Vaccination for hepatitis B virus (HBV) in the setting of hepatitis C virus (HCV) infection is recommended, but responses to vaccination are blunted when compared to uninfected populations. The mechanism for this failure of immune response in HCV-infected subjects remains unknown but is thought to be a result of lymphocyte dysfunction during chronic viral infection. We have recently demonstrated that PD-1, a novel negative immunomodulator for T cell receptor (TCR) signaling, is involved in T and B lymphocyte dysregulation during chronic HCV infection. In this report, we further investigated the role of the PD-1 pathway in regulation of CD4+ T cell responses to HBV vaccination in HCV-infected individuals. In a prospective HCV infected cohort, a poor response rate to HBV vaccination as assayed by seroconversion was observed in HCV-infected subjects (53%), while a high response rate was observed in healthy or spontaneously HCV-resolved individuals (94%). CD4+ T cell responses to ex vivo stimulations of anti-CD3/CD28 antibodies or hepatitis B surface antigen (HBsAg) were found to be lower in HBV vaccine non-responders compared to those responders in HCV-infected individuals who had received a series of HBV immunizations. PD-1 expression on CD4+ T cells was detected at relatively higher levels in these HBV vaccine non-responders than those who responded, and this was inversely associated with the cell activation status. Importantly, blocking the PD-1 pathway improved T cell activation and proliferation in response to ex vivo HBsAg or anti-CD3/CD28 stimulation in HBV vaccine non-responders. These results suggest that PD-1 signaling may be involved in impairing CD4+ T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this negative signaling pathway might improve success rates of immunization in the setting of chronic viral infection.

T regulatory (TR) cells suppress T-cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also has a pivotal role in regulation of T-cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating TR-cell functions that inhibit T-cell responses during virus-associated malignancy, TR cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4+ CD25+ and CD8+CD25 + TR cells, as well as high levels of PD-1 expressions on these TR cells were found in the peripheral blood of subjects with HCV-L compared with those from non-HCV-L or HCV alone or HS. TR cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of TR cells in peripheral blood mononuclear cells from HCV-L improved T-cell proliferation. Additionally, the suppressed T-cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in TR-cell number and the ability of TR to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating TR cells that suppress T-cell functions in the setting of HCV-associated B-cell lymphoma.

Background microRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis-induced immunosuppression. Methods Mice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)-induced sepsis. Apoptosis in spleens and apoptotic signals were investigated, and survival was monitored. T-cell immunoreactivities were examined during late sepsis. Nuclear factor B (NF-B)-inducing kinase (NIK), tumor necrosis factor (TNF)-receptor associated factor 1 (TRAF1), and X-linked inhibitor of apoptosis protein (XIAP), the putative targets of miR-23b, were identified by a dual-luciferase reporter assay. Results miR-23b expression is upregulated and sustained during sepsis. The activation of the TLR4/TLR9/p38 MAPK/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased the number of spleen cells positive by terminal deoxynucleotidyl transferase dUTP nick-end labeling and improved survival. miR-23b inhibitor restored the immunoreactivity by alleviating the development of T-cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 and interleukin 4 during late sepsis. We demonstrated that miR-23b mediated immunosuppression during late sepsis by inhibiting the noncanonical NF-B signal and promoting the proapoptotic signal pathway by targeting NIK, TRAF1, and XIAP. Conclusions Inhibition of miR-23b reduces late-sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.

Homeostasis of human T cells is regulated by many factors that control proliferation, differentiation of effector cells and generation of memory. Our current knowledge of the mechanisms controlling human T cell homeostasis in vivo is based on experiments in small animal models. However many differences exist between immune systems of mice and humans, including cell composition, function, and gene expression. Humanized mouse models have shown great value in the study of human immunobiology. I have used novel humanized mouse models to examine the role of human MHC (HLA) and human IL2 in CD8 T cell and CD4 regulatory T cell (Treg) homeostasis. To study human CD8 T cells I engrafted CD8 T cells from healthy donor PBMC into NOD-scid IL2rgnull (NSG) mice that lacked expression of murine MHC and that expressed HLA-A2. My data demonstrate that CD8 T cell survival and effector function required the presence of HLA-A2, helper function from human CD4 T cells and exogenous human IL2. To study human Treg homeostasis I used NSG mice engrafted with human fetal thymus and hematopoietic stem cells (BLT model). NSG-BLT mice support the growth of human thymic tissue and enable the efficient development of HLA-restricted Treg and conventional T cells. Using an AAV vector to express human IL2, I demonstrated that functional human Treg but not conventional T cells increased in number in NSG-BLT mice and that this coincided with increases in activated human NK cells. Overall my research has revealed that HLA and human IL2 have an essential role in human T cell survival and function in vivo.

