Rozprawy doktorskie na temat „T-helper”

Humoral immunity provides protection against subsequent infections. Antigen-specific, high-affinity, class-switched antibodies are produced by B cells through rounds of proliferation, B cell receptor rearrangement and selection in the germinal centres (GC). T cells play an essential and indispensable role in this process and in the recent years the term T follicular helper cells (TFH) was coined to describe this cell subset. The aim of my thesis is to investigate whether there is more than one type of T cells within the TFH population and whether it has important functional consequences. Firstly, I use sheep red blood cell immunisation (SRBC) and Salmonella enterica infection to show phenotypical differences between TFH expressing high and low level of surface molecule PD-1. In order to investigate the relationship between different TFH populations gene profiling was carried out on the microarray platform. Detailed transcriptome analysis revealed the discrete nature of isolated TFH cell subsets and provided an overview of their genetic landscape. Secondly, I have investigated the dependence of TFH subsets on cognate interactions with B cell in SRBC model by generating BM chimeras. I have demonstrated that generation of PD-1HI TFH, but not of PD-1LO TFH, depends on antigen presentation by B cells. Furthermore, I have shown that provision of wild-type but not MHC II knock-out B cells rescues PD-1HI formation in BM chimeras after SRBC immunisation. Finally, I have explored plasticity within TFH subsets and showed that none of the populations is in a terminally differentiated state, as they can convert into one another. Thirdly, experiments with S. enterica model revealed that the absence of PD- 1HI TFH is independent of the splenic architecture disruption present within the first week of the response. Surprisingly, co-immunisation studies showed that PD-1HI population is not only present but even enhanced in the group which received both SRBC and S. enterica when compared to single immunisations. The work presented in the thesis documents that there is a significant and previously unappreciated heterogeneity within TFH subset. This knowledge is important for designing optimal vaccine strategies and treating autoimmune diseases, as in both processes the antibody production plays a crucial role and its manipulation (either enhancing or blocking antibody production, respectively) can significantly improve clinical interventions.

Beta-lactam antibiotics are used to treat recurrent opportunistic infections that occur in patients with cystic fibrosis. However, the incidence of reported hypersensitivity in these patients is greatly higher than the healthy population. Due to the delayed nature of beta-lactam hypersensitivity, a T-cell mediated immune response is implicated. Type IV drug hypersensitivity is a major clinical concern, with a cutaneous aetiology driven through antigen specific T cells. Pro- and anti-inflammatory cytokines can be detected in reactions to β-lactam drugs in responsive T cells from hypersensitive patients. Classical Th1/Th2 phenotypes are currently used to classify the reactions, though these do not accurately characterise the function of the immune cells. In addition, this classification does not reference the newer Th subsets such as Th9, Th17 and Th22. To characterise the nature of the T-cell response observed in piperacillin hypersensitive patients with cystic fibrosis, T-cell clones from both blood and inflamed skin of hypersensitive patients were generated, then characterised in terms of phenotype and function. Additional investigations into PBMC responses were conducted. Naive T cells from healthy donors were also primed to piperacillin, as well as attempted regulation of the cytokine response through modulation of a selected nuclear receptor involved in the progression of a Th22 mediated response. Drug-specific clones were generated from both blood (n=570, 84% CD4) and skin (n=96, 83% CD4) samples obtained from patients hypersensitive to piperacillin. All clones secreted high levels of IFN-γ and IL-13. Interleukin-22, perforin and granzyme B were secreted in over 50% of clones, with none from either blood or skin showing any detectable level of IL-17A. Naive T cells primed to piperacillin via autologous dendritic cells showed proliferative responses (p=0.001, SI > 2). Clones generated from primed T-cells showed similar patterns of cytokine secretion when compared alongside clones generated from hypersensitive patients. Significant differences in chemokine receptor expressions were observed between blood-derived piperacillin-specific clones, skin-derived piperacillin-specific clones and skin-derived non piperacillin-specific clones. CLA, CXCR6 and CCR1 expression was higher on piperacillin-specific skin derived clones when compared to non-piperacillin specific skin derived clones (p=0.01). CCR2, CCR4, CXCR1 and E-cadherin were higher on skin specific clones when compared to blood specific clones (p=0.01). Piperacillin specific clones isolated from blood and skin of hypersensitive patients, as well as healthy donor PBMC migrated in the presence of chemokines specific to their respective cell surface receptors, with migration to CCR4 and CCR10 most prevalent. In addition, modulation of the aryl hydrocarbon receptor showed that an abrogation of the cytokine response was observed in cells treated with an AhR inhibitor. This abrogation was only observed in the secretion of interleukin-22, a key cytokine in a Th22 response. Currently, T-cell mediated hypersensitivity involving drug binding to the HLA molecule has only been shown for HLA class I molecules; no data has provided evidence for HLA class II interactions with drugs being able to activate CD4+ T-cells. To investigate whether HLA class II molecules binding to drugs could activate T-cells, an investigation into the COX-2 specific NSAID lumiracoxib was performed. Lumiracoxib was withdrawn from use after incidences of liver toxicity were reported in 2008. Utilising a naive T-cell dendritic cell co-culture assay, attempts were made to generate drug-specific responses from lymphocytes to either lumiracoxib or its major/minor metabolites. No positive responses were generated, with all assays performed showing no activation of drug-specific T-cells following re-challenge with the drugs. In conclusion, the data generated over the course of this thesis has shown that there is a subset of piperacillin-specific T-cells in hypersensitive patients with cystic fibrosis that secrete IL-22, IFN-γ, perforin and granzyme B in response to antigen challenge. No cells secreted IL17, suggesting a strong Th22 phenotype rather than Th17. In addition, The AhR signalling pathway is also heavily implicated in the development of hypersensitivity, giving further evidence for the role of both IL-22 and Th22 lymphocytes in beta-lactam hypersensitivity.

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Las Células Madre Mesenquimales (MSCs) se han convertido en un interesante campo de estudio. Inicialmente fueron estudiadas por su capacidad de diferenciarse a células de diversos tejidos mesodermales, sin embargo, con el paso de los años se determinó que las MSCs tenían una amplia capacidad de escapar del reconocimiento de células del sistema inmune. Más tarde se descubrió que las MSCs no solo tienen la capacidad de escapar del reconocimiento, sino que también son capaces de inhibir la activación y proliferación de células del sistema inmune. Diversas enfermedades autoinmunes y proinflamatorias están mediadas por linfocitos T, principales células efectores del sistema inmune adaptativo, por tanto, dadas las características inmunosupresoras de las MSCs, han sido propuestas como nueva estrategia terapéutica para el tratamiento de estas enfermedades. Con este objetivo, varios autores han enfocado sus estudios para determinar el mecanismo por el cual las MSCs ejercen este efecto inhibitorio. Algunos autores postulan que el contacto celular, entre MSCs y linfocitos, es indispensable para producir este efecto, sin embargo otros postulan que las MSCs secretan una amplia variedad de factores solubles inmunosupresores que son suficientes para producir el efecto inmunosupresor. Dentro de las estirpes linfocitarias sobre las cuales las MSCs podrían ejercer su efecto inmunosupresor encontramos a los linfocitos T helper (TH), las cuales son células efectoras que se clasifican principalmente en: linfocitos TH1 que participan en la respuesta contra bacterias intracelulares, linfocitos TH2 que participan en la respuesta contra parásitos, linfocitos TH17 que participan en la respuesta contra bacterias extracelulares y hongos y, finalmente, los linfocitos T reguladores (Treg) cuya función es mantener la homeostasis del sistema inmune y promover la tolerancia inmunológica frente a antígenos propios. Durante más de una década fue ampliamente descrita la capacidad inmunosupresora de las MSCs sobre linfocitos T, sin embargo en los últimos años se ha descrito que bajo ciertas condiciones las MSCs producen un efecto estimulador sobre algunas de estas estirpes linfocitarias. El objetivo de este estudio fue determinar si las MSCs suprimen la proliferación y diferenciación de linfocitos TH1 y TH17. Estos linfocitos fueron estudiados ya que se ha descrito que diversas enfermedades autoinmunes se caracterizan por un aumento o desbalance de estas estirpes. Para esto, investigamos si es que el efecto inmunomodulador de las MSCs era dependiente del estado de activación y diferenciación de linfocitos, del ratio MSCs:CD4+, del contacto celular o de factores solubles. Dado que ha sido ampliamente descrito que las MSCs son capaces de secretar basalmente IL-6, la cual corresponde a una citoquina que cumple diversas funciones sobre el sistema inmune, entre ellas promover la secreción de citoquinas y factores de crecimiento necesarias para la respuestas de linfocitos T y que además son capaces de promover la estirpe, altamente proinflamatoria, TH17, y que esta secreción aumenta cuando las MSCs se encuentran frente a estímulos proinflamatorios como IFN-γ o TNF-α, postulamos que la IL-6 secretada por las MSCs es el principal factor soluble involucrado en la inmunomodulación ejercida por las MSCs. Las MSCs fueron obtenidas a partir de médula ósea de ratones C57BL/6 y caracterizadas por el patrón de expresión de antígenos de superficie y por su capacidad de multidiferenciación. Los linfocitos T CD4+ fueron obtenidos a partir de bazo de ratones C57BL/6, purificados mediante kit comercial y cultivados en presencia de citoquinas que favorecen la diferenciación hacia la estirpe TH1 o TH17. Las MSCs fueron agregadas a los cultivos de linfocitos TH1 o TH17 a distintos tiempos de cultivo celular en presencia o ausencia de contacto celular, contacto MSCs-linfocito. La diferenciación de los linfocitos fue medida por medio de la detección de citoquinas intracelulares características de ambas estirpes, IFN-γ e IL-17 para linfocitos TH1 y TH17 respectivamente, por medio de citometría de flujo. Demostramos que las MSCs son capaces de inhibir a linfocitos TH1 independiente del estado de activación y del ratio MSCs:CD4+. Determinamos que el efecto inmunosupresor está presente incluso en condiciones donde no existe contacto celular y que este efecto es independiente de la IL-6 secretadas por las MSCs. A diferencia de lo que ocurre con linfocitos TH1, las MSCs sólo son capaces de inhibir a linfocitos TH17 cuando estas son agregadas a tiempo temprano al cultivo celular y este efecto es dependiente del contacto celular, mientras que cuando las MSCs son agregadas al cultivo a tiempos tardíos, al día 4 de cultivo celular, promueven la diferenciación de linfocitos TH17. Concluimos que el efecto inmunomodulador que ejercen las MSCs sobre linfocitos TH1 y TH17 es por medio de mecanismos diferenciales. El efecto inmunosupresor sobre linfocitos TH1 es independiente de IL-6, sin embargo, no ha sido posible determinar el efecto real que ejerce la IL-6 secretada por las MSCs sobre linfocitos TH17 ya que la diferenciación de linfocitos TH17 requiere de IL-6 en el medio de cultivo. Sin embargo, determinamos que las MSCs en baja concentración no solo pierden su capacidad inhibitoria cuando se encuentran con linfocitos TH17 diferenciados sino que son capaces de promover su diferenciación. Observamos también que la IL-6 proveniente de MSCs podría, bajo ciertas, de revertir este efecto
Mesenchymal Stem Cells (MSCs) have become an interesting field of study. Initially MSCs where studied for their capacity to differentiate into various cell types from different tissues from the mesoderm, however, over the years was determined that MSCs have a large capacity to escape from the recognition by cells from the immune system. Later it was discovered that MSCs not only have the capacity to escape recognition, but are also able to inhibit the activation and proliferation of immune cells. Several autoimmune and proinflammatory diseases are mediated by T cells, major effectors cells of the adaptive immune system, therefore, given the immunosuppressive properties of MSCs they have been proposed as a new therapeutic strategy for treating these diseases. To this end, several authors have focused their studies to determine the mechanisms by which MSCs exert this inhibitory effect. Some authors postulate that cell contact between MSCs and lymphocytes is essential to produce this effect, while others postulate that MSCs secrete a wide variety of immunosuppressive soluble factors that are sufficient to produce the immunosuppressive effect on T lymphocytes. MSCs can exert their immunosuppressive effect on lymphocytes called T helper (TH), which are an effectors cell line mainly classified into: TH1 cells that participate in the response against intracellular bacteria, TH2 cells that participate in the response against parasites, TH17 cells that participate in the response against extracellular bacteria and fungi. Finally there are T regulatory (Treg) cells which main function is to maintain immune system homeostasis and to promote immunologic tolerance against self antigens. For over a decade, it was widely described the immunosuppressive capacity of MSCs on T lymphocytes, however in recent years it has been described that under certain conditions MSCs may produce a stimulatory effect on some of these lymphocytes strains. The aim of this study was to determine whether MSCs are able to suppress TH1 and TH17 proliferation and differentiation. These cells were studied because it has been previously described that many autoimmune disease are characterized by and increase or imbalance of this two strains. For this purpose, we investigated whether de immunomodulatory effect of MSCs was dependent on lymphocytes activation and differentiation state, MSCs: CD4+ ratio, cell contact or soluble factors. It has also been highly described that MSCs are able to secrete basal amounts of IL-6, cytokine which has a variety of function on the immune system, among them, to promote cytokine and growth factor secretion necessary for T cell response, and to promote differentiation of the highly proinflammatory TH17 cells. These IL-6 basal secretion is augmented when MSCs are in presence of proinflammatory stimulus like IFN-γ or TNF-α, thus we postulate that MSCs secreted IL-6 main soluble factor involved in MSCs immunomodulation. MSCs were obtained from mice bone marrow and characterized by their surface antigen expression pattern and their capability of multilineage differentiation. CD4+ T cells were obtained from mice splenocytes, purified by a commercial Kit and cultivated with cytokines that promote the differentiation to TH1 or TH17 cells. MSCs were added to TH1 or TH17 cultures at early or late time points and in the presence or absence of cell to cell contact, MSCs-T cell contact, mediated by a transwell system. The differentiation of TH1 or TH17 cell was measured by the detection of intracellular cytokines characteristics for each population, IFN-γ and IL-17 for TH1 and TH17 cells respectably, by flow cytometry. We demonstrated that MSCs are capable to suppress TH1 cells differentiation despite on their state of activation or MSCs:CD4+ ratio. We determined that the immune suppressor effect of MSCs is present even in the absence of cell to cell contact and that this effect is independent of MSCs secreted IL-6. In contrast with TH1 cells, MSCs are only capable to suppress TH17 cells when added at early time points of cell culture and that this effect requires cell to cell contact, while promoting TH17 differentiation when added at later time points, at day 4 of cell culture. We concluded that the immune modulator effect of MSCs on TH1 and TH17 cells is mediated by differential mechanism. The immune suppressor effect on TH1 cells is independent from IL-6, though it was not possible to determined de real effect of MSCs secreted IL-6 over TH17 cells, since the TH17 differentiation media requires IL-6. However, we determined that low concentration of MSCs in the coculture, fail to inhibit TH17 differentiation more over they promote an augmentation of TH17 cells. We also observed that under certain culture conditions MSCs secreted IL-6 may revert this effect

T helper (Th) cell activation and development is one of the most critical events in regulating the adaptive immune response. Understanding its regulation could be of great therapeutical value as many severe diseases are associated with failure in controlling T cell activation and development. However, the regulation of T cell activation appears to be one of the most complex set of cellular and molecular interactions known in the immune system. There is therefore an urgent need for tools to unravel this complexity, and to make use of the quantitative experimental data. To address this issue, mathematical and computational models, based on rigorous biophysical and kinetic data, were developed to study the specific role of some of the major costimulatory molecules involved in Th cell activation, and others developed to investigate proposed theories about mechanisms involved in Th cell differentiation. The simulations of costimulation reveal new implications for the function of the costimulatory molecules CD28 and CTLA-4, and their ligands B7-1 and B7-2, and show how binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects, all profoundly influence complex formation at the immunological synapse. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be, as has been previously proposed, due to the different binding properties of B7-1 and B7-2. Simulations of Th cell development reveal that both instructive and selective processes are likely to be involved in Th cell differentiation. In addition, further simulations indicate that Th2 cells are more likely to become dominant by inhibiting Th1 cells (negative selection), rather than selecting their own growth (positive selection). This thesis also includes an experimental work in which the immunomodulatory role of the bacterial signalling molecule N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) was analysed. This study strongly suggests that OdDHL suppresses Th cell activation and development, and that it is likely targeting the intracellular signalling events involved in the early stages of Th cell activation.

CD4+ T cells can be subdivided on the basis of their lymphokine repertoire produced on activation resulting in TH1 like and TH2 like populations. The purpose of this study was to measure the intraocular expression of cytokines as a means of defining the CD4+ lymphocyte subsets present during the development of uveoretinitis. Lewis rats were immunised with retinal antigen and pertussis toxin resulting in early signs of disease activity evident by day 9 with increasing severity evident by day 12. Extensive clinical and histological damage was observed by day 14 with a reduction in severity through to end stage disease at day 24. In this study, the failure to establish pure populations of retinal antigen specific T lymphocyte cell lines and the observation of the lack of sensitivity of Northern hybridization to signals expressed at low levels resulted in the more sensitive technique of RT-PCR being utilized. Both IL2 and IFN mRNA expression was detected at all stages of disease with highest levels being present early in uveitis. In contrast IL4 mRNA levels increased with disease progression. This study suggests a pathogenic role for TH1 like cells and a protective role for TH2 like cells in this autoimmune disease. In order to provide an insight into alternative treatment strategies of the disease, immunomodulation of EAU was carried out using the immunosuppressive drugs CsA, FK506 and rapamycin and the resultant cytokine mRNA profiles examined. The results indicated that CsA and FK506 downregulated the TH1 response having suppressive effects on the levels of IL2 and IFN mRNA respectively. In contrast rapamycin was found to modulate the TH2 response enhancing IL4 levels. From this data, a drug based strategy employing rapamycin appears to be the most favourable approach.

In the human immune system, T-helper cells are able to differentiate into two lymphocyte subsets: Th1 and Th2. The intracellular signaling pathways of differentiation form a dynamic regulation network by secreting distinctive types of cytokines, while differentiation is regulated by two major gene loci: T-bet and GATA-3. We developed a system dynamics model to simulate the differentiation and re-differentiation process of T-helper cells, based on gene expression levels of T-bet and GATA-3 during differentiation of these cells. We arrived at three ultimate states of the model and came to the conclusion that cell differentiation potential exists as long as the system dynamics is at an unstable equilibrium point; the T-helper cells will no longer have the potential of differentiation when the model reaches a stable equilibrium point. In addition, the time lag caused by expression of transcription factors can lead to oscillations in the secretion of cytokines during differentiation.

T helper 17 (Th17) cells potently produce interleukin (IL)-17, which is essential for Th17 cell-mediated pathogen clearance. Failure to regulate Th17 cells can increase Th17 cell numbers and IL-17 production, and is associated with autoimmune disease pathology. Therefore, understanding how Th17 cell responses are controlled may improve treatments in instances of Th17 cell dysregulation. Investigations in mice and humans have mainly studied the cytokine signals that determine Th17 cell responses. However, the strength of TCR signalling has previously been shown to be a further factor capable of determining effector T cell development. The central hypothesis of my thesis is, therefore, that the strength of TCR stimulation is also capable of regulating Th17 cell responses. I also investigated if T-cell density, a parameter often overlooked in investigations, can also affect Th17 cell responses. Cell density has been shown previously to be capable of modulating many parameters including the expression of certain Th17 cell-related transcription factors. To assess the effect of T-cell stimulation strength on Th17 cell responses, human CD4+ T-cells were activated with high or low strength stimuli administered by bead-bound antibodies which activate the TCR/CD3 complex and the costimulatory molecule CD28, or by monocyte-derived dendritic cells pulsed with decreasing superantigen concentrations. Experiments were performed in the presence of pro-Th17 cell cytokines IL-1β, TGFβ and IL-23. In both systems low strength TCR stimulation profoundly and significantly promoted Th17 cell responses, both proportionately and absolutely. The enhancement of Th17 cell responses by low TCR stimulation only occurred in the presence of co-stimulation through CD28. Furthermore, it was revealed by chromatin immunoprecipitation that low strength stimulation promoted Th17 cell responses by allowing binding of a Ca2+ regulated transcription factor NFATc1 to the IL-17 promoter in a Ca2+ dependent manner. To investigate how low strength T-cell stimulation might promote human Th17 cell responses in vivo, 20 healthy donors were genotyped for a single nucleotide polymorphism within the gene Protein Tyrosine Phosphatase-N22 (PTPN22), which is highly associated with the autoimmune diseases type I diabetes and rheumatoid arthritis. PTPN22 encodes a TCR signalling molecule, Lyp, which in minor allele carriers confers both reduced TCR and Ca2+ signalling. Culture of genotyped memory CD4+ T-cells with anti-CD3/anti-CD28 stimulation and pro-Th17 cell cytokines revealed a trend indicating that the presence of the minor T allele promoted both IL-17 and IFN-g production but decreased regulatory IL-10 secretion. Regulation of Th17 cells by T-cell density was explored by culturing memory CD4+ T-cells at decreasing T-cell densities in the presence of either pro-Th17 or pro-Th1 cell cytokines. Low T-cell densities profoundly promoted Th17 cell responses both proportionately and absolutely. No effect was observed on the IFNy response within cultures containing pro-Th1 cell cytokines, suggesting that T-cell density specifically affects Th17 cell responses. STAT3 activation, important for IL-17 expression, can be regulated by cell density. Analysis of STAT3 activation by western blot revealed higher STAT3 activation in low density cultured T-cells compared to high density cultured T-cells, which may provide an explanation for the increased Th17 cell responses observed. The data within this thesis provide interesting and novel mechanisms by which human Th17 cell responses are regulated. I have demonstrated that Th17 cell responses are favoured by both low strength TCR stimulation and low T-cell density. These data highlight the diversity of factors capable of affecting Th17 cell responses in vitro; factors of which in the majority of studies have been overlooked.

Respiratory Syncytial virus (RSV) remains one of the most important causes of severe respiratory infection worldwide. Natural immunity is only partially protective, and no safe and effective vaccine has so far been found. CD4+ T cells play an essential role in shaping cellular and humoral immune responses. They have been shown to provide signals that are necessary for full differentiation of CD8+ T cells and B cells, as well as exhibiting direct anti-viral functions. However, their role in human RSV infection is poorly understood. Among the reasons for this are restrictions on sampling during the course of human infection and a resultant lack of tools to allow the study of antigen-specific CD4+ T cell responses. Further understanding of their role in the generation of effective immunity is essential for the development of better vaccines. Using an established experimental human challenge model, we inoculated 49 healthy adult volunteers with live, virulent RSV (Memphis 37). We analysed viral load by quantitative PCR (qPCR) on nasal lavage samples. Subjects were deemed infected if RSV was detectable on two consecutive days between day 2 and day 10 post inoculation. RSV infection developed in 26/49 (53%) of volunteers. In those infected, viral load peaked at day 7 and occurred at the same time as the peak symptoms score. Analysis of peripheral blood mononuclear cells (PBMCs) and BAL cells revealed the peak of polyclonal CD4+ T cell responses at day 10, characterised by the expression of the activation marker CD38 and proliferation marker Ki-67. At this time-point, RSV-specific CD4+ T cells were producing Th1 cytokines IFN- and TNF- To understand this response in greater detail, we generated a custom RSV M37 peptide library covering the complete RSV proteome. Using IFN- ELISpot, candidate epitopes were found in the F, G, M and N proteins. Of these, the most immunodominant CD4+ T cell epitopes were within the major surface glycoproteins F and G. Their class II restriction was established by depletion of CD8+ T cells from PBMCs and were confirmed by functional analysis of PBMCs from HLA class II matched donors. Using these, MHC class II-peptide tetramers using F and G peptides were constructed for the first time. RSV-specific CD4+ T cells were detectable in PBMCs of infected subjects at day 10, and exhibited the phenotypic markers of effector and effector memory T cells. These were highly activated but showed less evidence of proliferation than RSV-specific CD8+ T cells generated at the same time. Thus, the T cell response to RSV in healthy adults displays broad specificity but immunodominant CD4+ T cells primarily recognize peptides from the surface proteins F and G. Our studies imply the importance of Th1 and follicular helper T cells in the response to infection and provide novel tools that will allow further characterisation of RSV-specific CD4+ T cells in peripheral blood and lower airway.

Despite considerable progress in conventional cancer therapies, major challenges persist in the treatment of patients with advanced stage malignancies. Cancer immunotherapy (harnessing the immune system against tumors) has demonstrated limited success to date, partially due to the immunosuppressive environment generated by tumors. The mechanisms of cancer-induced immune suppression are multiple and include the promotion of immunosuppressive cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). MDSC expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. Different approaches have been explored to negatively impact MDSC, each associated with specific pitfalls. In this study, we demonstrated that the anthracycline doxorubicin selectively eliminates MDSC in the spleen, blood and tumor beds. Furthermore, five days after doxorubicin treatment residual MDSC exhibited impaired suppressive function, which correlated with reduced reactive oxygen species (ROS) production, and down-regulation of arginase-1 and indoleamine 2,3-dioxygenase (IDO) expression. Of therapeutic relevance, the frequency of effector lymphocytes (CD4⁺ and CD8⁺ T cells) or natural killer cells (NK) to suppressive MDSC ratios was significantly increased following doxorubicin treatment of tumor-bearing mice. Importantly, the proportion of natural killer (NK) and cytotoxic T cells (CTL) expressing perforin and granzyme B and of CTL producing IFNγ was augmented following doxorubicin administration. The mechanism of doxorubicin-mediated elimination of MDSC was partly mediated by the increase of ROS production in MDSC at earlier time points after doxorubicin treatment. Consistently, MDSC isolated from gp91-/- mice were less sensitive to doxorubicin in vitro, and doxorubicin effects on MDSC in gp91-/- tumor-bearing mice were reduced. Of clinical significance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease, resulting in better overall survival. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. Our results therefore indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T cell-based immunotherapy.

Cluster of Differentiation (CD) 4+ T-helper (Th) cells are critical in regulating protective immune responses to an array of pathogens. In order to generate robust effector CD4+ T cell populations, T cells need to rapidly integrate multiple signals in the Secondary Lymphoid Organs (SLOs). The interaction between a peptide Major Histocompatibility Complex (MHC) II molecule (pMHC-II) on Antigen Presenting Cells (APCs) and T Cell Receptors (TCRs) on T cells provides signals that drive T cell activation, cell division and clonal expansion. In addition, cytokine cues provided by APCs tailor the effector functions of CD4+ T cells to the microbe encountered. To this end, during infection with Mycobacterium tuberculosis (M.tb), an intracellular bacterium and the causative agent of Tuberculosis (TB), innate Interleukin (IL) 12 is known to instruct Th1 differentiation required for inducing the microbicidal function of macrophages. While TCR affinity is known to influence robust T cell expansion and Th cell differentiation, the relative contribution of TCR signals to cytokine cues for the generation of effective Th1 responses is unclear. To address this question, I developed an in vitro T cell co-culture system and an in vivo adoptive transfer model, employing two recently generated TCR transgenic mouse lines. CD4+ T cells in these mice recognise the same epitope of a dominant mycobacterial antigen with differing binding affinities. Both the in vitro and in vivo approaches revealed that in contrast to the clonal expansion of CD4+ T cells, which is driven predominantly by TCR affinity, the acquisition of Th1 effector function is critically dependent on the presence of IL-12. However, TCR affinity determines the pace of Th1 differentiation by controlling the cell division-dependent sensitivity of activated CD4+ T cells to IL-12 instruction. These findings suggest that the temporal regulation of Th1 differentiation and expansion by TCR signals determine the quality of the CD4+ T cell response in SLOs. In this way, high affinity CD4+ T cells may play a critical role in pathogen control during early stages of infection. Although the importance of Th1 responses in controlling mycobacterial infection is well-established, the contribution of follicular Th (Tfh) cells to the defence against M.tb infection is incompletely understood. In the second part of this thesis, I discovered that aerosol M.tb infection induced a disproportional increase in B cells in the lung-draining lymph nodes. Dysregulated B cell expansion prevented the optimal control of M.tb infection in the lymph node. Furthermore, the accumulation of B cells in M.tb-infected lymph nodes was associated with the development of CD4+ T cells expressing molecules traditionally linked to both Th1 and Tfh cells. The generation of Tfh-like cells required both B cells and the master regulator of Tfh cells, B Cell Lymphoma (BCL) 6. Surprisingly, CD4+ T cell-expressed BCL6 plays an opposing role in resistance to M.tb infection, depending on the infection stage. BCL6 expression during acute M.tb infection is detrimental, as it impairs the generation of Th1 effectors and host resistance. However, its expression during chronic infection is beneficial for the maintenance of CD4+ T cell responses and antimicrobial resistance. These findings suggest that the expression of BCL6 may delay the generation of Th1 effectors in the LN. They also indicate that interactions between T and B cells in the lymph node play a previously unrecognised role in determining the outcome of M.tb infection in mice. Taken together, my findings reveal how TCR affinity regulates the quality of Th1 responses during mycobacterial infection and describe how interactions between Tfh-like cells and B cells have important consequences for the control of M.tb infection. These findings uncover the protective mechanisms regulating regional immunity in peripheral and lymphoid tissues.

T-helper (Th) 17 cells are a subset of CD4+ T cells defined through the release of the cytokine interleukin-17a (IL-17a). Activation of these cells is critical for protection against some extracellular bacterial and fungal pathogens. However, a dysregulated Th17 response targeted against self is thought to play an important role in the immunopathology of a number of autoimmune conditions including Inflammatory Bowel Disease (IBD), Multiple Sclerosis (MS) or inflammatory arthritides. Further understanding of the mechanisms that influence the development of Th17 cells may aid future therapeutic targeting of these cells. Whilst the role of the cytokine milieu in Th cell polarisation is relatively well characterised, the degree of signalling through the TCR can also shape the form of the Th cell response. Both the density of antigenic peptide available and the affinity of the antigenic peptide for a particular TCR can contribute to the degree of TCR signalling. The hypothesis of this project was that TCR signal intensity could alter the development of Th17 cells from a naive precursor population. In particular, it was of interest to determine how citrullination of a putative TCR contact amino acid in an antigenic peptide could alter the Th cell response observed. The 5/4E8 T-cell receptor transgenic (TCR Tg) mouse provides a model in which >80% of T-cells specifically recognise an immunodominant epitope derived from the G1 domain of aggrecan – peptide-84-103 (p84-103). This model allowed for the examination of these factors and the underlying mechanism ex vivo using a purified naive CD4+ T cell population in coculture with LPS-matured dendritic cells (mDCs). The data presented in this thesis show the activation, proliferation and effector responses of naive 5/4E8 TCR Tg T cells to alterations in both cognate peptide (p89- 103) density and affinity through citrullination of a putative TCR contact residue (R93Cit). Interestingly, by reducing TCR signal strength the observed response shifts from one dominated by the Th2 phenotype to Th17 cells. Linking the degree of TCR activation to Th cell phenotype was the intensity of IL-2 signalling that in turn shaped the balance between phosphorylated STAT3 and STAT5. Compared to p89-103-primed T cells, T cells responding to R93Cit produced less IL-2, expressed lower levels of the ILiii 2 receptor subunit CD25, and showed reduced levels of STAT5 phosphorylation, whilst STAT3 activation was unaltered. IL-2 blockade in p89-103-primed T-cells selectively reduced STAT5 but not STAT3 phosphorylation, and concomitantly enhanced Th17 development. In summary, this work indicates the impact that changes to the intensity of TCR signalling can have on the murine Th17 response. Indeed, these data illustrate how a disease-relevant post-translational modification such as citrullination can promote Th17 development by altering the balance between STAT5 and STAT3 activation in responding T cells.

Adaptive immunity is controlled by CD4+ T cells that develop into functionally distinct T helper (Th) cell subsets. The paradigm that cellular immunity is mediated by Th1 cells and humoral immunity by Th2 cells has been revised upon the discovery of a third Th lineage which produces Interleukin (IL)-17 (Th17 cells). Th17 cells exhibit a unique cytokine profile compared to other Th subsets and are important for host defense against extracellular pathogens. However, aberrant expansion of Th17 cells is linked to several autoimmune diseases including rheumatoid arthritis, psoriasis, inflammatory bowel disease and multiple sclerosis. Due to their association with dysregulated immune responses, there has been intense interest in understanding factors regulating Th17 cell development, phenotype and function. The findings herein identify several mechanisms which regulate this lineage. First, I identified a central role for a transcription factor known as retinoic acid-related orphan receptor C isoform 2 (RORC2) in the development of human Th17 cells. In order to study in vivo derived Th17 cells, a flow cytometry-based sorting method was established to isolate Th17 cells directly from peripheral blood. After defining the phenotype of these bona fide Th17 cells, I investigated their interactions with T regulatory (Treg) cells. Contrary to prior findings and RORC2 overexpression studies, Treg cells were potent inhibitors of in vivo-derived Th17 cell proliferation and suppressed their inflammatory mediators. To examine the stability of this lineage, a comprehensive study of the epigenetic phenotype of Th17 cells was performed. Further to defining Th17 cell’s epigenetic profile, examination of epigenetic marks following exposure to potent polarization conditions suggests Th17 cells are resistant to lineage conversion. Taken together, these findings provide an in-depth examination of this inflammatory T cell subset, provide new tools to study Th17 cells and elucidate mechanisms that regulate Th17 cells, thereby providing insights to aid development of therapeutic strategies for Th17-associated disorders.

The intestinal IgA response has features that are different to those of the systemic humoral response, which is dominated by IgG. Although the IgA response, like the IgG response, includes an antigen specific component, it is also associated with polyspecificity and autoimmunity. The profile of intestinal immunoglobulins changes in inflammatory bowel disease (IBD) where there is a disproportionate increase in IgG production and in ulcerative colitis (UC), this includes the production of autoantibodies. In this thesis, two immunoregulatory T cell subsets that could influence the intestinal B cell response have been studied; T follicular helper cells (TFH) and regulatory T cells (Treg). Results in chapter 3 show that there is a higher density of TFH in gut associated lymphoid tissue (GALT) compared to peripheral lymphoid tissue due to a higher density of CD57- TFH- The expression of cytokines and CD40L was almost comparable between CD57+ and CD57- TFH- However, culturing experiments suggest that CD57-TFH may develop into CD57+ TFH and there is a constant turnover of TFH in the gut. Experiments in chapter 4 attempted to seek evidence for a developmental relationship between TFH and Treg by analysis of T cell receptor sequences. No evidence of plasticity between these subsets was observed. Experiments in chapter 5 set out to characterise TFH in IBD. In the appendix of UC patients, nearly all TFH were CD57+ and at a high density within germinal centres (GC). This thesis concludes that TFH are more phenotypically diverse and denser in GALT compared to peripheral lymphoid tissue. This may reduce the threshold for GC B cell survival in the gut permitting the propagation of plasma cells that secrete polyspecific and autoreactive IgA. TFH are denser still in the small GC in UC appendix. This may be relevant to the production of autoantibodies and disease pathogenesis.

DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response. Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge. The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation. One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells. Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.

Chez les mammifères, les lymphocytes T CD4 sont essentiels à la défense de l’organisme contre des infections par des pathogènes ou le développement de tumeurs. Après activation, les lymphocytes T CD4 naïfs ont la capacité de se différencier en divers lymphocytes T helper (Th1, Th2, Th17…) en fonction des signaux reçus. Le choix du lignage permet d’adapter le phénotype et la fonction des cellules au type de danger détecté. Le processus de différenciation des lymphocytes T helper implique l’établissement de programmes d’expression des gènes distincts. La dynamique et la stabilité de ces programmes sont notamment régulées par l’activité d’éléments cis-régulateurs. Le but de ma thèse était de comprendre les mécanismes épigénétiques qui contrôlent la programmation des lymphocytes T CD4. Dans cet objectif, nous avons étudié le rôle de la H3K9 méthyl-transférase SETDB1 dans la différenciation des lymphocytes T CD4 en Th1 et Th2, deux lignages T helper fortement antagonistes. Nous avons découvert que SETDB1 réprime de manière critique le programme d’expression des gènes Th1. En effet, en l’absence d’expression de Setdb1, la différenciation Th1 est exacerbée. De plus, lorsqu’elles sont exposées à un signal pro-Th1, les cellules Th2 franchissent les barrières de lignage et se transdifférencient en Th1. De manière surprenante, SETDB1 ne cible pas directement les enhancers Th1. Au contraire, l’enzyme dépose de manière type cellulaire spécifique la marque répressive H3K9me3 au niveau d’un set restreint de rétrovirus endogènes (ERVs). Des analyses bio-informatiques ont indiqué que les rétrotransposons ciblés sont fortement associés à des gènes impliqués dans les processus immunitaires. La suite de ces analyses a indiqué que ces ERVs flanquent et répriment l’activité d’éléments cis-régulateurs des gènes Th1, ou agissent eux même comme des enhancers du lignage. En conclusion, la déposition de H3K9me3 par SETDB1 garantit l’intégrité des lymphocytes T helper en réprimant un panel d’ERVs qui ont été exaptés en modules cis-régulateurs pour façonner et contrôler le réseau de gènes Th1
CD4 T lymphocytes play a central role in the defense of mammal organisms against infections by pathogens and the development of tumors. Upon activation, naïve CD4 T cells differentiate into distinct helper cell subsets depending on environmental cues. T helper cells are key players of the immune system as they finely orchestrate immune responses in a danger-adapted manner. The process of T helper differentiation relies on the establishment of complex and lineage-specific gene expression programs. The dynamics and stability of these programs are regulated at the chromatin level through epigenetic control of cis-regulatory elements. My thesis objective was to investigate the epigenetic pathways involved in the regulation of enhancer activity in CD4 T cells. In this purpose, we studied the role of the H3K9 specific methyltransferase SETDB1 in the differentiation of Th1 and Th2 cells, which are strongly antagonistic. We report that SETDB1 critically represses the Th1 gene expression program. Indeed, Setdb1-deficient naïve T cells show exacerbated Th1 priming. Moreover, when exposed to a Th1-instructive signal, SETDB1-deficient Th2 cells cross lineage boundaries and transdifferentiate into Th1 cells. Surprisingly, SETDB1 does not directly target Th1 enhancers to heterochromatin. Instead, SETDB1 deposits the repressive H3K9me3 mark at a restricted and cell type specific set of endogenous retroviruses, strongly associated with genes involved in immune processes. Further bioinformatic analyses indicated that these retrotransposons flank and repress Th1 gene cis-regulatory elements or behave themselves as Th1 gene enhancers. Thus, H3K9me3 deposition by SETDB1 ensures T cell lineage integrity by repressing a repertoire of ERVs that have been exapted into cis-regulatory modules to shape and control the Th1 gene network

Les mastocytes ont longtemps été associés aux réponses allergiques, néanmoins de nombreuses études les décrivent comme des acteurs de la réponse inflammatoire. Les mastocytes sont souvent retrouvés en étroite apposition au sein des tissus avec les lymphocytes T Helper (TH). L'objet de ma thèse est de caractériser la nature de l'interaction mastocyte/TH et notamment la capacité des mastocytes à jouer le rôle de cellules présentatrices d'antigène. Dans une première étude, nous avons démontré que des lignées primaires de mastocytes murins (PCMC) peuvent acquérir un phénotype de cellules présentatrices d'antigènes, lorsqu'elles sont traitées en présence d'IFN-gamma pendant 72 heures. Ainsi conditionnés, les PCMC capturent, apprêtent et présentent l'antigène à des lymphocytes T CD4+ activés, mais restent incapables d'activer leurs homologues naïfs. Durant ce phénomène, une véritable synapse immunologique est mise en place où le lymphocyte TH polarise son TCR et toute sa machinerie sécrétoire vers l'aire de contact intercellulaire. En retour, le mastocyte s'active et baisse significativement le seuil d'activation des récepteurs FcepsilonRI en réponse à une stimulation par des IgE. Ces résultats indiquent que les mastocytes peuvent établir une coopération spécifique avec les lymphocytes TH. Dans une seconde étude, nous avons déterminé les conséquences fonctionnelles d'une telle coopération sur la biologie des lymphocytes TH humains. In vitro les mastocytes humains forment des synapses immunologiques fonctionnelles avec des lymphocytes T CD4+ activés. Ces interactions productives favorisent la génération de lymphocytes TH produisant de grandes quantités d'IL-22 (à la fois de purs TH22 et des TH produisant simultanément de l'IL-22 et de l'IFN-gamma) à partir d'un pool de lymphocytes TH mémoires. Cette différenciation vers un tel profil de TH est dépendante de l'action de deux cytokines que sont l'IL-6 et le TNF-alpha. L'analyse microscopique détaillée d'échantillons de peaux psoriasiques révèle un enrichissement en lymphocytes TH produisant simultanément de l'IL-22 et de l'IFN-gamma, lors qu'ils sont au contact des mastocytes. Ces résultats indiquent que les mastocytes peuvent être de véritables partenaires fonctionnels pour les lymphocytes TH au sein des tissus, leur fournissant des signaux d'activation et les orientant vers la production d'une cytokine supplémentaire l'IL-22
Mast cells have been classically associated to allergic disorders, however several studies describe their involvement in the inflammatory response. Mast cells are often observed in tissues in close apposition with CD4 T cells (TH). The aim of my thesis is to better characterize the nature of mast cell/TH interaction and in particular the mast cell capacity to act as antigen presenting cell and the consequences for the TH cell response. We first addressed this question using mouse primary mast cell lines derived from peritoneal progenitors (PCMC) used as antigen presenting cell to activate cognate OTii T cells with OVA peptide. Our results show that in vitro treatment of PCMC with IFN-gamma and IL-4 induced surface expression of mature MHC class II and costimulatory molecules. When IFN-gamma primed PCMC were used as antigen presenting cells for Helper T (TH) cells, they induced activation of effector T cells but not of their naive counterparts. Confocal laser scanning microscopy showed that TH cells formed immunological synapses and polarized their secretory machinery towards antigen-loaded PCMC. Finally, upon cognate interaction with TH cells, mast cells lowered their threshold of activation via FcepsilonRI. These results show that mast cells can specifically cooperate with class II restricted TH cells. In a second part, we investigated the functional impact of mast cell antigen presentation on human T cell-mediated responses. In vitro, human mast cells form productive immunological synapses with antigen-experienced TH cells. These interactions promote the generation of interleukin 22 (IL-22) producing TH cells (either pure TH22 or IL-22 plus IFN-gamma-producing TH cells) from the circulating CD4+ memory T cell pool via a tumor necrosis factor (TNF-alpha) - and IL-6-dependent mechanism. Three-color immunohistochemistry and confocal laser scanning microscopy reveal a rich infiltrate of IL-22+ TH cells in psoriatic skin biopsies. Most of the IL-22+ TH cells are found in contact with mast cells and co-express IFN-gamma, suggesting that IFN-gamma+IL-22+ TH cells are generated in vivo upon interaction with mast cells. Our results reveal a novel functional trait of mast cells by showing that they can shape the inflammatory potential of human TH lymphocytes in tissues, by providing activating signals and skewing the TH cell response toward the production of IL-22

Summary of the work

Abdominal aortic aneurysm (AAA) is a chronic degenerative disease that usually affects men over 65 years with an estimated prevalence of 5%. Aneurysm rupture represents a catastrophic event which carries a mortality rate of almost 90%. Current therapeutic options for AAAs measuring 5.5 cm in diameter or larger are based on prophylactic surgery, including conventional open reconstruction and endovascular stent-graft insertion. For patients with small asymptomatic AAAs (4.0 up to 5.5 cm in diameter), evidence from two recent large randomized controlled trials indicates no long-term survival benefit from immediate elective surgical repair as compared to imaging surveillance until aneurysm expands to 5.5 cm. This highlights the need for development of novel medical management strategies, including selective pharmacologic approaches, directed at preventing aneurysm expansion. In this regard, it is expected that a detailed knowledge of the pathobiology of human AAA lesion and a better understanding of pathophysiological mechanisms underlying initiation and progression of aneurysmal degeneration, particularly the specific involvement of T lymphocytes, will have special relevance to this challenging issue.

Inflammatory and helper T-cell responses in abdominal aortic aneurysm :controversial issues

Innate and inflammatory responses to endovascular versus open AAA repair. The occurrence of early acute systemic inflammatory responses after conventional open AAA repair is widely recognized and is thought to lead to the development of organ dysfunction and multiple organ failure, responsible for a large proportion of morbidity and mortality associated with aortic surgery. New therapeutic strategies designed to avoid ischemia-reperfusion injury related to aortic cross-clamping and to minimize the degree of tissue damage have thus been developed recently. Specifically, the advent of endovascular techniques has radically extended management options for patients with AAA. Although the method is believed to offer a clear short-term benefit over open repair, notably as regards restricted perioperative haemodynamic parameter fluctuations, reduced blood loss, briefer duration of surgery, shorter hospital stay, and lower 30-day mortality and complication rates, conflicting data are available regarding the exact nature and extent of the inflammatory events arising after such endoluminal procedures ;while several authors have indeed reported that endovascular AAA repair can determine a less intense and extensive inflammatory response, others have unexpectedly observed that the method may elicit a strong inflammatory response, the so-called « postimplantation syndrome ».

Adaptive cellular immune responses in human aneurysmal aortic lesion.

The inflammatory nature of AAA disease has long been suggested by the presence of a great number of CD4+ T lymphocytes in the outer media and adventitia of human AAA lesion. Interestingly, such infiltrating T-cell populations may have significant implications in the process of aneurysm dilation, since cytokines produced by T cells, notably IFN-gamma, have previously been shown to modulate production of matrix-degrading enzymes by resident macrophages and to induce apoptosis of medial SMCs. Through these key pathological mechanisms, T cells could potentially contribute to orchestrate aortic wall connective tissue disordered remodeling and degradation, and promote extensive disruption of elastic media, ultimately leading to aneurysmal degeneration. Nevertheless, despite their relative abundance in human AAA wall tissues, there is limited and controversial information as regards the functional profile of lesional lymphocytes, the exact nature of aortic wall adaptive cellular responses, and the etiologic role of T cells and their cytokines in initiation and progression of the aneurysmal process. Indeed, both Th1-type and Th2-type responses have been identified in human studies and experimental animal models of AAA.

Aims of the work

The main objectives of our work were to explore the innate and adaptive cellular immune responses in human AAA. In the first part of our work, we aimed to examine prospectively innate and inflammatory responses arising in a non-randomised cohort of patients undergoing endovascular versus open AAA repair. In the second part of our work, we focused our efforts on characterizing the nature of adaptive cellular immune responses and the phenotypic and functional repertoire of T cells in human AAA wall tissues obtained from a consecutive series of patients undergoing open AAA repair. Specifically, we sought to determine whether type 1 or type 2 responses occur predominantly in advanced AAA lesion.

Main experimental findings

Limited inflammatory response after endovascular AAA repair. Serial peripheral venous blood samples were collected preoperatively, immediately after declamping or insertion of endograft, and after 1, 3, 6, 12, 24, 48, and 72 hours. We first examined the acute phase reaction and liberation of complement cascade products using turbidimetric method and nephelometry. We found that endovascular repair produced lower postoperative CRP, leucocytosis, neutrophilia, and C3d/C3 ratio as compared to open surgery. We next analyzed surface expression of activation markers on peripheral CD3+ T cells using flow cytometry. We observed a strong upregulation of CD38 after open but not endovascular repair. Analysis of CD69 and CD25 molecules revealed no perioperative fluctuations in any group. We then investigated release of various circulating soluble cell adhesion molecules, proinflammatory cytokines, and chemokines using enzyme-linked immunosorbent assays. We demonstrated that both procedures are characterized by similar increases in ICAM-1 and IL-6 levels. Finally, tendency towards high levels of TNF-alpha and IL-8 was detected in endovascular repair, but data failed to reach statistical significance.

Predominance of type 1 CD4+ T cells in human aneurysmal aortic lesion. We have developed a tissue enzymatic digestion and cell extraction procedure to isolate intact mononuclear cells from aortic wall segments. This original cell isolation protocol enabled us to examine ex vivo the presence, phenotype, and cytokine secretion profile of infiltrating T lymphocytes freshly isolated from human AAA tissues for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4+ and CD8+ T cells display a unique activated memory phenotype, as assessed by an increased expression of CD69 and HLA-DR activation antigens, downregulation of CD62L molecule, and predominant expression of the CD45RO isoform characteristics of memory cells. In addition, we identified the presence in human aneurysmal aortic wall lesion of CD4+ T cells producing high levels of IFN-gamma but not IL-4, reflecting their type 1 nature. In an additional series of experiments, cytokine gene expression was determined in whole aneurysmal and non-diseased aortic samples using LightCycler-based quantitative real-time reverse transcription-polymerase chain reaction. The molecular basis of type 1 or type 2 dominant responses was further specified by analyzing mRNA levels of transcription factors specifically involved in Th1 or Th2 differentiation such as T-bet and GATA-3. We demonstrated that aneurysmal aortic specimens exhibit high transcript levels of IFN-gamma but not IL-4, and consistently overexpressed the IFN-g-promoting cytokine IL-12 and the type 1-restricted transcription factor T-bet, further establishing the prominent type 1 nature of aortic wall responses. Moreover, such selective tissue expression of IL-12 and T-bet in the vessel microenvironment points to a potential role for these signals in directing aortic wall responses towards a type 1 phenotype.

Conclusions

Our findings indicate that endovascular AAA repair is associated with a lesser degree of acute phase reaction, peripheral T-cell activation, and release of complement proteins as compared to conventional open surgery, suggesting that the innate and inflammatory responses to AAA repair are significantly attenuated by the endovascular approach as compared to the traditional open reconstruction. These results support the view that the endoluminal procedure represents an attractive alternative to open surgery for the treatment of large aneurysms. On the other hand, we have demonstrated that Th1 cell infiltrates predominate in human end-stage AAA lesion. These observations are relevant for helping clarify the pathobiology of human AAA tissues and defining prospects for the prevention of aneurysm expansion. Indeed, identification of such infiltrating populations of IFN-gamma-producing CD4+ T cells not only provide new insights into the pathogenesis of the disorder, but could also serve as a basis for the development of novel medical management strategies directed at preventing aneurysm formation and progression, including therapeutic approaches based on the modulation of aortic wall responses and designed to selectively target T-cell activation and cytokine production. In this respect, the present work provides experimental evidence in support of the emerging concept that, although multifactorial, aneurysm disease may be regarded as a Th1-driven immunopathological condition, and suggests that strategies targeting IFN-gamma could be a particularly exciting and fruitful avenue for further investigation. Ongoing clinical and basic research in these areas can be expected to yield design of promising pharmacologic approaches to control AAA expansion. From a clinical perspective, such efforts have the potential to dramatically influence both the outcome and management of this common and life threatening condition.

Doctorat en sciences médicales
info:eu-repo/semantics/nonPublished

microRN'As are a class of short RNA molecules that mediate post-transcriptional regulation of gene expression thereby controlling cell fate. The importance of microRN'As in normal T helper cell function has been highlighted by recent studies in which T cells that are globally deficient in microRNAs exhibit major abnormalities of homeostasis and differentiation. However the identities of those individual microRNAs that are required for normal T cell function remain unclear, and there is a lack of data regarding the cellular processes that are regulated by microRNAs in T cells. This thesis examines the hypothesis that specific microRNAs are of critical importance for normal T helper cell function and differentiation. microRNA expression was profiled in highly-polarised T helper cell subsets, and analysed in order to detect differentially expressed microRNAs. A candidate microRNA, mir-142, was selected on the basis of these data for further functional analysis. Novel constitutive and conditional mir-I42-deficient mice were generated and systematically analysed for T cell functional defects. mir-142 deficiency was found to profoundly affect T cell homeostasis, with a significant reduction in the size of the peripheral T cell pool and impaired proliferation and survival in vivo. T helper cell differentiation was also affected, with default adoption of a Th1 phenotype and impaired stability of non-Th1 lineages. Analysis of predicted targets revealed that both retinoic acid receptor (RAR)-γ and T -bet are targets of mir-142 that are functionally responsible for these abnormalities. Deficiency of mir-142 in T helper cells was also found to prevent the development of disease in a T cell-­mediated colitis model. In summaiy, this work identifies mir-142 as a critically important microRNA for normal T helper cell function and differentiation. Future studies will continue to analyse the function of mir-142 and may investigate this microRNA as a potential therapeutic target in immune-mediated disease.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 118-125).
The adaptive immune system is an extraordinarily diverse inventory comprised of highly specialized cells, the differentiation of which requires numerous lineage specifications at various developmental stages. The precise control of immune cell differentiation and the delicate balance of their population composition are crucial for effective protection against infectious environmental agents, without triggering autoimmune responses or allergies. It is therefore important to understand at the molecular level in individual cells how lineage commitment is regulated. I explored the heterogeneous gene expression during the lineage specification of single T helper cells, by quantitatively measuring mRNA and protein levels. I have discovered a paradigm of cell lineage specification governed by the signaling interplay between extracellular cues and intracellular transcriptional factors, where the strength of extracellular signaling dominates over the intracellular signaling components. In the presence of extracellular cues, T helper cells stochastically acquire any intermediate Thl/Th2 states. The states of T helper cells can be gradually tuned by depriving availability of extracellular cytokines, which are produced stochastically by a small subpopulation of cells. When extracellular cues are removed, the weak intracellular signaling network reveals its effect, leading to classic mutual exclusion of antagonistic transcriptional factors.
by Miaoqing Fang.
Ph.D.

During the course of an immune response, CD4+ T helper cells differentiate into a number of subsets including: T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. The functional diversity of CD4+ T effector cells results in a coordinated, pathogen-specific immune response. For example, the production of IFNγ by TH1 cells is vital for the clearance of intracellular pathogens, while TFH cell engagement with cognate B cells is required for germinal center (GC) formation and the generation of pathogen- and vaccine- induced antibody production. The development of CD4+ subsets is contingent on extracellular signals, in the form of cytokines, and downstream transcriptional networks responsible for promoting the unique gene expression profile for each subset while simultaneously suppressing alternative cell fates. However, the exact composition of, and stage-specific requirements for, these environmental cytokines and transcription factor networks in the governance of TFH cell differentiation remain incompletely understood. The work in this dissertation seeks to understand how cell-extrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Here, we demonstrate that in response to decreased IL-2 and constant IL-12 signaling, T helper 1 (TH1) cells upregulate a TFH-like phenotype, including expression of the TFH lineage defining transcription factor Bcl-6. Intriguingly, our work established that signals from IL-12 were required for both the differentiation and function of this TFH-like population. Mechanistically, IL-12 signals are propagated through both STAT3 and STAT4, leading to the upregulation of the TFH associated genes Bcl6, Il21, and Icos, correlating with increased B cell helper activity. Conversely, exposure of these TFH-like cells to IL-7 results in the STAT5-dependent repression of Bcl-6 and subsequent inhibition of the TFH phenotype. Finally, we describe a novel regulatory mechanism wherein STAT3 and the Ikaros zinc finger transcription factors Ikaros and Aiolos cooperate to regulate Bcl-6 expression in these TFH-like cells. Collectively, the work in this dissertation significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines.
Ph. D.
Specialized cells called T helper cells serve as a critical interface between the innate (first line of defense) and adaptive (specialized and long-term) immune systems. During the course of an infection, T helper cells are responsible for orchestrating the immune-mediated elimination of invading viruses, bacteria, and parasites. This wide breadth of functionality is achieved through the formation of distinct T helper subsets including T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. Individual subsets have distinct developmental requirements and have unique functions within the immune system. For example, TFH cells are required for the production of effective antibodies that recognize invading pathogens, leading to their subsequent elimination. This naturally occurring process is the basis for a number of modern medical therapies including vaccination. Conversely, aberrant generation of antibodies that recognize host tissues can result in the onset of various autoimmune diseases including lupus, multiple sclerosis, and crohn’s disease. Due to the importance of TFH cells to human health, there is intense interest in understanding how these cells are formed. It is recognized that the generation of these therapeutically important immune cells is mediated by numerous cell-extrinsic andintrinsic influences, including proteins in their cellular environment called cytokines, and important proteins inside of the cell called transcription factors. However, as this is a complicated and multi-step process, many questions remain regarding the identity of these cytokines and transcription factors. The work in this dissertation seeks to understand how cellextrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Collectively, this body of work significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines.

The Rhesus (Rh) D antigen is important in transfusion medicine and is the major target in haemolytic disease of the newborn (HDN). The aims of this project were to characterise the helper-T (Th) cell response that drives anti-D alloantibody production in HDN as a first step towards developing an improved or alternative strategy to the current programme of prophylaxis, based on the manipulation the T-cell response. Peripheral blood mononuclear cells (PBMC) were obtained from 22 individuals, who had developed anti-RhD alloantibodies following natural or deliberate immunisation, and from 22 RhD negative and from 12 RhD positive control donors who had not been immunised with RhD. A panel of 69 overlapping synthetic 15-mer peptides, spanning the sequence of the RhD protein, was screed for the ability to stimulate recall proliferation and cytokine production from T-cells in cultures of the PBMC. T cells from all 22 of the alloimmunised donors proliferated in response to RhD peptides and typically multiple peptides were stimulatory. In contrast, only a few minor responses were observed in the control donors. RhD peptides 6, 13, 17, and 28 were identified as immunodominant peptides that stimulated proliferation in over 50% of the alloimmunised donors. Each of these peptides were promiscuous in their ability to stimulate T-cells from donors of the common HLA allotypes in this study. Each immunodominant peptide contains multiple core epitopes and multiple sets of MHC/TCR contacts. Preliminary findings indicate that neither peptides shorter than 15mer length nor analogues can be designed to boost or tolerise alloimmunised donors. The RhD peptides induced a complex pattern of cytokine production from alloreactive T cells. Both IFN-gamma and IL-4 could be produced to the RhD peptides indicative of a Th0 response. In addition, particular peptides elicited the production of the potentially inhibitory cytokines IL-10 or TGFb and not proliferation.

Autoimmune haemolytic anaemia provides an opportunity to characterise the T-cell response to an autoantigen of known pathological relevance. In most cases, AIHA is characterised by the production of autoantibodies to the Rh proteins. The aims of this work were to confirm that human AIHA is T-helper cell dependent and whether specific immunotherapy could be developed to target the T-helper cell response. Peripheral blood mononuclear cells (PBMC) were isolated from patients with either primary AIHA and stimulated in vitro with a panel of 42 15-mer peptides with 5 mer overlaps spanning the entire sequence of the RhD and CE proteins. PBMC with 22/27 patients proliferate in response to stimulation with at least one of these peptides compared to only 4/14 healthy donors. The proliferating cells were identified as T-helper cells by flow cytometry analysis and abrogation of the response using MHC class II blocking antibodies. There is a predominance of HLA-DRB*1501 and HLA-DQB*06 haplotypes whilst the HLA-DRB1*07 haplotype is under represented amongst the panel of primary AIHA patients. Antibodies directed against HLA-DR and HLA-DQ can inhibit the T-cell response to peptides from the Rh proteins (anti-HLA-DR>anti-HLA-DQ) suggesting that these MHC class I molecules play an important role in presenting the synthetic Rh peptides. The pathogenic T-helper cell response to RhD protein in vivo, may depend upon the balance of pathogenic and regulatory epitopes that are processed and presented by antigen presenting cells.

A T helper cell clone, referred to as clone 9, was derived from an allogeneic mixed lymphocyte culture. Clone 9, as well as supernatant factor(s) derived from it, could help the cytotoxic T lymphocyte (CTL) responses of H-2 Db (Db) responder cells to alloantigens, or they could help the CTL responses of non- Db responder cells to Db alloantigens. Clone 9 cells or their factor(s) were active only when added during the first 24 hours of a five-day culture period. Clone 9 or its factor(s) could also synergize with interleukin-2 (IL-2)-containing medium in mounting cytotoxic responses to alloantigens. The helper activity in clone 9 supernatant was not due to IL-2 and it was specifically absorbed out by Db -spleen cells. The characterization of the Db -specific helper factor(ASHF) was facilitated by the isolation of a T hybridoma clone (clone 25), obtained from fusion of clone 9 cells with the T cell lymphoma, BW5147, and a B cell hybridoma that produced an IgM monoclonal antibody (clone 30 IgM) which bound ASHF. An additional monoclonal antibody (F23.1), which recognizes a determinant of the Vβ8 family of the T cell receptor, was also particularly useful for the characterization of ASHF. Analysis with these reagents showed that both clone 30 IgM and F23.1 immunoadsorbents could retain ASHF activity. Preabsorption of the ASHF with Db spleen cells prior to affinity purification over a clone 30 IgM column resulted in the absorption of Db-specific helper activity as well as the loss of a 50,000 molecular weight (MW) band on SDS-PAGE under reducing conditions. Furthermore, affinity purification of ASHF over the F23.1 immunoadsorbent, but not an irrelevant monoclonal antibody (mAb) column, also yielded a 50,000 MW molecule. Taken together, these findings suggest that the 50,000 MW molecule is a component of the ASHF and it is intimately related to the B chain of the T-cell receptor. The mode of action of clone 9 and its products in the induction bfCTL responses was also investigated. It was found that clone 9 and ASHF could help CTL responses by inducing IL-2 production in B6-stimulated cultures. In addition to ASHF, clone 9 cells also produced an additional factor(s) which participated in the induction of CTL responses. This additional factor(s) was referred to as IL-X. IL-X synergized with excess human recombinant IL-2 in the activation of CTL precursors (CTL-P) in the absence of antigenic stimulation. A model which involves the participation of ASHF, T helper cells, IL-2 and IL-X in the induction of CTL responses is proposed.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate