by intracellular staining. Measurement of secretory factors ELISA: Cell secretions in supernatants from cocultures were measured by ELISA, Luminex or intracellular cytokine staining. The principle of ELISA is much same as ELISPOT with the only difference being the impossibility in estimating the number of cells actually secreting the analyst of interest. Levels of p24, IL-10 and TGF-β in supernatants from our cocultures were measured using ELISA kits as instructed by the manufacturer (eBioscience). Supernatants from day 8 of cocultures were also analyzed using the BioPlex Cytokine Luminex assay for IL-10, IL-2, IL-4, IL-5, IL-13, IL-17, TNF-α, G-CSF, TGF-β, IFN-γ, RANTES, PDGF-β, MCP-1, IP-10, bFGF, eotaxia, IL-9, IL-8, IL-6, IL-1R-α, VEGF, and IL-1Rβ following manufacturer’s instructions. 29 Methods Intracellular cytokine staining: The benefit of intracellular cytokine staining is the simultaneous analysis of surface molecules and intracellular antigens or proteins at a cell to cell level. The cells are first stained for surface expression, fixed with a fixative (e.g. formaldehyde) and permeabilized with a detergent (e.g. saponin) to allow antiantigen or cytokine antibodies to stain intracellularly. To measure cytokine levels in in vitro stimulated cells, blockade of cytokine secretion with protein transport inhibitors e.g. monensin or Brefeldin A (BFA) solution at the final hours of stimulation is important. In our experiments, intracellular cytokine staining for the assessment of cytokine/chemokine production was performed on primed T cells. Primed CD4+ and CD8+ T cells were initially stimulated with HIV-1 MDDCs for 6 hours and in the presence of 10 μg/mL of BFA. Fluorochrome-conjugated antibodies against IL-2, TNF-α, MIP-1β, and IFN-γ were used. Intracellular cytokine staining for FOXP3 was performed according to manufacturer’s instruction (eBioscience) and granzyme/perforin expression measured from the allogeneic cocultures after day 7 restimulation (day 8). Samples were acquired on a FACSCalibur or LSRII and data was analyzed using Cell Quest or FlowJo software. T cell proliferation analysis T cell proliferation is an important measurement for T cell response to particular stimuli. Various methods are available but herein in this thesis have we described two methods consistently. T cell proliferation was measured by either monitoring the degree of dilution of the dye carboxyfluorescein succinimidyl ester (CFSE) by flow cytometry or monitoring the levels of radioactive 3H-Thymidine incorporated in the proliferated cells within the last 16 hours of coculture. CFSE is a dye that will spontaneously and irreversibly binds to the amino acid lysine in both intracellular and cell surface proteins of a cell. When the T cells divide, CFSE is equality distributed between the daughter cells and as a result, the fluorescence intensity of the dye will be halved as the number of cell cycles doubles. Halving of cellular fluorescence intensity marks each successive generation in a population of proliferating cells and is readily measured by flow cytometry (247) (Figure 10). In this thesis, naïve T cells (pellet) were resuspended in 10μM CFSE in 0.1%BSA for 10 minutes at 37oC. Cells were washed and cocultured with mock or HIV-1 exposed DCs. At the end of culture, CFSE dilution was measured in expanded T cells. The measurement of T cell proliferation by 3H-Thymidine was an alternative method used in this thesis. During cell division, cell DNA replicates and thus their precursors adenines, cytosine, guanine and thymidine are added. 30 Methods Figure 10: CFSE dilution, a measurement of T cell proliferation. The parent cells are coupled with CFSE dye and each cell division decreases the intensity to half as it is divided up between the parent and the daughter cells. (A) Represents a histogram for allogeneic T cell activation with two peaks. The peak to right represents parent/undivided cells with the strongest intensity and the peak to the left represent the daughter cells with weaker intensity. (B) Shows a histogram for PHA activated T cells with clear peaks and each peak represents a successive cycle which is not visible in allogeneic reactions. Measurement of the fluorescence intensity from parent to daughter cells gives a measurement of T cell proliferation. Radioactive thymidine (3H-Thymidine), will therefore be incorporated into the DNA as the cells proliferate and levels will be measured with a scintillation counter. In this thesis, 4μCi of 3H-Thymidine was added to the cocultures during the last 16 hours. Cells were harvested to a membrane, using a cell harvester, and membrane washed 5 times with water and kept to dry. The membrane was then laminating in 5ml β-plate scintillation fluid and loaded to the beta counter in a cassette. The level of radioactivity was measured and shown to correlate positively with T cell proliferation. Fluorescence activated cell sorter (FACS) The principle guiding FACS is the rapid separation of cells in suspension based on the size, granularity and color of their fluorescence. Antibodies towards a target(s) in suspension are conjugated with a fluorescence dye. A cell suspension is acquired through a nozzle permitting a stream of single cells to pass through. A laser beam directed towards the stream excites the targets as each labeled cell passes through resulting in fluorescence, which is detected by a detector. The cells are first separated depending on light scatter (forward scatter measures cell size and side scatter measures cell granularity). Different fluorochromes on different cell types or molecules on the same cell will be recorded at different wavelength and depicted as quantitative measurements for cell types, numbers or percentage of protein expressed on the cell. In our experiments, 31 Methods antibody-cell-suspensions were incubated for about 30 minutes in FACS buffer (0.5% BSA), washed and collected in FACS tubes for acquisition. Flow cytometric acquisition was performed on a four color FACSCalibur or LSRII. Data were analyzed using Cell Quest or FlowJo software. Gene expression by real time PCR (SYBR® Green) Real-time PCR allows the detection of amplified DNA in real time and therefore, measures the kinetics of the reaction as compared to traditional PCR that measures only the final phase of the reaction. The SYBR® Green technique is an alternative real time method whereby the measurement is based on the intensity of fluorescence emission of a dye (SYBR® Green). During amplification, dsDNAs are formed within which SYBR® Green intercalates resulting in fluorescence. The intensity of fluorescence emission is proportional to the amount of dsDNA formed, which depends on initial starting material. A house keeping gene is required to normalize the final values thereby checking for differences in starting concentrations. In our assays, mRNA was isolated using a commercial Spin technology according to the manufacturer’s protocol (Qiagen). First strand cDNA was made from 1 μg of mRNA in a 20 μL reaction volume. Twenty micro liters of reaction mix containing 1 μL of oligo dT (50 μM), dNTP (10 mM), SuperScriptTM III (200 U/mL), DTT (0.1 M), RNaseOUTTM (40 U/mL), 4 μL of 5 x First-Strand buffer and 1 μg of mRNA in 11 μL of nuclease-free water. β-actin was used as our endogenous control and qRT-PCRs was performed using Prism 7900HT. Samples were run in triplicate each, a 10 μL reaction mixture that contained 5-10nM (1 μL each) of forward and reverse primers, 5 μL Fast SYBR® Green Master Mix, 4 nM (2 μL) template DNA and 1 μL of water. Results were expressed as ΔΔCt and the values normalized. Statistical analysis Statistical significance was measured using GraphPad Prism 4.0 software (GraphPad, La Jolla, CA, USA). The Wilcoxon non-parametric matched pairs test was used to compare between paired groups showing decline in specific HIV-1 responses over time as in paper I, whereas the unpaired t test and One way ANOVA-Tukey’s test was used to compare between the groups as of paper II, III and IV. Of note, due to considerable degree of variability within the experiments, we obtained statistical values after normalizing/transforming the data whereby each data set was divided by the sum total of all values in the data set. Each value within a group is presented as percentage. Differences were considered significant when *P<0.05, **P<0.005 and ***P<0.001. 32 Findings and Discussion FINDINGS AND DISCUSSION Paper 1 Background The progression to AIDS is eminent in most cases of HIV-1 infected subjects worldwide if not treated with ART (90). This is owed to insufficient immune responses generated and loss of CD4+ T cells. HIV-1 specific cellular immune responses have been reported in rapid progressors and shown to wane as the disease progresses (175, 185). In connection to this, HIV-1 favorably infect HIV-1 specific CD4+ T cells partially explaining why the cellular responses rapidly disappeared over time (184). LTNPs control viral replication and delay disease progression owing in part to efficient priming and persistent HIV-1 specific T cells (172-174). These subjects have prompted intense interest to understand the mechanisms under which sustained immune responses are generated (90). DCs are professional APCs and even though they are responsible for viral dissemination to T cells (129, 179) they also prime HIV-1 specific cellular responses (172, 173). Drawing from a vaccine study by Lu et al (248), using autologous DCs pulsed ex vivo with aldrithiol-2 (AT-2) inactivated HIV-1, they showed increase levels of HIV-1 specific T cells and decreased viral load and an eventual control of viral load in chronically infected subjects. Different HIV-1 antigenic constructs have been used to pulse DCs and shown to prime HIV-1 specific responses in vitro (249-253). However, the capacity of DCs pulsed in vitro with whole virions; the most physiological source of HIV-1 antigens, to prime T cells in vitro had not been fully investigated. Nevertheless, animal studies using whole HIV-1 to prime HIV-1 specific responses had been conducted (254, 255). The aim of this study was to examine whether whole HIV-1 could serve as an efficient source of antigens for DCs to prime HIV-1 specific T cells from naïve cells in vitro. Of note, we used both infectious and noninfectious AT-2 HIV-1 to allow us to compare the nature of responses generated between these antigen sources, findings which could be of great interest for vaccine design. Principal Findings The study investigated the effects exerted by HIV-1 on DC phenotype and functionality and showed that HIV-1 did not affect the phenotypic profile of mature MDDCs, which is an important component needed for naïve T cell stimulation. In addition, MDDCs retained their ability to process, present, and activate HIV-1 specific CD4+ and CD8+ T cells. Further to investigate if MDDCs could prime HIV-1 specific T cell responses from naïve T cells, DCs were loaded with AT-2 or infectious HIV-1 and cultured with naïve T cells in vitro. Priming with noninfectious AT-2 HIV-1 yielded a slightly higher response compared to infectious HIV-1. MDDCs pulsed with HIV-1 activated significantly more 33 Findings and Discussion HIV-1 specific CD4+ than CD8+ T cell responses. Assessment of the primed responses by ELISPOT assays using a matrix of HIV-1 overlapping peptides showed that the epitopes recognized by the primed T cells were from the env-gp41, env-gp120, gag-p17, gag-p24, pol-INT, and pol-RT. The in vitro primed specific T cells secreted high levels of effector cytokines (IL-2, IFN-γ, MIP, and TNF-α) and were comparable with responses existing in infected individuals (256-259). We showed that five out of the ten CD4+ T cell responses primed in vitro matched the responses seen ex vivo in HIV-1 infected individuals at different stages of disease (256-259). The unmatched responses (ELKKIIGQVRDQAEHLK pol-IN157-173, MTKILEPFRKQNPDIVIY pol-RT163-181, AVLSIVNRVRQGYSPLSF env-gp41700-717, LELDKWASLWNWFNITNW envgp41601-678, and RPVVSTQLLLNGSLA env-gp120252-266 were considered novel as these epitopes had not been reported before. We were able to detect three out of five of these novel responses, i.e. pol-IN157-173, env-gp120252-266 and env-gp41700-717, in acutely infected HIV-1 individuals suggesting that these responses arise very early in infection and disappear even before they are detected. Although less frequent, HIV-1 specific CD8+ T cell responses were also observed and they all matched the responses seen ex vivo in HIV-1 infected individuals at different stages of disease (256-259). Improved priming of specific CD8+ T cell responses were achieved after depletion of CD4+ T cells indicating the possible dominance of CD4+ T cells over CD8+ T cells within the priming cultures. Discussion/Conclusions DCs help disseminate whole viral particles to T cells (129, 177-179), but can also prime HIV-1 specific T cell responses in HIV-1 infected individuals (172-174, 179, 184, 185, 256-259). The ability of MDDCs to prime broad HIV-1 specific T cell responses in vitro can be used in vivo to prime and also to activate HIV-1 specific memory T cells towards the control of infection. Our study confirmed this notion showing that DCs pulsed with infectious or AT-2 inactivated HIV-1 could prime T cell responses that arise naturally in vivo in HIV-1 infected individuals and demonstrates that AT-2 HIV-1 was an excellent source of antigens, a finding that may be of great importance in the quest for vaccine development (248). The differences in responses between infectious and AT-2 HIV-1 remains unclear but could be attributed to direct effects of viral replication in the cells or negative immunoregulatory effects exerted by HIV-1 on both DCs and T cells (180, 260). All in vitro primed CD8+ T cell responses were confirmed in vivo in chronically infected patients as compared to CD4+ responses where only five out of ten were confirmed. Of interest, our studies in acutely infected HIV-1 individuals revealed that three out the five of the novel epitopes existed initially in them but declined overtime, a finding that confirmed the theory of rapid decline of CD4+ specific T cell responses during the course of infection and the importance this may play in HIV-1 pathogenesis (175, 184, 185, 187, 261). Long-term studies in these infected subjects revealed both loss in gag, pol, env and nef CD4+ specific T cell responses as well as IL-2 and IFN-γ secretion. This is true as 34 Findings and Discussion HIV-1 specific T cell responses are preferential targets for elimination by HIV-1 (184) and also as a consequence of chronic immune activation due partly to bacterial translocation (41, 262). The overwhelming CD4+ T cell responses seen in vitro consisted with the strong and broad CD4+ T cell responses we observed in individuals with acute HIV-1 infection in vivo, which were greatly reduced or disappeared within three months after onset of infection. Studies in LTNP have revealed the importance of HIV-1 specific responses as they have increased and persistent levels for many years of infection (79, 90, 91, 172-174). Focus on enveloped based vaccines aimed at inducing neutralizing antibody responses for sterilizing immunity (263, 264) has gradually shifted towards cellmediated responses (265-267), which have been shown to control viral load in asymptomatic infected individuals (80-84, 86-89). Therefore, in depth understanding of mechanisms for efficient priming of HIV-1 specific responses can be of paramount benefit to foster the development of cell mediated therapeutic vaccines. On the other hand, detailed studies to investigate the mechanisms behind the rapid decline in the CD4+ and CD8+ specific T cell responses in rapid progressors is crucial for the maintenance of the eventual vaccine induced responses and which will be prime focus of our subsequent investigations. Paper II Background HIV-1 dissemination has been linked to DCs and T cells. DCs pick up HIV-1 at the sites of infection, and migrate to the lymph nodes where they present viral peptides to T cells thereby triggering specific immune responses (172-175) but at the same time transfer whole viral particles to the CD4+ T cells (177-179). HIV-1 has adapted myriad ways of evading and hijacking the immune system to establish a persistent infection in humans (188). This includes viral disguise, general activation of the immune system, induction of general immune suppressive mechanisms, and direct or indirect killing of infected and uninfected cells (189). The majority of infected individuals with high viral loads have both diminished levels and functionally impaired DC and CD4+ T cells (191, 192), which reveals that the presence of high viral burden exerts negative and deleterious effects on the host’s immune cells. Individual HIV-1 proteins, such as nef, vpr, and tat exert negative effects on immune cells. Nef is associated with decreased surface expression of MHC class I, CD80, and CD86 molecules on infected cells (194). In addition, nef upregulates TNF-α production and Fas ligand (FasL) expression on DCs, resulting in cytotoxic DCs with an impaired ability to activate CD8+ T cells (268). Vpr down regulates the expression of costimulatory molecules on DCs (195). Given the myriad effects observed when studying individual HIV-1 proteins, we wanted to study the effects of the whole virion, i.e. the physiological source of HIV-1 antigens, on immune cell functionality. Our aim therefore was to study the effects of whole HIV-1, infectious and 35 Findings and Discussion noninfectious AT-2 HIV-1 BaL, on DCs ability to prime T cell responses. We were also interested in pinpointing the main factors responsible for the immunomodulation. Principal Findings The results showed that in vitro exposure of DCs to both infectious HIV-1 and noninfectious AT-2 HIV-1 impaired DC’s ability to prime naïve T cells leading to decreased T cell proliferation. The involvement of T regulatory cell cytokines, i.e. IL-10 and TGF-β, was eliminated as there were no significant differences in their levels between mock and HIV-1 exposed cocultures. We therefore postulated a contact dependent suppressive mechanism as shown for naturally occurring regulatory T cells (NTregs) (210, 214). Noteworthy, our findings showed that the addition of HIV-1 primed T cells into new DC-T cell priming assays suppressed T cell proliferation. This indicated that NTregs could be involved in T cell impairment but when the NTregs were depleted from the naïve T cell pool, the impaired T cell proliferation persisted. The failure to reverse T cell impairment in the absence of NTregs prompted an inquiry for inducible suppressor T cells. To understand this concept, HIV-1 primed T cells were immunophenotyped for inducible suppressor markers PD-1, CTLA-4, and TRAIL (221, 226, 227, 269). We showed increased expression of TRAIL, PD-1, and CTLA-4 on HIV1 primed T cells compared to mock T cells. Blocking TRAIL, PD-1, and CTLA-4 individually demonstrated a slight but insignificant restoration in T cell proliferation but when simultaneously blocked did the restoration of T cell proliferation reach significant levels. Discussion/Conclusion HIV-1 exposed DCs do not only infect T cells interacting with them (177, 178, 184) but also, as we have shown in this study, induce immune impairment by giving rise to increased expression of inhibitory molecules CTLA-4, TRAIL, and PD-1 on the primed T cells. The T cells primed in the presence of HIV-1 induced impairment of other T cells in a contact dependent manner. The mechanism of immune suppression by Tregs/suppressor T cells varies depending on the microenvironment. Suppressor T cells are recruited to DC areas to regulate antigen specific naïve T cell activation and are more efficient in their interaction with DCs than naïve T cells due to their high expression of adhesion molecules such as LFA-1 (210, 214). Suppressor T cells may as well induce suppression by downregulating CD80/86 expression on DCs via a CTLA-4 dependent mechanism. Certain suppressor T cells can directly kill or inactivate responder T cells by secreting granzyme/perforin or immunosuppressive cytokine such as IL-10 and TGF-β (210, 214). In our system, secretion of IL-10 and TGF-β was not responsible for T cell impairment, rather a contact dependent interaction between cells. HIV-1 therefore has profound immunomodulatory effects on DCs using them as vehicles of transport to the lymph 36 Findings and Discussion nodes and infection of T cells (129, 177-179) as well as modulating DCs to prime contact-dependent suppressor T cells. Paper III Background Chronic viral infections, including HIV-1, are characterized by impaired immune responses. HIV-1 affects the immune system in myriad ways but general immune suppression is of critical importance for viral pathogenesis (189). Immune suppressive mechanisms include the induction of immunosuppressive molecules on activated T cells and expansion of Tregs (219, 221, 226, 269). Tregs dependent suppression may function through the secretion of immunosuppressive cytokines, e.g. IL-10, and TGF-β (208), or via cell-to-cell contact mechanisms such as granzyme and perforin (270, 271) killing and/or through the delivery of negative signals to responder cells (272). Tregs may also function by direct functional modification or killing of APCs, (214) downregulation of costimulatory molecules on APCs in a CTLA-4 dependent mechanism and/or stimulate DCs to express indoleamine 2, 3 dioxygenase (IDO), an enzyme catabolizing the essential amino acid tryptophan to kynurenines and this substance is toxic to T cells (214). In addition to expression of CTLA-4 on Tregs, HIV-1 primed T cells expressing PD-1 and TRAIL (219, 221, 226, 227). We showed previously that TRAIL in combination with CTLA-4, and PD-1 significantly impaired T cell proliferation in the presence of HIV-1 and their simultaneous blockade drastically restored the T cell proliferation. In this study we postulated other possible inhibitory molecules on T cells primed by HIV-1 exposed DCs. LAG-3 and B and T lymphocyte attenuator (BTLA) have been shown to be involved in regulation of cell cycle activity (216, 236) and functionality of regulatory T cells (236). In addition, TIM-3 inhibited TH1 responses and promoted immunological tolerance (229) and has been reported to be overexpressed in HCV infected individuals (273). In addition to PD-1, CTLA-4, TRAIL, we therefore examined the expression of LAG-3, BTLA, and TIM-3 on HIV-1 primed T cells. We also examined the expression of CD160, as this is a potent immune regulator in disease conditions (274). Furthermore, we studied the transcriptional factor BLIMP-1, which has also been reported as a transcriptional repressor in CD8+ T cell exhaustion (217, 275) and T cell homeostasis (242) and compared the molecular expression with mock primed T cells. Principal Findings Our findings demonstrated that presence of HIV-1 during DC priming of allogeneic naïve T cells significantly increased expression of CTLA-4, PD-1, TRAIL, LAG-3, TIM-3, and CD160 on the T cells primed by HIV-1 pulsed DCs. The gene expression levels of the transcription repressors BLIMP-1 and FOXP3 were also significantly upregulated and correlated with expression of inhibitory molecules. We further showed that the 37 Findings and Discussion suppressor T cells coexpressed TIM-3, LAG-3, TRAIL, and/or CTLA-4 with PD-1. The functional attributes of the HIV-1 primed T cell i.e; cytolitic proteins like perforin, and granzyme, and cytokines; TNF-α, IFN-γ, and IL-2 were decreased compared to mock primed T cells. This could be due to reduced T cell activation in the HIV-1 primed T cells or impared abilty of production. Of note, increased expression of inhibitory molecules correlated with BLIMP-1 expression and decreased T cell proliferation. Discussion/Conclusions We demonstrated expression of a broad array of negative costimulatory molecules and elevated gene levels of transcription repressors BLIMP-1 and FOXP3 in HIV-1 primed T cells and a correspondent decrease in functional attributes. These results correlated with in vivo studies that showed positive correlations of TIM-3 (230), CD160 (232, 233), LAG-3 (234, 235), CTLA-4 (227) PD-1 (221, 226, 227), and TRAIL (269) with viral load and CD4+ T cell numbers. Suppressor T cells are known to induce suppression in a contact dependent manner partly due to secretion of perforin and granzyme (210, 214, 270, 271). At the early phase of infection, HIV-1 specific CD4+CD25+ effector cytotoxic T cells are generated that express increased levels of granzyme and perforin to control viral replication (261) as well as IFN-γ and IL-2 (171), but the IFN-γ and IL-2 responses declined overtime. Our suppressor T cells had decreased secretion of granzyme and perforin which could be in part a reason behind the declining immunological functions in HIV-1. In the same light, total levels of IFN-γ, TNF-α and IL-2 were also reduced which could be a consequence of the decreased T cell activation or actually be due to impaired production and immune activity induced by HIV-1. The secretion level per cell is therefore important and needs further investigation for these cytokines. The transcriptional repressor BLIMP-1 has been linked to regulate CD8+ T cell exhaustion and correlates positively with expression of PD-1, LAG-3, CD160, and 2B4 during chronic LCMV infections (217). We found that the inhibitory molecules we described above correlated positively with BLIMP-1 concurring with the findings in LCMV infections. Therefore, priming of T cells with HIV-1 exposed DCs in vitro leads to expansion of suppressor T cells armed with numerous inhibitory molecules, which could be part of the immune deficiencies seen in HIV-1 infected individuals. Paper IV Background HIV-1 infections modulate the immune system to their advantage partly via induction of negative inhibitory molecules on the T cell surfaces (221, 226, 227, 230, 232, 233, 269). In vitro studies show that allogeneic HIV-1/DC-T cell cocultures lead to increase expression PD-1, CTLA-4, TRAIL, LAG-3, TIM-3, CD160, and transcriptional factors BLIMP-1, and FOXP3 (219, 220). The mechanisms under which HIV-1 initiates the 38 Findings and Discussion prolific expression of these inhibitory molecules are not understood. Hence, the understanding of these pathways could be a milestone in deciphering the path through which vaccine studies could embark on. Owing to the role of DTX1 in T cell anergy (218), NFAT in regulation of PD-1 (240) and CTLA-4 (241) or CD8+ T cell exhaustion (276), MAPK in PD-1 induction (277), and the role of STAT3 in the downmodulation of TH1 responses, induction of immunosuppressive genes (278-281), and generation of T cells beneficial for tumor survival (281), we set out to study these pathways and their involvement in immune modulation in HIV-1 primed T cells. Principal Findings We have shown previously that HIV-1 impairs DCs ability to prime naïve T cell responses (219, 220) and further established that similar impairment occurred in memory T cells but to a lesser extent. Viral access to the DC cytosol was necessary to elicit the impaired T cell proliferation and increased gene and protein expression of inhibitory molecules. We subsequently investigated the role of P38MAPK, STAT3, and NFATc signaling pathways in the induction of impaired T cell proliferation and enhanced expression of inhibitory molecules. The findings showed that HIV-1 utilized the STAT3 pathway to induce inhibitory molecules as blockade of this pathway significantly reduced gene and protein expression of inhibitory molecules as well as rescued T cell proliferation. The mechanism of STAT3 involvement was unclear as we did not detect increased IL-6 and IL-10 secretion, which are known stimulators of the STAT3 pathway (282, 283). Neither were the levels of other known extracellular STAT3 activators, e.g. PDGF, G-CSF, EGF, and VEGF (284-286), increased in supernatants. Therefore we suggested the involvement of the intracellular STAT3 regulator P38MAPK (287, 288). Of interest, blockade of P38MAPK drastically curtailed expression of inhibitory molecules and rescued T cell proliferation showing a mechanism of induction of suppressor T cell by STAT3 through the P38MAPK route. We were able to show that PD-1 expression was also regulated via NFATc (240) as well as the DTX1 (218) as blockade reduced their expression but in the contrary gave slight increases in expression of other inhibitory molecules. To confirm that these findings could be translated into an autologous system did we use DCs pulsed with the superantigen Staphylococcus enterotoxin B (SEB) to activate T cells in the presence or absence of HIV-1. This autologous system gave similar increased expression of inhibitory molecules and decreased proliferation of the primed T cells when HIV-1 was present as seen in our allogeneic system and thus confirming the immunomodulatory effect of HIV-1. Blockade of STAT3 and P38MAPK in the SEB activated autologous system also increased T cell proliferation significantly. 39 Findings and Discussion Discussion/Conclusions Increased expression of PD-1, CTLA-4, TRAIL, CD160, TIM-3, and LAG-3 have been documented in HIV-1 infected individuals (221, 227, 230, 233, 269, 289). Furthermore, TN cells primed in the presence of HIV-1 in vitro expressed elevated levels of these negative costimulatory molecules (220) and blockade of PD-1 and CTLA-4 dramatically improved T cell functions in vitro (145, 221, 226, 227). The mechanisms and molecular events underlying the upregulation of these molecules both in vivo and in vitro remained elusive. Herein, we established that HIV-1 entry into the DCs cytosol is the first step towards the induction of negative costimulatory molecules in addition to well known transcriptional repressors BLIMP-1 (217, 220, 242, 243), DTX1 (218), and FOXP3 (290). We showed in this study for the first time that these inhibitory molecules are regulated by P38MAPK/STAT3 pathways as blockade of these signaling cascades significantly reduced expression of inhibitory molecules and rescued T cell proliferation. STAT3 may be a potential target for HIV-1 to modulate the immune system seeing that STAT3 has been reported in cancer as a potent inducer of immunosuppressive genes (133, 279, 281). We eliminated the involvement of IL-6, IL-10, G-CFS, EGF, VEGF, and PDGF, which are all known to induce STAT3 signaling (282-286, 291) and postulated an intracellular mechanism through which HIV-1 mediated events (292, 293) that activated STAT3 through the P38MAPK pathway (287, 288). The interruption of trafic through the P38MAPK pathway significantly suppressed the expression of inhibitory molecules and this proved to be the pathway HIV-1 utilized to induce inhibitory molecules in vitro and possibly in vivo. We confirmed these findings in an autologous system whereby DC-T cell cultures were activated with the superantigen SEB. We showed a significant rescue in T cell proliferation when the P38MAPK/STAT3 pathway was interrupted. However, we maintained the allogeneic system throughout our studies since this system best mimics the mechanism of a precise T cell activation. T cell activation with superantigen has been shown to circumvent the normal peptide/MHC-TCR interaction mechanism for T cell activation (294, 295). In conclusion, presence of HIV-1 in the DCs cytosol condition these cells to induce signaling via P38MAPK and subsequent activation of STAT3 leading to transcriptional activation of inhibitory genes and proteins, which render the primed T cells immunosuppressive. 40 Overall Findings/Conclusions OVERALL FINDINGS/CONCLUSIONS The immune system responds to HIV-1 by mounting both humoral and cellular specific responses, which control viral replication. These responses are highly dependent on both viral and host factors. These responses decline as the disease progresses due to massive loss of CD4+ T cells and leads to reestablishment of viremia. Hence, the generation of HIV-1 specific responses and persistence of these responses is a fundamental requisite to control HIV-1 infections. We have shown that HIV-1 specific T cell responses can be generated in vitro by pulsing MDDCs with infectious or non infectious HIV-1 and coculturing them with autologous naïve T cells. The in vitro primed responses were confirmed in vivo in HIV-1 infected individuals and it was clear that these responses declined fast as the disease progressed. We next established in vitro a possible mechanism through which immune responses to HIV-1 could be impaired as the disease progressed. HIV-1 impaired DCs ability to activate functional allogeneic naïve T cells. The T cells primed in the presence of HIV-1 had upregulated inhibitory molecules CTLA-4, PD-1, TRAIL, and FOXP3 and had the ability to impair other naïve T cell responses. The neutralization of the inhibitory molecules CTLA-4, PD-1, and TRAIL restored T cell proliferation. The next point of focus was the possible involvement and expression of other inhibitory molecules and was consequently screened on the HIV-1 primed T cells for well established inhibitory molecules, LAG-3, TIM-3, BTLA and CD160, and transcription repressor BLIMP-1. We reported increased expression of all these molecules except for BTLA on HIV-1 primed T cells. HIV-1 primed T cells also produced reduced levels of granzyme and perforin as well as TNF-α, IL2 and IFN-γ. These finding reaffirmed the decline in immune responses that occur in vivo in HIV1infected as the disease progresses. The mechanisms under which HIV-1 significantly enhanced the inhibitory molecules expressed on primed T cells remained unknown. We established that STAT3 was involved in the increase of inhibitory molecules on HIV-1 primed T cells. STAT3 was not activated through the well established STAT3 stimulants, IL-6, IL-10, EGF, PDGF, VEGF, or G-CFS, rather through the intracellular P38MAPK pathway. (See figure 11 for a schematic summary of inhibitory molecules). 41 Overall Findings/Conclusions Figure 11: Summary of HIV-1 induced inhibitory molecules in DC T cell primed responses. HIV-1 negatively modulates DC function by inducing increase expression of inhibitory molecules on primed T cells, which can act both on other APCs and on T cells. HIV-1 binding and fusion to DCs is an important requirement to render the DCs capable of inducing expression of inhibitory molecules on the primed T cells. 42 Recommendations and Future Challenges RECOMMENDATIONS AND FUTURE CHALLENGES The prime aim or motive of HIV research; be it epidemiological or scientific research is to contribute to knowledge that can open the way forward towards the discovery of vaccines or drugs that can cure this disease. Therefore, the major challenge today is the quest towards an effective vaccine that will successfully eliminate HIV-1 and the numerous emerging subtypes. This cannot be achieved without an in depth understanding of HIV-1 structure and pathogenesis or the mechanisms of action. In this study, we have focused on the type of specific T cell immune responses generated against HIV-1 both in vitro and in vivo. This knowledge has revealed a possibility that HIV-1 specific T cell immune responses can be generated in vitro and therefore, has opened a window for future studies at improving the generation of in vitro specific responses and how these responses could be adapted in vivo in patients to ameliorate or cure their conditions. We also identified a broad array of inhibitory molecules thought to be partially responsible for the decline of immune functions in HIV-1 and the underlying mechanisms responsible for the enhanced expression of these inhibitory molecules. Future studies are needed to understand these attributes and possibly develop methods to prevent the upsurge of inhibitory molecules or prevent cell responses as a result to these molecules. These mechanisms are very important to understand rather than developing blockers for these inhibitory molecules as these numerous molecules may be difficult to block simultaneously in patients without causing severe systemic damage to the host. The mechanisms under which these molecules elicit their inhibitory effects on other cells are also of prime importance to study as this may shed more light on possible preventive tactics implored to abrogate their activity. 43 Recommendations and Future Challenges 44 Lay language Summary LAY LANGUAGE SUMMARY Human Immunodeficiency Virus type 1 (HIV-1) is the virus that causes AIDS known in full as Acquired immune deficiency syndrome. HIV-1 infects the body primarily through unprotected sexual intercourse and till date there is no cure for this infection, only drug regimens that keep the infection under control. White blood cells are one part of the host defense against diseases (the immune system) and these cells recognize and fight infections and have failed to successfully kill the virus and free the body from HIV-1. However, some individuals are able to live with the HIV-1 infection for a long time without any symptoms and they are called long term non progressors. These individuals’ white blood cells are able to control the disease by blocking the production of new viruses. This protects them from obtaining other infections (opportunistic infections), such as tuberculosis, diarrhea, fungi, parasites, and cancers, which normally are easy to acquire due to the HIV-1 infection. This gives evidence that a strong immune response mounted against HIV-1 infection is beneficial as it can control infection. Therefore, finding ways of creating strong immune responses in infected individuals could greatly control the disease. When HIV-1 enters the body, it is picked up by specialized white blood cells called dendritic cells. These cells are experts in activating the body’s defense against infections. The dendritic cells will degrade and expose pieces of HIV-1 on their surface to another type of white blood cell called T cells and instruct them what kind of responses they should mount to fight the infection. The aim of this thesis was to understand the type of T cell responses generated by dendritic cells during the HIV-1 infection and to understand why the immune system becomes dysfunctional and fails to eradicate HIV-1 from the body. We showed that when we exposed dendritic cells to HIV-1, the dendritic cells activated T cells that recognized HIV-1 and these T cells had the potential to kill HIV-1 infected cells. These cells responded to HIV-1 by producing factors (IL-2, MIP-1, TNF-α, and IFN-γ) with the ability to suppress this virus. The T cell responses in the HIV-1 infected individuals gradually declined as the disease progressed. We went on to show that one reason behind the impaired immune responses was the increased expression of suppressive molecules (PD-1, CTLA4, TRAIL, LAG3, TIM3, BLIMP-1, FOXP3, and DXT1) on the T cells. HIV-1 induction of these suppressor molecules was dependent on signaling events (P38MAPK/STAT3) inside the T cells. 45 Lay language Summary These findings show that it is possible for dendritic cells to generate strong immune responses against HIV-1 outside the body and this knowledge can be applied in vaccine design. Our results also suggested that the decline in the T cell functionality was partly due to increased expression of immune suppressive molecules. The impairment could be reversed by blocking the effects of these suppressive molecules or even better by preventing the signaling events that lead to their production. 46 Acknowledgements ACKNOWLEDGEMENTS I cannot conclude without acknowledging the support of a few people who stood by me to enable this dream come true. To my supervisor Prof. Marie Larsson, I will forever remain indebted to you for the wonderful opportunity you gave me. You saw in me what myself and others could not see and you help tap out those qualities for which I am very thankful today. Marie, working with you did not only improve my academic career but also upgraded a moral side in me. You are not only a great scientist and supervisor, but also a wonder role model. To my cosupervisors Prof. Jormah Hinkula, I will always remember your willingness to accommodate me from the first day we met in the department. Since then, you have supported me academically and morally throughout these years. And to you Prof. KarlEric Magnusson, thank you for being there and ready to attain to me and thanks for your constant curiosity on the prospects of my work. To my coworkers in Marie’s group, Veronica, Vegard, Shankar, Rada, Sundar and all the masters’ students who have been in and out of the group, I appreciate every moment we spent together. Veronica, you have been a source of comfort and encouragement. We shared common difficulties and due to that, you could best understand my frustrations and made it a duty to help when you could. To you Vegard, your kindness and simplicity eased the working environment and enabled the freedom to share whatever I had in mind with you. This quality was just what I needed to function properly from the first to the last day at work. To you Shankar, coming to the lab was a blessing as you help in the experiments and your special skills in editing the manuscripts. I will always be thankful to have worked with you. Rada, I thank God for, making you join Marie Larsson’s group. You have been an asset not only to the group but also to the entire department with your exotic computer skills. You never said no to my requests and always did your work with a lot of passion. Thank you for the wonderful pictures you made for this thesis. And to Sundar, thanks for your support in the lab and editing of the manuscripts. Your curiosity in understanding the subject matter enabled me to learn many more things as we discussed science together. To you Carolin Jonson and Lena Svensoon, you have not only been of help in the lab but also outside the lab. You have consistently been there for the last 5 years and showed no signs of resilience in your efforts to help me succeed. Your interest in the well being of my family has been a corner stone for my admiration towards you. I owe my Swedish skills to you Lena 47 Acknowledgements To my fellow work mates whom I now call friends in Lennart svensson’s Lab, Johan I am truly grateful to have had you in my life. Elin, thanks for the chats we had at work and parties we attended. Your sharpness truly shaped me to be a better person. Beatice, you are truly a nice person and I enjoyed each moment of our discussions. To Marlin, your composure during your work and the writing of your thesis truly inspired me to be strong as my turn approached. To Marie Hagbom, thanks for your friendship and concern over my family. To Filemon, we had wonderful moments each time you visited Sweden to complete your PhD and I am truly grateful for having you as a friend. To Sumit alias Tum Casiho, thanks for your friendship and helping me with some Hindi. To crown it all, special thanks Prof Lennart svensson. “Learning without playing makes Jack a doll boy”. Under your guide, you made sure we had good working conditions and the refreshments badly needed to rekindle the brains. To Lotta and Pia, I thank you for your kind help with micro arrays and western blotting and your persistence to make my Swedish better. I owe my Swedish skills to you. To Hong, Sasan, Magnus, Daniel, Ryan, Anika, Lisa, Olivia, Gosia, Helen, Matthias and Anna, I have enjoyed every moment I spent with you at work and the parties we attended and am grateful for that. To you Chi Celestine, Alain Asongwec and wife Matilda, Gilbert Nche, Muluh Clifford, Ngeh Jonathan and Akenji Valentine, you occupy a special place in my heart for what we share as friends and family. To my “other brother” Xavier Ekolle NdodeEkane, life in Sweden would have been miserable without you as we went through thick and thin in the same boat but still kept our cool for each other. Your calmness and confidence for the future has truly guided me during times of indecision. Good luck with the last phase of your PhD too. To all the “Paysans”, with whom we have had special moments here in Linköping, Fidelis, Elvis, Eric, Pride, Leon, Justice, Laurence, Gerald, Sam, Atem, Pascal, Paul, Ebot, Alisine, Pauline, Challote and Millat, thanks for making me feel Ndzemabeah was very close even though I was far away in Sweden.. To my Family, special thanks go to my Father (RIP) and mother Akumah Shadrack and Magdalene respectively who gave me the life and education I have now. Daddy, this is it!!! and you should smile down on us as we celebrate this day for it is for you. To my step mothers Mami and Mangieh, I owe you my loyalty for the wonderful support I have always received from you. My brothers Ernest for the incredible moments we have had while growing up and thanks for being an example of hard work which has also inspired the rest of our siblings. To Hilarious for being such a wonderful “big” brother teaching me to be happy even when there was nothing to be happy about. Special thanks to Watson and Sanna. Watson we share the world together and that has helped us to 48 Acknowledgements grow stronger in spirit and I hope we keep the candle light glowing. To Addison, Kenneth, Sebastian, Elvis, Mabi, Akumah, and mankah thanks for being such wonderful and supportive siblings. Million thanks go to my uncles and wives Mr. and Mrs. Akumah Gideon for assuming the role of a father and mother and special thanks for the support during my college days. I will always remain indebted to you. To Mr. and Mrs. Akumah Ruben, you have always been there each time I needed you and no words can express how thankful I am for that. Uncle, you told me not to return to Cameroon without a PhD and today, I am proud to have respected that order. To the Engstroms, Rosens, Nordins, Gogina Mores and Kakembo, you have been my family here in Sweden. Words cannot express how thankful I am for your hospitality and love. To Tina and Bjorn, I thank God for making us friends. Doing things with you Tina Rosen and Tina wood has helped feel many holes in my life as I have learned so much from you. Special thanks to all my choir members as singing with you helped release tension and made my focus at work much better To crown it all, special thanks to my wife Suzan Che. Wards cannot express how much you mean to me as a wife, friend, and coach and it is my singular pleasure to have you in my life. Excuse my stubbornness and irrationality sometimes for that has simply been a source of strength for both of us. To my sons Mbumah Karl-Jeremy Che and FruMbonneh Desmond Che to whom I dedicate my thesis to, you are why I do the things I do and you mean everything to me. 49 Acknowledgements 50 References REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. Barre-Sinoussi F, Chermann JC, Rey F, et al. 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