The relative contribution of T cell receptor-dependent stimulation versus TcR-independent bystander stimulation in the massive increase in the number of activated proliferating CD8+ T cells seen during acute many acute viral infections is unclear. To determine if this increase was the result of TcR-independent bystander activation and proliferation, anti-viral cytotoxic T lymphocytes were induced in vivo via DNA immunization so that the anti-viral immune response could be examined in the absence of the high levels of cytokines generated during acute infection. After a single immunization with a plasmid encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV) a nearly 2 log10 reduction in viral titers in the spleen was observed 3 days after LCMV infection. After 2 or 3 immunizations a greater that 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. After intracerebral challenge vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. By limiting dilution analysis LCMV-specific CTL precursors were detected in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at days 4 after LCMV challenge. However, the numbers of CTL precursors elicited by DNA vaccination was too low to determine if cytokine-mediated TcR-independent bystander activation and proliferation had taken place. HY-specific TcR-transgenic mice, which have a restricted TcR repertoire, and LCMV-carrier mice, which are tolerant to LCMV, were used to determine the extent of TcR-independent bystander activation and proliferation during acute LCMV infection. LCMV infection of C57BL/6 mice induced CTL that lysed uninfected H-2k and H-2d allogeneic targets, but, LCMV-induced CTL from HY- transgenic mice lysed only the H-2k-expressing cells. The HY-mice generated both anti-H-2k and anti-H-2d CTL in mixed lymphocyte cultures, strongly suggesting that the generation of allospecific CTL during acute LCMV-infection is antigen specific. During the LCMV infection there was blastogenesis of the CDB+ T cell population, but the HY-specific T cells remained small in size, and did not alter their expression of the activation molecules CD44 and MEL-14. In order to examine the potential for bystander stimulation under conditions of a very strong CTL response, T cell chimeras were made between normal and HY-transgenic mice. Even in the context of a normal vicus-induced CTL response, no stimulation of HY -specific T cells was observed, and HY-specific cells were diluted in number by day 9 post-infection. In LCMV-carrier mice in which donor and host T cells could be distinguished by Thy 1 allotypic markers, adoptive transfer of LCMV-immune T cells into LCMV-carrier mice, whose T cells were tolerant to LCMV, resulted in activation and proliferation of donor CDB cells but little or no activation of host CDB+ T cells. These results show that TcR-independent bystander activation of non virus-specific T cells is not a significant component of an anti-viral T cell response and support the hypothesis that the massive polyclonal CTL response to LCMV infection is virus-specific. T cells activated during potent anti-viral immune responses are sensitized to undergo apoptosis after strong TcR-stimulation in a process known as activation-induced cell death (AICD). To determine if T cells, not participating in the immune response were also subject to AICD, LCMV-carrier mice were used. Using TUNEL flow cytometry, it was shown that after reconstitution of Thy 1.2+ LCMV-carrier mice with spleen cells from Thy 1.1+ LCMV-immune mice, the Thy 1.2+ host T cells which were not specific for the virus and did not proliferate in a bystander fashion, were rendered sensitive to TcR-induced apoptosis in vitro. This bystander sensitization to AICD was shown not to be dependent on the continued presence of activated proliferating donor cells during the in vitro culture period. Bystander sensitization to AICD was not the result of an antigen presenting cell defect, but rather was the result of an in vivo conditioning of the T cells themselves. The mechanism of this sensitization was, at least, partially dependent on the ability of host T cells to respond to IFNγ, and on the expression of Fas ligand on the activated, proliferating donor cells. This bystander sensitization to AICD may explain why memory T cell responses are so poor during acute viral infection and can serve as a potential mechanism for virus-induced immunosuppression.

Transcriptional regulation of CD8+ T cell differentiation during acute and chronic viral infections is an intricate web made up of many of transcription factors. While several transcription factors have been elucidated in this process, there are still many more that remain elusive. In this work, we look into the role of two transcription factors, IRF4 and Runx2, and their role in CD8+ T cell terminal effector cells and memory precursor cells during acute LCMV-Armstrong infection. We found that IRF4 expression was regulated by TCR signal strength during infection, and that IRF4 expression levels directly correlated with the magnitude of the effector cell response. IRF4 was also shown to regulate T-bet and Eomes, two transcription factors critical for CD8+ T cell differentiation into effector and memory cells. From these results, we were interested in the potential role of IRF4 during chronic LCMV-clone 13 infection, where ratios of T-bet and Eomes are critical for viral clearance. We found that haplodeficiency of IRF4 in the T cell compartment lead to an increase in the ratio of Eomes to T-bet in T cells, which in turn affected the proportion of Eomeshi versus T-bethi cells and resulted in a loss in ability to clear viral infection. Irf4+/-Eomes+/- compound heterozygous mice were generated to test if decreasing Eomes expression would rescue the Irf4+/- phenotype. Irf4+/-Eomes+/- mice were phenotypically similar to WT mice in terms of Eomes to T-bet ratios, and were able to clear viral infection, demonstrating a critical role of IRF4 in regulating T-bet and Eomes during chronic viral infection. Next we looked into the role of Runx2 during acute LCMV-Armstrong infection and found that Runx2-deficient pathogen-specific CD8+ T cells had a defect in the total number of memory precursor cells compared to WT controls. We further showed that Runx2 was inversely correlated with TCR signal strength, and that Runx2 expression was repressed by IRF4. From these work, we have introduced two more transcription factors that are critical for CD8+ T cells differentiation during acute and chronic viral infection. Given the sheer number of transcription factors known to regulate these processes, having a full understanding of the transcriptional network will allow us to find the best targets for therapeutic intervention for treatments ranging from vaccine development and autoimmunity to cancer immunotherapy and treatment of chronic viral infections.

The overall goal of this thesis is to examine how the T cell receptor (TCR) repertoire evolves during and after viral infections. Previous studies had examined TCR usage of selected virus-specific T cell clones, but little was known about how a diverse T cell repertoire changes during the transition between an acute infection and a memory response. It was also unclear how the T cell repertoire evolves under conditions of persistent infections associated with clonal exhaustion. To address these issues I used as a model system the lymphocytic choriomeningitis virus (LCMV) infection of mice, for which the T cell response is well-characterized. LCMV, strain Armstrong (LCMV-ARM), infection induces a strong CD8+ T cell response, which clears the virus and converts to a memory response. In contrast, high doses of LCMV clone 13 leads to persistent infections associated with T cell clonal exhaustion. These two extremes of T cell responses enable one to compare the evolution of the TCR repertoire under conditions where an acute T cell response converts to a memory response with that of T cell clonal exhaustion. In this thesis I analyzed the TCR repertoire usage directly ex vivo by the technique of CDR3 length spectratyping throughout the acute LCMV infection, into memory, after modulation by subsequent heterologous and homologous viral infections, and under conditions of T cell clonal exhaustion. Kinetic studies on the frequencies of precursor cytotoxic T lymphocytes (PCTL) to the three LCMV immunodominant peptides had suggested that the virus-specific T cell repertoire becomes fixed by day 7 postinfection, when the virus is cleared. The pCTL data also showed that a high frequency of the LCMV-specific memory T cells remained stable throughout the lifetime of the mouse. To examine any changes of the TCR repertoire usage that may develop during the acute LCMV infection and into memory, the Vβ8 population was subjected to spectratype analyses, because Vβ8 represented a substantial amount of the LCMV-induced CD8+ T cells recognizing several LCMV-encoded peptides. Analyses of the Vβ8.1 spectratype showed that genetically identical mice generated remarkably different T cell responses, as reflected by different spectratypes and different TCR sequences in same-sized spectratype bands; a conserved CDR3 motif was, however, found within some same-sized bands. This indicated that meaningful studies on the evolution of the T cell repertoire required longitudinal studies within individual mice instead of comparisons between mice. Such longitudinal studies with peripheral blood (PB) samples showed that the virus-induced T cell repertoire changed little after viral clearance and during the silencing phase of the T cell response and that dominant spectratype peaks were preserved into long term memory. To determine the effect of secondary LCMV infection on the spectratype, the recalled LCMV-induced spectratypes were analyzed. Most of the dominant peaks detected in the primary infections remained present in the secondary infection. Some new peaks were also detected for the first time in the secondary infection, suggesting a further selection of the virus-induced T cell repertoire. The spectratype data support the concepts that the LCMV-induced T cell repertoire remains unchanged during the silencing phase after clearance of the virus and that the LCMV infection dramatically skews the host T cell repertoire in the memory state long after the virus is cleared. Studies had shown that high doses of LCMV clone 13 induce a transient anti-viral CTL response followed by clonal exhaustion of T cells. To determine how the TCR repertoire evolves under conditions of persistent infections associated with T cell clonal exhaustion, the Vβ8.1 spectratypes were analyzed at various time points after the infection. In contrast to the stable LCMV-induced spectratype after viral clearance, continuous selection of the T cell repertoire occurred under conditions of persistent infections, as the T cell clones appeared and disappeared at different rates. The T cell repertoire ultimately returned to a Gaussian distribution under conditions of clonal exhaustion, indicating that clonal deletion occurs in the great majority of the virus-induced T cells. To test the stability of the LCMV-induced TCR repertoire under conditions of subsequent heterologous viral infections, the recalled LCMV-induced spectratypes were examined in the presence or absence of intervening heterologous viruses. The results showed that the intervening heterologous viruses disrupted the recalled Vβ8.1-Jβ1.3 spectratype on secondary LCMV infection; this otherwise remained stable in the absence of intervening heterologous viruses. This result supports the hypothesis that subsequent heterologous viral infections disrupt the stable LCMV-induced T cell repertoire. To detennine whether a subset of the memory T cells was deleted by the IFN-induced apoptosis of memory T cells, the LCMV-immune spectratypes were analyzed before and after the injection of the IFN inducer, poly I:C. The LCMV-immune spectratypes remained relatively stable after poly I:C injection, suggesting that there is no selective protection or deletion of discrete memory T cell clones during the IFN-induced apoptosis. In summary, the data in this thesis show that (i) the virus-induced T cell repertoire changes little after viral clearance and during the silencing phase of the T cell response, (ii) the LCMV infection dramatically skews the host T cell repertoire in the memory state, (iii) the evolution of the T cell repertoire occurs during secondary infections and under conditions of clonal exhaustion associated with persistent infections, (iv) genetically identical hosts generate different T cell responses to the same virus, and (v) intervening heterologous viral infections disrupt the recalled LCMV-induced T cell repertoire, but the LCMV-immune repertoire remained relatively stable upon the treatment of the IFN inducer, poly I:C.

Des études récentes menées chez l’homme et la souris ont suggéré que la diversité du répertoire TCR pourrait jouer un rôle dans la protection contre des pathogènes à haut pouvoir mutagène. Afin d’étudier le répertoire des lymphocytes T CD4, nous avons utilisé un modèle de souris TCRβ transgéniques exprimant une chaine β spécifique du peptide env122-141 dans le contexte du MHCII. Suite à l’immunisation des souris TCRβ transgéniques avec des cellules dendritiques pulsées avec le peptide env, une rapide prolifération et une restriction du répertoire des lymphocytes T Vα2 CD4 spécifiques est observée. L’analyse de la diversité du répertoire de ces cellules par séquençage à haut débit, a montré l’émergence d’un répertoire plus divers dans des souris déplétées en lymphocytes T régulateurs. Ces résultats suggèrent qu’en plus du rôle des Tregs dans le contrôle de la magnitude de la réponse immunitaire, ces cellules pourraient également contrôler la diversité du répertoire des lymphocytes T suite à une stimulation antigénique. / Recent studies conducted in mice and humans have suggested a role for the TCR repertoire diversity in immune protection against pathogens displaying high antigenic variability. To study the CD4 T cell repertoire, we used a mouse model in which T cells transgenically express the TCRβ chain of a TCR specific to a MHCII-restricted peptide, env122-141. Upon immunization with peptide-pulsed dendritic cells, antigen-specific Vα2+ CD4+ T cells rapidly expand and display a restricted TCRα repertoire. In particular, analysis of receptor diversity by high-throughput TCR sequencing in immunized mice suggests the emergence of a broader CDR3 Vα2 repertoire in Treg-depleted mice. These results suggest that Tregs may play a role in the restriction of the CD4 T cell repertoire during an immune response, raising therefore the possibility that in addition to controlling the magnitude of an immune response, regulatory cells may also control the diversity of TCRs in response to antigen stimulation. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17951号 / 医博第3835号 / 新制||医||1000(附属図書館) / 30781 / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 生田 宏一, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第17979号 / 医科博第48号 / 新制||医科||4(附属図書館) / 80823 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 五十嵐 樹彦, 教授 長田 重一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM