Introduction: Sickle cell anemia (SCA) is one of the most common genetic disorders in the world. However, until recently, only the direct consequences of the deoxyHbS polymerization was used to explain the pathophysiology of the disease. Currently, it is known that the clinical repercussions of FA involve complex interactions between the erythrocyte, endothelium and leukocytes: cytokines secreted by inflammatory cells are involved in SCA crisis and in the maintenance of a systemic inflammatory status, suggesting that T cells (Helpers and Cytotoxics) and NK have a fundalmental role in the clinical phenomena of SCA. This study aims to evaluate the profile of T and NK lymphocytes in patients with AF and compare them with the profile of individuals with sickle cell trait and individuals without hemoglobinopathies. Materials and methods: Peripheral blood was collected from 13 individuals; 7 with SS hemoglobinopathy (SS group), 5 with sickle cell trait (AS group) and 5 normal (AA group). The lymphocytes isolation for analysis was performed using Ficoll-Hypaque solution. Immunophenotyping for the determination of lymphocyte subtypes was performed by flow cytometry, using eight-color cytometer and eight BD Biosciences antibodies. Data were analyzed using Flowjo software and tabulated in SPSS IBM 22.0. The results referring to the numerical variables were expressed through measures of central tendency. Results: A lower frequency of T lymphocytes and a greater frequency of NK cells were observed in sickle cell patients.The variation of TCD4 + found between the SCA and the AA group was significant (p = 0.04). Lower frequency tendency in patients of the SS group remained for B lymphocytes. A higher frequency of NK cells was observed in patients with sickle cell anemia (mean: group AA = 15.63%, group AS = 14.82% and group SS = 23.34%) with statistically significant variation. Conclusion: SCA patients present a lower frequency of CD4 + CD8 + T cells and TNK cells when compared with HbAS individuals and individuals without hemoglobinopathies. An increasing and progressive frequency of NK cells is observed between groups AA, AS and SS. We believe that further studies are needed to understand the role of these cells in the genesis of systemic inflammation of SCA. This understanding may contribute to the development of new therapeutic strategies. / INTRODUÇÃO: A anemia falciforme (AF) é uma das desordens genéticas mais comuns do mundo. Entretanto, até pouco tempo atrás, apenas as consequências diretas da polimerização da desoxiHbS explicava a fisiopatogenia da doença. Atualmente, sabe-se que as repercussões clíncas da AF envolvem interações complexas entre o eritrócito, endotélio e leucócitos: citocinas secretadas por células inflamatórias estão envolvidas nas crises e na manutenção de um status inflamatório sistêmico, o que sugere que as células T (auxiliares e citotóxicas) e NK têm papel fundalmental nos fenômenos clínicos da AF. Este estudo visa avaliar o perfil de linfócitos T e NK em portadores de AF e comparar com o perfil de indivíduos com traço falciforme e indivíduos sem hemoglobinopatias. MATERIAIS E MÉTODOS: Foi coletado sangue periférico de 17 indivíduos; 7 com hemoglobinopatia SS (grupo SS), 5 com traço falciforme (grupo AS) e 5 normais (grupo AA). Todos confirmados por eletroforese de hemoglobina. Amostras de pacientes hemotransfundidos 30 dias antes ou em uso de antiinflamatórios 2 dias antes da coleta foram excluídas. O isolamento dos linfócitos para análise foi realizado utilizando solução de Ficoll-Hypaque. A imunofenotipagem para determinação dos subtipos linfocitários foi realizada por citometria de fluxo, utilizando citômetro de oito cores e oito anticorpos da BD Biosciences. Os dados foram analisados com auxílio do software Flowjo e tabulados no SPSS IBM 22.0. Os resultados referentes às variáveis numéricas foram expressos através de medidas de tendência central: média e valores mínimos e máximos. RESULTADOS: Globalmente foi observada menor frequência de linfócitos T e maior frequência de células NK nos pacientes falcêmicos, com média de 31,2% de TCD4+ contra 43,47% do grupo AS e 45,37% do grupo AA. Para os linfócitos TCD8+, o grupo SS obteve média de 12,64% contra 17,1% do AS e 16,42% do AA. A variação de TCD4+ encontrada entre os pacientes AF e o grupo AA foi significativa (p=0,04). Tendência de menor frequência em pacientes do grupo SS se manteve para os linfócitos B. Foi observada maior frequência de células NK em pacientes portadores de anemia falciforme (média do grupo AA=8,83%; AS=11,2% e grupo SS=19,6%), respectivamente, com diferença significativa entre o grupo AA e o grupo SS (p=0,040). CONCLUSÃO: Portadores de AF apresentam tendência de menor frequência de linfócitos T, com diferença significativamente menor comparado ao portador de traço e o paciente portador de AF, para linfócitos T CD4+ verificou-se redução significativa deste subtipo celular entre grupo SS e AA. Neste estudo também foi evidenciado uma frequência crescente e progressiva de células NK entre os grupos AA, AS e SS. Acreditamos que mais estudos são necessários visando compreender o papel destas células na gênese da inflamação sistêmica da AF. Este entendimento possivelmente contribuirá para o desenvolvimento de novas estratégias terapêuticas. / Aracaju, SE

A persistência de células T de memória funcionais é importante para garantir uma imunidade protetora na infecção pelo Vírus da Imunodeficiência Humana (HIV). As células T de memória têm sido subdivididas em memória central (TCM), memória efetora (TEM) e memória efetora altamente diferenciada (TEMRA) com base na expressão de moléculas de superfície como CCR7 e CD45RA, e na capacidade de produzir citocinas e proliferar. Recentemente, identificamos 18 peptídeos derivados de seqüências do consenso B do HIV-1, ligadores de múltiplas moléculas HLA-DR e amplamente reconhecidos por linfócitos T de sangue periférico de pacientes infectados pelo HIV. Diante disso e considerando a importância das células T de memória na manutenção da resposta imune específica, nosso objetivo foi caracterizar fenotípica e funcionalmente as subpopulações de células T de memória de indivíduos infectados pelo HIV envolvidas no reconhecimento in vitro desses epitopos. Foram incluídos 14 indivíduos controles sadios e 61 pacientes HIV+ com contagem de linfócitos T CD4+ maior que 250 células/mm3. Os pacientes HIV+ foram divididos em seis diferentes grupos clínicos de acordo com o estágio da infecção, carga viral (CV) plasmática e uso de terapia anti-retroviral (ART): não progressores por longo tempo (LTNP), avirêmicos em uso de ART (AV-ART), virêmicos em uso de ART (VI-ART), virêmicos sem uso de ART (VI sem ART), virêmicos recéminfectados sem uso de ART (VI-RI) e controladores. Células mononucleares do sangue periférico dos indivíduos do estudo foram estimuladas com o conjunto de peptídeos do HIV-1 e com um conjunto de peptídeos do Citomegalovírus (CMV). A freqüência de células de memória produtoras de IFN- e IL-2 e a proliferação celular antígeno-específica foram detectadas por citometria de fluxo de multiparâmetros. Nossos resultados mostraram que o conjunto de peptídeos do HIV-1 foi capaz de ativar subpopulações funcionais de memória TCM, TEM e TEMRA secretoras de IFN- e IL-2 em 100% dos pacientes HIV+ dos diferentes grupos clínicos. O conjunto de peptídeos do HIV-1 também induziu proliferação das subpopulações de linfócitos T de memória. As freqüências de TEMRA CD4+IFN-+, TEMRA CD4+IFN-+ total, TCM CD8+IFN-+, TCM CD8+IFN-+ total, TEM CD8+IFN-+, TEM CD8+IFN-+ total e TEMRA CD8+IFN-+ correlacionaram-se negativamente com a carga viral do HIV em pacientes virêmicos. Esses dados sugerem que essas subpopulações de memória funcionais são importantes no controle da viremia. Comparando as respostas HIV e CMVespecíficas observamos freqüências mais elevadas de células T de memória produtoras de IL-2, IFN-/IL-2 e IFN- em respostas ao pool de peptídeos do HIV. Esses dados sugerem que esse conjunto de peptídeos derivados de seqüências do HIV-1 ativa respostas polifuncionais de subpopulações de linfócitos T de memória. Nossos resultados mostraram que o conjunto de peptídeos do HIV-1 foi capaz de estimular diferentes subpopulações distintas de linfócitos T de memória produtores de IFN-, IFN-,/IL-2 e IL-2 de indivíduos em diferentes estágios da infecção pelo HIV e sugerem o envolvimento de subpopulações de memória funcionais no controle da viremia. Estes achados fortalecem a possibilidade de uso desses peptídeos em uma formulação vacinal bem-sucedida em humanos / The persistence of functional memory T cell is important to ensure a protective immunity to Human Immunodeficiency Virus (HIV) infection. Memory T cells have been subdivided into central memory (TCM), effector memory (TEM) and highly differentiated effector memory (TEMRA) based on the expression of surface molecules such as CCR7 and CD45RA, and the ability to produce cytokines and proliferate. Recently, we identified 18 peptides derived from B consensus sequences of HIV-1 that bind to multiple HLA-DR molecules and are widely recognized by peripheral blood T lymphocytes from HIV-infected patients. Given this and considering the importance of memory T cells in the maintenance of specific immune response, our objective was to characterize phenotypic and functionally memory T cell subsets from HIV-infected individuals involved in the recognition of these epitopes in vitro. The study included 14 healthy control subjects and 61 HIV+ patients with CD4+ lymphocytes counts higher than 250 cells/mm3. The HIV+ patients were divided into six different clinical groups according to the stage of infection, plasma viral load (VL) and antiretroviral therapy use (ART): long-term non-progressors (LTNP), aviremic under ART (AV-ART), viremic under ART (VI-ART), viremic without using ART (VI without ART), recently infected viremic without using ART (VI-RI) and controllers. Peripheral blood mononuclear cells from study subjects were stimulated with HIV-1 peptide pool and with a cytomegalovirus (CMV) peptide pool. The frequencies of IFN- and IL-2 producing memory cells and antigenspecific cell proliferation were detected by multiparametric flow cytometry. Our results showed that the HIV-1 set of peptides was able to activate TCM, TEM and TEMRA functional memory subsets that secrete IFN- and IL-2 in 100% of the HIV patients from the different clinical groups. The HIV-1 set of peptides also induced memory T lymphocyte subsets proliferation. TEMRA CD4+IFN-+, total TEMRA CD4+IFN-+, TCM CD8+IFN-+, total TCM CD8+IFN-+, total TEM CD8+IFN-+, TEM CD8+IFN-+ and TEMRA CD8+IFN- + frequencies negatively correlated with HIV viral load in viremic patients. These data suggest that these functional memory subsets are important to control the viremia. When comparing the HIV and CMV-specific responses we observed higher frequencies of IL-2, IFN-/IL-2 and IFN- producing memory T cells in response to HIV peptide pool. These data suggest that this set of HIV sequence derived peptides activates polyfunctional response of memory T lymphocyte subsets. Our results showed that the HIV-1 peptide set was able to stimulate different IFN-, IFN-/IL-2 e IL-2 producing memory T lymphocytes from individuals in different stages of HIV infection and suggest the involvement of functional memory subsets in the control of viremia. These findings strengthen the possibility of using these peptides in a successful vaccine formulation in humans

Ce travail de thèse reposait sur l’hypothèse que la variabilité génétique des protéines « cibles » des médicaments immunosuppresseurs de la famille des inhibiteurs de la calcineurine (ICN ; ciclosporine et tacrolimus) pourrait expliquer une partie de la variabilité observée dans leur efficacité et toxicité. Une revue de la littérature nous a permis de lister un panel de variants génétiques au sein de la voie de la calcineurine, considérés comme étant de bons candidats pour des études en transplantation. Ces variants n’ont pas été associés au risque de rejet aigu ou d’infection grave dans une étude incluant 381 patients transplantés rénaux suivis durant un an après la transplantation. La variabilité pharmacodynamique des ICN a ensuite été explorée au travers des régulations épigénétiques. Une analyse de la méthylation de l’ADN après exposition médicamenteuse a été menée sur deux modèles. Premièrement, la lignée cellulaire JURKAT a été utilisée pour développer la méthode d’immunoprécipitation de l’ADN méthylé (MeDIP). Chez des souris traitées par ciclosporine et tacrolimus durant 3 mois, nous avons ensuite isolé les cellules cibles des médicaments, les lymphocytes T CD4 puis, après immunoprécipitation de l’ADN méthylé et analyse par séquençage pangénomique haut débit (MeDIP-seq, séquençeur Ion Proton), nous avons recherché les régions du génome présentant des différences de méthylation induites par le traitement. L’analyse différentielle bio-informatique a été menée à l’aide des outils SAMtools (Li et col., 2009), BEDtools (Quinlan and Hall, 2010), MACS2 (Zhang et col., 2008) et Diffbind (Stark and Brown, 2011 - Bioconductor). Sur l’ensemble du génome, nous n’avons identifié que 24 régions présentant un niveau de méthylation modifié par l’exposition au tacrolimus. Le promoteur du gène Calm2, codant pour l’isoforme 2 de la calmoduline, semble être davantage méthylé chez les souris traitées. Ces résultats préliminaires semblent prometteurs pour la découverte de biomarqueurs épigénétiques de la réponse thérapeutique aux immunosuppresseurs. / Inter-individual genetic variation might account for diverse efficacy and toxicity of calcineurin inhibitors (cyclosporin and tacrolimus). In particular, some variants located within genes coding for proteins of the calcineurin pathway can explain part of this variability. In this manuscript, a panel of candidate genes was selected based on bibliographic review and tested in a pharmacogenetics study encompassing 381 renal transplants followed for one year after surgery. None of these candidates was associated with the acute rejection or serious infection risks. Furthermore, the pharmacodynamic variability of these drugs was also investigated, exploring the use of epigenomics profiling as proximal readout of the calcineurin inhibition treatment. In particular, we investigated the impact of drug exposure on DNA methylation in two experimental models. Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq, Ion Proton technology) was deployed in JURKAT cell line, used as in vitro model, and in CD4 T lymphocytes isolated from mice treated with either cyclosporin or tacrolimus for three months. After sequencing, the differentiated methylated regions caused by drug exposure were analyzed. Bioinformatics analyses were performed using SAMtools (Li et al., 2009), BEDtools (Quinlan and Hall, 2010), MACS2 (Zhang et al., 2008) and Diffbind (Stark and Brown, 2011 - Bioconductor). Overall, the genome-wide analysis revealed only 24 regions with a differentiated enrichment in DNA methylation after three month-tacrolimus treatment, indicating a targeted effect of these treatments on a subset of key genes. Of note, CALM2 promoter, coding for the calmodulin isoform 2 protein, showed significant hypermethylation in tacrolimus-treated mice. These preliminary results corroborate the interest in using DNA methylation as promising approach to identify candidate biomarkers for therapeutic drug monitoring in calcineurin inhibitor treatments.

Les lymphocytes T Vγ9Vδ2 sont impliqués dans la réponse immunitaire contre de nombreux pathogènes et contre les cellules tumorales. Ils reconnaissent des antigènes solubles, non peptidiques, ayant une faible masse moléculaire, qui sont appelés phosphoantigènes. Dans ce contexte, l’isopentenyl pyrophosphate (IPP) et un métabolite de l’ATP (ApppI) sont des phosphoantigènes qui s’accumulent dans les cellules suite à l’inhibition par les amino-bisphosphonates de l’activité d’une enzyme clé de la voie du mévalonate, la farnesyl pyrophosphate synthase (FPPS). Les bisphosphonates sont utilisés en clinique dans le traitement et la prévention des complications liées aux ostéolyses malignes. Les études pré-cliniques montrent que les bisphosphonates (et en particulier les amino-bisphosphonates) ont également une activité anti-tumorale qui peut être directe et/ou indirecte. Ils peuvent bloquer différentes fonctions des cellules tumorales (adhésion, invasion, prolifération) en inhibant l’activité de la FPPS. Ils agissent aussi sur les cellules endothéliales et inhibent l’angiogenèse tumorale. Les amino-bisphosphonates activent aussi l’activité cytotoxique des lymphocytes T Vγ9Vδ2 vis-à-vis des cellules tumorales. Les mécanismes moléculaires sous-jacents responsables de cette activation sont par contre méconnus. Les travaux réalisés dans cette thèse montrent tout d’abord que les aminobisphosphonates (zolédronate et risédronate) induisent l’accumulation d’IPP et d’ApppI dans différentes lignées humaines de cancer du sein et qu’il existe une corrélation entre le niveau de production d’IPP/ApppI et la capacité des lymphocytes T Vγ9Vδ2 à détruire ces cellules tumorales in vitro. Nous avons ensuite montré que le traitement de souris immunodéficientes NOD-SCID avec des aminobisphosphonates stimule la différenciation des lymphocytes T Vγ9Vδ2 humains à partir des cellules du sang périphérique lorsque celles-ci sont injectées par voie intrapéritonéale aux animaux. Par ailleurs, ces lymphocytes T vont ensuite venir infiltrer des tumeurs mammaires sous-cutanées qui ont été préalablement greffées chez les animaux traités. Cette infiltration lymphocytaire survient uniquement lorsque les tumeurs produisent de l’IPP/ApppI. Il en résulte alors une régression des tumeurs chez les souris NOD-SCID. L’infiltration des tumeurs par les lymphocytes T Vγ9Vδ2 humains s’explique par le fait que l’IPP et l’ApppI stimulent la migration des lymphocytes T. Les lymphocytes T Vγ9Vδ2 interagissent alors avec les cellules tumorales par le biais de mécanismes faisant intervenir ICAM-1, puis les lymphocytes sécrètent des facteurs (interféron γ, perforine) qui sont cytotoxiques pour les cellules tumorales. L’ensemble de nos travaux montre donc que les amino-bisphosphonates pourraient être utilisés en immunothérapie dans le traitement des cancers du sein. / Vγδ9Vδ2 T lymphocytes are involved in the immune response against several pathogens and tumoral cells. They recognize non-peptidic, low molecular weight soluble antigens, so called phosphoantigens. In this context, isopentenyl pyrophosphate (IPP) and a metabolite of ATP (ApppI) are phosphoantigens which accumulate in many cells following the inhibition by amino-bisphosphonates of farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate pathway. Bisphosphonates are commonly used in the clinic for the treatment and prevention of complications associated with malignant osteolysis. Preclinical studies demonstrate that bisphosphonates (in particular amino-bisphosphonates) also have direct and indirect anti-tumor activity. They are able to block different functions in tumor cells, such as adhesion, invasion and proliferation, by inhibiting FPPS activity. Bisphosphonates also act on endothelial cells and inhibit tumor angiogenesis. Amino-bisphosphonates also activate the cytotoxic activity of Vγδ9Vδ2 T lymphocytes against tumor cells. However, the underlying molecular mechanisms responsible for this activation are still unknown. In the present thesis, we first demonstrate that amino-bisphosphonates (e.g. zoledronate and risedronate) induce the accumulation of IPP and ApppI in different human breast cancer cell lines and that a correlation exists between the levels of IPP/ApppI production and the capacity of Vγδ9Vδ2 T lymphocytes to destroy tumor cells in vitro. We then demonstrate that the treatment of immunodeficient NOD-SCID mice with amino-bisphosphonates stimulates the differentiation of Vγδ9Vδ2 T lymphocytes from human peripheral blood mononuclear cells injected intraperitoneally in animals. Following their expansion, the T lymphocytes are then able to infiltrate subcutaneous mammary tumors, which were grafted in treated animals. This infiltration of γδ T lymphocytes arises only when the tumors produce IPP/ApppI, resulting in the regression of tumor growth in NOD-SCID mice. The infiltration of tumors by human Vγδ9Vδ2 T lymphocytes is explained by the capacity of IPP and ApppI to stimulate the migration of γδ T lymphocytes. Vγδ9Vδ2 T lymphocytes then interact with tumor cells through a mechanism involving ICAM-I and several secretion factors (i.e. interferon γ, perforine) which are cytotoxic for tumor cells. Thus, our work demonstrates that amino-bisphosphonates could be useful for breast cancer immunotherapy.

Skeletal muscle is prone to damage from a range of stimuli. The muscle repair process that ensues is complex, involving several phases and requiring the participation of many different cell types. Among the cells involved are various immune cells including neutrophils, macrophages, monocytes, and eosinophils. More recently, T cells were added to this list of immune cells known to participate in effective muscle repair from traumatic injuries in mice. We recently published data showing that T cells also accumulate in human muscle following contraction-induced damage. These data suggested that T cells might be involved an adaptation known as the repeated bout effect that renders muscle protected from future damage after an initial exposure. This document contains research on the role of the immune system, particularly T cells, in the "repeated bout effect."

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.

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.

No description available.

In the United States, individuals of Hispanic ethnicity receive disproportionately lower-quality healthcare. These healthcare disparities exacerbate unequal access to quality healthcare services, including disparities in cardiovascular disease (CVD) and human immunodeficiency virus (HIV) care. Research on the role of ethnicity on the CVD outcomes of people living with HIV (PLWH) has been limited. We hypothesize that immunological (CD4+ cell count) and virological (HIV viral load) control may play a role in the development of acute myocardial infarction (AMI) among PLWH, and that Hispanic ethnicity may worsen these outcomes. To verify our hypotheses, we conducted a retrospective cross-sectional study to investigate the strength and direction of association between CD4+ cell count (immunological cohort, n=513) and HIV viral load (virological cohort, n=261) on AMI among respondents of the All of Us Research Program. Hispanic and non-Hispanic respondents for both cohorts were comparable in terms of demographic characteristics, except for a significantly different distribution by race. While we identified increased proportion of non-Hispanic individuals with AMI in the immunologic (6.0% vs. 1.0%; P=0.04) and virologic (5.8% vs. 0%; P=0.007) cohorts, we were not able to identify CD4+ cell count or viral load as significant predictors significantly increasing the likelihood of AMI. Potential explanations discussed include self-selection bias resulting in incomplete laboratory data and an underpowered sample size. While the sample in this study did not support an increased likelihood of AMI by ethnicity, the results should be interpreted carefully in light of the limitations and the established pathophysiological and epidemiological associations posited, underscoring the importance of future research efforts that better represent ethnic minorities and the associations between HIV infection and CVD.

Abstract : Long-term survival of T lymphocytes in a quiescent state is essential to maintain their cell numbers in secondary lymphoid organs. Interaction of the T cell antigen receptor (TCR) with self-peptide/MHC synergizes with IL-7-induced anti-apoptotic signals to promote T cell survival. These extrinsic stimuli are also implicated in T cell metabolism and survival by regulating several signaling pathways including the phosphatidyl-inositol-3 kinase (PI3K)/Akt pathway. In mice and in rats, loss of functional GTPase of the immune associated nucleotide binding protein 5 (GIMAP5) causes peripheral T lymphopenia due to spontaneous death of T cells. The underlying mechanisms responsible for the pro-survival function of GIMAP5 in T lymphocytes remain largely unknown. Previous work from my laboratory has shown that T cells from GIMAP5-deficient rats show reduced influx of calcium (Ca[superscript 2+]) from the extracellular milieu following stimulation of the TCR complex. In this thesis, I characterized the mechanism by which GIMAP5 regulates Ca[superscript 2+] homeostasis, and elucidated the signaling pathways modulated by GIMAP5 to facilitate the survival of T cells. Firstly, I investigated if GIMAP5 prevents apoptotic death of T lymphocytes by affecting the Ca[superscript 2+] buffering capacity of mitochondria, which is required for sustained Ca[superscript 2+] influx via the plasma membrane channels. I observed that mitochondrial Ca[superscript 2+] accumulation following capacitative Ca[superscript 2+] entry is defective in T cells from Gimap5 deficient rats. Disruption of microtubules, but not the actin cytoskeleton, abrogated Ca[superscript 2+] sequestration by mitochondria in T cells from control but not Gimap5 deficient mice. Similarly, mice lacking functional GIMAP5 displayed defective T cell development and Ca[superscript 2+] influx. Furthermore, I observed that the proximal signaling events following TCR stimulation was reduced and was accompanied by defective proliferation in T cells from Gimap5 deficient mice. Additionally, IL-7-induced STAT5 phosphorylation was decreased in CD4[superscript +] T cells from Gimap5 deficient mice. I also showed that loss of functional Gimap5 results in increased basal activation of mammalian target of rapamycin (mTOR), independent of protein phosphatase 2A (PP2A) or AMP-activated protein kinase (AMPK). Instead, the constitutive activation the PI3K pathway contributed to the spontaneous high mTOR activation. Collectively, my observations suggest that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to the regulation of diverse signaling pathways in a context dependent manner. GIMAP5 also facilitates microtubule-dependent mitochondrial buffering of Ca[superscript 2+] following capacitative entry. GIMAP5 is required to integrate the survival signals generated following activation through TCR and IL-7R. / Résumé : La survie à long terme des lymphocytes T en état de repos est essentielle pour maintenir leurs nombres dans les organes lymphoïdes secondaires. Le récepteur antigénique des cellules T (TCR) en contact avec les peptides du soi / CMH et en synergie avec l'IL-7 induit des signaux anti-apoptotiques pour favoriser la survie des cellules T. Ces stimuli extrinsèques sont également impliqués dans le métabolisme et la survie des cellules T grâce à la régulation de plusieurs voies de signalisation dont la voie phosphatidyl-inositol-3 kinase (PI3K) /AKT. Chez la souris et chez le rat, la perte de l’activité de GIMAP5 (GTPase of Immune Associated Nucleotide Binding Protein 5), provoque une lymphopénie T périphérique en raison de la mort spontanée des cellules T. Le mécanisme sous-jacent responsable de la fonction de survie de GIMAP5 dans les lymphocytes T reste largement inconnu. Nous avons observé que les cellules de rats déficients en GIMAP5, après stimulation par complexe TCR, montrent un afflux de calcium (Ca[indice supérieur 2+]) réduit provenant du milieu extracellulaire. Dans cette thèse, J’ai caractérisé le mécanisme d’action de GIMAP5 dans la régulation de l'homéostasie du Ca[indice supérieur 2+], ainsi que les voies de signalisation modulées par GIMAP5 pour faciliter la survie des cellules T. Tout d'abord, j’ai étudié si GIMAP5 empêche l’apoptose des lymphocytes T en affectant la capacité des mitochondries à réguler la concentration du Ca[indice supérieur 2+], ce qui est nécessaire pour soutenir l’influx de Ca[indice supérieur 2+]. J’ai trouvé que l’accumulation du Ca[indice supérieur 2+] mitochondrial après l’entrée capacitive de Ca[indice supérieur 2+] est défectueuse dans les cellules T de rat déficientes en Gimap5. La disruption des microtubules, mais pas du cytosquelette d'actine, abroge la séquestration du Ca[indice supérieur 2+] mitochondrial dans les cellules T primaires de rat, mais pas dans les cellules T déficientes en Gimap5. J’ai observé que les cellules T provenant de souris déficientes en Gimap5 démontrent une diminution de l’entrée de Ca[indice supérieur 2+]. De plus, la prolifération des cellules T déficientes en Gimap5 est diminuée suite à la stimulation du TCR. En outre, la phosphorylation de STAT5 induit par l'IL-7 est diminuée dans les cellules T CD4[indice supérieur +] de souris déficientes en Gimap5. Également, la perte de Gimap5 aboutit à une activation accrue de la cible mammalienne de la rapamycine (mTOR), indépendamment de la protéine phosphatase 2A (PP2A) ou de la protéine kinase activée par l'AMP (AMPK). Au lieu de cela, l'activation constitutive de la voie PI3K contribue à une forte activation spontanée de mTOR. Collectivement, la fonction de survie de GIMAP5 dans les lymphocytes T peut être liée à la régulation de différentes voies de signalisation. GIMAP5 facilite la fonction, microtubule dépendant, des mitochondries dans leurs actions de régulation du Ca[indice supérieur 2+] après l’entrée capacitive de Ca[indice supérieur 2+]. GIMAP5 est nécessaire pour intégrer les signaux de survie produits suite à l'activation du TCR et de l’IL-7R, qui pourrait être associée à la régulation de l'activité PI3K / AKT / mTOR.