INTRODUÇÃO: A atuação do sistema nervoso parassimpático em células imunes é conhecida como \"Via Anti-inflamatória Colinérgica\". Trabalhos prévios demonstraram que a estimulação vagal reduz a inflamação e melhora a sobrevida em modelos experimentais com sepse. Neste estudo avaliamos se o uso do anticolinesterásico piridostigmina: altera o número de linfócitos T (CD4+ e CD8+) convencionais (CD25+Foxp3-) e reguladores (CD25+Foxp3+) no sangue periférico, no baço e no miocárdio; modifica a concentração de citocinas (interleucina 1, interleucina 6, TNFalfa) no miocárdio; e influencia a função ventricular após infarto agudo do miocárdio experimental (IAM) em ratos. MÉTODOS: Utilizamos ratos machos adultos da linhagem Wistar, com peso variando entre 200 e 250 g, divididos em 3 grupos de 20 animais cada: grupo controle (GC), grupo infartado sem tratamento (IC) e grupo infartado tratado com piridostigmina (IP). O infarto agudo do miocárdio (IAM) foi obtido com a técnica da ligadura da artéria coronária esquerda, e o grupo IP recebeu piridostigmina na dose de 40mg/kg/dia na água de beber, iniciada 4 dias antes do IAM. Todos os animais foram submetidos à canulação da artéria femoral no dia seguinte ao IAM para registro das curvas de pressão arterial, e posterior análise dos componentes da variabilidade da freqüência cardíaca (VFC), domínio do tempo (SDNN e RMSSD) e da freqüência (componentes LF e HF); o estudo ecocardiográfico foi realizado no segundo dia pós IAM. No terceiro dia pós IAM, os ratos foram divididos em subgrupos de 10 animais, e sacrificados de forma específica para coleta de materiais: 500 ul de sangue periférico e baço fresco para realização da técnica de citometria de fluxo; ventrículo esquerdo para dosagem de citocinas pela técnica de ELISA; e ventrículo esquerdo para realização de imunohistoquímica. Foram usadas as técnicas padronizadas e de uso corrente nos laboratórios. Os resultados foram avaliados por análise de variância (ANOVA) multifatorial, usando o programa GraphPad Prism com teste post hoc de Tukey. RESULTADOS: O grupo IC comparado ao grupo controle apresentou queda significativa da pressão arterial e aumento da freqüência cardíaca. O grupo IP, comparado ao grupo IC, apresentou maior atividade vagal, caracterizada pela significante redução da FC e aumento da VFC (SDNN, 9,2±1,5 vs 5,2±0,5 p < 0,05). Os parâmetros ecocardiográficos avaliados evidenciaram presença de área hipo/acinética e redução da fração de ejeção do ventrículo esquerdo nos grupos infartados, de igual magnitude. Com relação ao número de linfócitos T, verificamos que o grupo IC, comparado ao grupo controle, apresentou número significativamente menor de linfócitos reguladores (CD25+Foxp3+) no sangue periférico (CD4+: 63,5 ±1,4 vs 70,6 ±3,2%, e CD8+: 68,3 ±1,9 vs 76,1 ± 2,8%). O grupo IP, comparado ao grupo IC, apresentou significativa redução do número de linfócitos T convencionais no sangue periférico (respectivamente, CD4+: 1,5 ±0,2 vs 2,2 ± 0,2 %; CD8+: 1,1 ± 0,1 vs 1,8 ± 0,9%), e no baço houve redução somente do tipo CD4+ (respectivamente, 1,4 ± 0,2 vs 2,2 ± 0,2%), com aumento do tipo CD8+ (respectivamente, 1,2 ± 0,1 vs 0,7 ± 0,1 %). O grupo IP também apresentou significativo aumento de linfócitos reguladores (CD25+Foxp3+) no sangue periférico (respectivamente, CD4+: 76,5 ± 2,9 vs 63,5 ± 1,4 %; CD8+: 75,1 ± 1,0 vs 68,3 ± 1,9 %), e não apresentou diferenças significativas no número dessas células no baço. O grupo IC comparado ao grupo controle apresentou significativa marcação de anticorpos para CD4 e CD8 nas áreas infartada e peri-infarto por meio da análise de imunohistoquímica. O grupo IP comparado ao grupo IC, apresentou significativo aumento de CD4+ (respectivamente, 20,9 ± 6,5 vs 12,2 ± 2,5, p < 0,05) e de CD8+ (respectivamente, 17,9 ± 2,8 vs 5,8 ± 1,1%, p < 0,05) na área infartada; observamos redução significativa na marcação de CD4+ (respectivamente, 6,0 ±1,2 vs 12,5 ±4,8) na área peri-infarto, sem alterações significativas na marcação de CD8+. CONCLUSÃO: O tratamento com piridostigmina em ratos com IAM está associado a aumento da atividade vagal, aumento do número de linfócitos reguladores (CD25+Foxp3+) no sangue periférico e maior mobilização de células inflamatórias (CD4+ e CD8+) para a área infartada no miocárdio, com redução de CD4+ na área peri-infarto, no entanto sem mudança de CD8+ nesta região. A mudança do perfil inflamatório decorrente do aumento da atividade vagal na fase aguda do IAM, pode ser um possível mecanismo para explicar os benefícios detectados no remodelamento cardíaco após o IAM, em especial, na redução da área de lesão e na melhora da função ventricular, com uso de anticolinesterásicos / INTRODUTION: The role of the parasympathetic nervous system in immune cells is known as \"Cholinergic anti-inflammatory pathway\". In previous work has demonstrated that vagal stimulation reduces inflammation and improves survival in experimental sepsis models. The aim of the present study evalued the use of anticholinesterase pyridostigmine: change the number of T lymphocytes (CD4+ and CD8+) conventional (CD25+Foxp3-) and regulatory (CD25+Foxp3+) in peripheral blood, spleen, and myocardium: modifies the concentration of cytokines (interleukin-1, interleukin-6, TNFalfa) in the myocardium, and influences ventricular function after experimental myocardial infarction (MI) in rats. METHODS: Adult male rats of Wistar strain, weighing between 200 and 250 g were divided into 3 groups of 20 animals each: control group (GC); untreated group without treatment (IC) and infarcted group treated with pyridostigmine (IP). Acute myocardial infarction (AMI) was obtained with the technique of ligation of the left coronary artery, and the IP group received pyridostigmine dose of 40 mg/Kg/day in drinking water starting 4 days before the AMI. All animals underwent cannulation of the femoral artery on the day following AMI to record the blood pressure curves, and subsequent analysis of the components of heart rate variability (HRV), the time domain (SDNN and RMSSD) and frequency (components LF and HF), the echocardiografic study was performed on the second day after AMI. On the third day post-MI, mice were divided into subgroups of 10 animals, and were sacrificed in order to collet specific materials: 500 ul of fresh peripheral blood and spleen technique for performing flow cytometry left ventricle for measurement of cytokine ELISA, and the left ventricle to perform immunohistochemistry. Techniques used were standardized and commonly used in laboraties. The results were evaluated by analysis of variance (ANOVA) multifactorial, using the GraphPad Prism with Tukey post hoc test RESULTS: The HF group compared to the control group showed a significant drop in blood pressure and increased heart rate. The IP group compared to the IC group showed higher vagal activity, characterized by a significant reduction in HR and increase HVR (SDNN, 9.2 ± 1.5 vs 5.2 ± 0.5, p < 0.05). The echocardiography parameters evaluated showed presence of area hypo/acinetic and reduced ejection fraction of the left ventricle in infracted groups of equal magnitude. Regarding the number of T lymphocytes, we found that the IC group compared with the control group showed significantly fewer lymphocytes regulators (CD25+Foxp3+) in peripheral blood (CD4+:63.5 ± 1.4 vs 70.6 ± 3.2% and CD8+ cells: 68.3 ± 1.9 vs 76.1 ± 2.8%). The IP group compared to the IC group showed a significant reduction in the number of conventional T lymphocytes in peripheral blood (CD4+:1.5 ± 0.2 vs 2.2 ± 0.2%; CD8+: 1.1 ± 0.1 vs 1.8 ± 0.9%) and was reduced only in the spleen of the type CD4+(1.4 ± 0.2 vs 2.2 ± 0,2%) with increased CD8+(1.2 ± 0.1 vs 0.7 ± 0.1%). The IP group also showed a significant increase of lymphocytes regulators (CD25+Foxp3+) in peripheral blood (CD4+: 76.5 ± 2.9 vs 63.5 ± 1.4%; CD8+:75.1 ± 1.0 vs 68.3 ± 1.9%), and no significant differences in the number of these cells in the spleen. The IC group compared to the control group showed significant labeling antibodies to CD4 and CD8 areas infarcted and peri-infarction by immunohistochemical analysis. The IP group compared to the IC group showed a significant increase in CD4 (20.9 ± 6.5 vs 12.2 ± 2.5, p < 0.05) and CD8 (17.9 ± 2.8 vs 5.8 ± 1.1%, p < 0.05) in the infarcted area, and we observed a significant reduction in the labeling of CD4 (6.0 ± 1.2 vs 12.5± 4.8) in the peri-infraction without significant changes in the marking of CD8. CONCLUSION: The treatment with pyridostigmine in rats with acute myocardial infarction is associated with increased vagal activity, increased number of regulatory lymphocytes (CD25+Foxp3+) in peripheral blood and increased mobilization of inflammatory cells (CD4 and CD8) to the infarcted myocardium, with reduction of these cells in the peri-infarction. The change of the inflammatory profile due to increased vagal activity may be a possible mechanism to explain the benefits in the evolution of myocardial infarction, especially in the improvement of cardiac remodeling and maintenance of ventricular function with anticholinesterase drugs

O desenvolvimento gradual e recente de uma epidemia mundial de obesidade alavancou sobremaneira o estudo dessa condição e de suas comorbidades metabólicas. No âmbito fisiopatológico, múltiplos estudos demonstraram a expressão aumentada de mediadores inflamatórios no tecido adiposo de animais e humanos obesos, o acúmulo local de macrófagos, e um papel central da inflamação no desequilíbrio da homeostase metabólica local e sistêmica na obesidade. A definição de um papel ativo dos macrófagos, e portanto da imunidade inata, na rede inflamatória do tecido adiposo, evocou a hipótese de que, similarmente a outras condições inflamatórias crônicas como a aterosclerose, a obesidade também contaria com a importante participação de elementos da imunidade adaptativa, como as células T e suas citocinas, em sua fisiopatologia. Com base nessas considerações, os objetivos principais desse estudo foram: 1) avaliar a presença das células T e o papel do interferon-gama (IFNy), clássica citocina T-helper 1 (ou Th1), na inflamação do tecido adiposo; e 2) estudar mecanismos de acúmulo das células T no tecido adiposo na obesidade, particularmente a participação do receptor CXCR3 nesse processo. Experimentos de citometria de fluxo mostraram que o tecido adiposo visceral de camundongos C57BL/6 obesos após consumo de dieta rica em gorduras apresentou maior número de macrófagos e também de células T, CD4+ e CD8+, em comparação a controles que receberam dieta pobre em gorduras. A expressão de I-Ab, marcador do complexo de histocompatibilidade principal classe II (MHC II) murino, também foi maior no tecido adiposo dos animais obesos, sugerindo a presença local da atividade de apresentação de antígeno com consequente ativação das células T. Quando estimuladas in vitro, células T derivadas do tecido adiposo de camundongos obesos produziram mais IFNy do que aquelas isoladas de controles, novamente sugerindo a ativação dessas células em um contexto de obesidade. Na análise das possíveis funções do IFNy no tecido adiposo, a estimulação da linhagem de células 3T3-L1 diferenciadas em adipócitos com IFNy recombinante resultou na produção aumentada de quimiocinas de macrófagos, como a proteína quimiotática de monócito (MCP-1), e de quimiocinas de células T, como a proteína 10 induzida por IFNy (IP-10) e monocina induzida por IFNy (MIG). A estimulação de adipócitos com o sobrenadante de células Th1 cultivadas in vitro, com abundante concentração de IFNy, também levou à produção aumentada de IP-10. Em análise mais ampla, através de microarray, dos possíveis efeitos do IFNy na expressão gênica de adipócitos, o tratamento dessas células com 100 U/ml de IFNy resultou na expressão aumentada de diversas quimiocinas e seus receptores em comparação ao grupo tratado com placebo. Similarmente à estimulação de células isoladas com IFNy, a incubação de tecido adiposo ex vivo de camundongos com essa citocina também resultou em secreção aumentada de IP-10, MIG e fator de necrose tumoral alfa (TNFy). A investigação do papel do IFNy na inflamação do tecido adiposo in vivo envolveu camundongos com deficiência de IFNy e controles, ambos os grupos submetidos a dieta rica em gorduras (obesos) ou pobre em gorduras (não obesos). Camundongos obesos deficientes em IFNy apresentaram expressão reduzida de mRNA de genes inflamatórios como TNFalfa e MCP-1 no tecido adiposo; acúmulo local reduzido de macrófagos; e melhor tolerância à glicose em comparação aos controles sob mesma dieta. Animais com deficiência de apolipoproteína E (ApoE) e também do receptor de IFNy também apresentaram em seu tecido adiposo a expressão reduzida de mRNA de genes inflamatórios, particularmente relacionados às células T, como IP-10, MIG, e o receptor CXCR3, em comparação aos controles com deficiência única de ApoE. Resultados in vitro e in vivo sugerem conjuntamente um importante papel do IFNy, e portanto, das células T e da imunidade adaptativa, na rede inflamatória do tecido adiposo na obesidade, com consequente impacto metabólico sistêmico. A presença de células T ativadas no tecido adiposo e seu acúmulo diferencial na obesidade motivaram também a pesquisa de potenciais mecanismos quimiotáticos reguladores desse processo. CXCR3, receptor das quimiocinas de células T, IP-10, MIG e quimiocina alfa de células T IFNy-induzida (I-TAC), é expresso preferencialmente em células T ativadas, e detém papel central na migração dessas células em outras condições inflamatórias crônicas, como a aterosclerose. Em camundongos com deficiência de CXCR3 e que receberam dieta rica em gorduras por 8 ou 16 semanas, o tecido adiposo apresentou significativamente menos células T, incluindo as células CD4+ e CD8+, em comparação a controles submetidos a mesma dieta. Os números similares de células T e outras populações de leucócitos no baço e sangue periférico dos animais deficientes em CXCR3 e controles fortalecem o conceito de um efeito do CXCR3 sobre o acúmulo de células T no tecido adiposo, independentemente do número de células circulantes e periféricas. Os camundongos deficientes em CXCR3 apresentaram também maior tolerância à glicose e expressão reduzida de mRNA de mediadores inflamatórios em seu tecido adiposo em comparação aos controles após 8 semanas de dieta rica em gorduras. No entanto, a diferença na tolerância à glicose entre os dois grupos tornou-se não significativa após 16 semanas de dieta gordurosa, coincidindo com redução substancial na expressão de mRNA de mediadores anti-inflamatórios (como interleucina-10 [IL-10] e Arginase 1), e número reduzido de células T regulatórias no tecido adiposo de camundongo s deficientes em CXCR3 em relação a controles. Esses resultados sugerem que o CXCR3 é capaz de regular o acúmulo de células T de diferentes subtipos, com perfil proinflamatório ou anti-inflamatório. Em conclusão, nossos resultados revelam um importante papel da citocina Th1 IFNy na rede inflamatória do tecido adiposo na obesidade em camundongos, sugerindo a participação fundamental das células T e portanto, da imunidade adaptativa nesse cenário. Além disso, o receptor CXCR3 contribui significativamente para o acúmulo das células T, incluindo as células T regulatórias, no tecido adiposo desses animais / The gradual and recent development of a worldwide epidemic of obesity greatly leveraged the study of this condition and its metabolic comorbidities. In the pathophysiologic context, multiple studies have demonstrated increased expression of inflammatory mediators in adipose tissue of obese animals and humans, the local macrophage accumulation, and a central role of inflammation in the imbalance of local or systemic metabolic homeostasis in obesity. The concept of an active role of macrophages and thus of innate immunity in the inflammatory network of adipose tissue, suggested the hypothesis that, similar to other chronic inflammatory conditions such as atherosclerosis, obesity also count on the participation of important elements of adaptive immunity such as T cells and their cytokines in its pathophysiology. Based on these considerations, the main objectives of this study were: 1) to evaluate the presence of T cells and the role of interferon-gamma (IFNy), classic T-helper 1 (Th1) cytokine, in adipose tissue inflammation, and 2) to study mechanisms of T cell accumulation in adipose tissue in the context of obesity, particularly the involvement of CXCR3 receptor in this process. Flow cytometry experiments showed that the visceral fat tissue of C57BL/6 obese mice fed a high fat diet showed a greater number of macrophages and also T cells, including CD4+ and CD8+ cells, compared to controls fed a low-fat diet. The expression of I-Ab, murine marker of class II major histocompatibility complex (MHC II), was also higher in adipose tissue of obese animals, suggesting the presence of local antigen presentation and consequent T cell activation. When stimulated in vitro, T cells derived from adipose tissue of obese mice produced more IFNy than those isolated from controls, again suggesting the activation of these cells in the context of obesity. In the analysis of possible functions of IFNy in adipose tissue, stimulation of 3T3 -L1 cells differentiated into adipocytes with recombinant IFNy resulted in enhanced production of macrophage chemokines, such as monocyte chemotactic protein-1 (MCP-1) and T-cell chemokines, such as interferon gamma-induced protein 10 (IP-10) and monokine induced by gamma interferon (MIG). The stimulation of adipocytes with the supernatant of in vitro cultured Th1 cells, with abundant levels of IFNy, has also led to increased IP-10 production. In a broader analysis, by microarray, of the possible effects of IFNy on adipocyte gene expression, treatment of these cells with 100 U/ml of IFNy resulted in increased expression of chemokines and their receptors in comparison to the placebo group. Similarly to the stimulation of isolated cells with IFNy, incubation of ex vivo adipose tissue with this cytokine also resulted in increased IP-10, MIG and tumor necrosis factor alpha (TNFalpha) secretion

Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

Type 1 diabetes mellitus (T1D) is an organ-specific autoimmune disease characterised by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. It is a long-term process initiated months or even years prior to the clinical onset. The main role in the pathogenesis is played by T lymphocytes but other cell types are involved as well. The presence of autoantibodies in the circulation is typical even before the disease onset. Nowadays, intensive research is focused on finding individuals at risk and developing an effective prevention. During my postgraduate studies I was involved mainly in the research of T1D prediction and prevention. We investigated the relationship of established autoimmune markers - autoantibodies - and the cellular reactivity to GAD65 and IA2 autoantigens. We discovered that the reaction to autoantigens is very individual and it is influenced by the patient's autoantibody profile. These results could be relevant in planning antigen-specific immunointervention studies and improving their efficacy. We also made an attempt to improve specificity and sensitivity of a beta cell destruction marker (specifically demethylated DNA), which would enable better understanding of the beta cell decline and identification of individuals at risk of T1D development. In...

Myasthenia Gravis (MG) is an autoimmune disease affecting neuromuscular transmission, in which the thymus is considered pathogenic organ. Earlier ideas suggesting that MG is only the receptors disease have been proven wrong. There are immunopathological changes in both target structures [specific receptors for acetylcholine (AChR] muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein 4 (Lrp4)], the thymus, as well as in peripheral lymphoid organs. Initial findings of the humoral immunity defect with the decisive role of the pathologic autoantibodies, were corrected with findings of the immune dysregulation at the level of T lymphocytes. According to today's knowledge, the development and maintenance of MG involves almost all cell types of immune function in the autoimmune inflammation: helper CD4+ T lymphocytes, cytotoxic CD8+ T lymphocytes, regulatory CD4+CD25+ T lymphocytes, Th17 lymphocytes, B lymphocytes and plasma cells. Thymus plays a dominant immunopathogenetic role in younger patients with MG, while extrathymic mechanisms are applied in older patients. As a result of that, the thymectomy (TE) is generally accepted as part of treatment for MG. However, there is still no data verified by a prospective controlled study, which would demonstrate a useful result of this treatment...

T lymphocytes are distinguished by the expression of αβ TCR or γδ TCR on their cell surface. The kinetic differences in the effector functions classifies γδ T cells as innate-like lymphocytes and αβ T cells as adaptive lymphocytes. Although distinct, αβ and γδ T cell lineages produce a common array of cytokines to mount an effective immune response against a pathogen. The production of cytokine IL-17 is a shared characteristic between the γδ T (Tγδ17) cells and the CD4 T (Th17) cells. γδ T cells develop into Tγδ17 cells in the thymus whereas CD4 T cells differentiate into Th17 cells in response to antigens in the peripheral lymphoid tissues. γδ T cells exported from the thymus, as pre-made effectors, are the early IL-17 producers compared with the late IL-17 producing Th17 cells. In this thesis we describe how TGFβ-SMAD2 dependent pathway selectively regulates Th17 cell differentiation but not Tγδ17 cells generation. We further illustrate the requirement of WNT-HMG box transcription factor (TF) signaling for the thymic programming of Tγδ17 cells. Cytokine TGFβ in co-operation with IL-6 induces the differentiation of Th17 cells. Conversely, TGFβ signaling also regulates the differentiation and maintenance of CD4+FOXP3+ regulatory T cells. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. While mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-/- T cells are impaired in their response to TGFβ in vitro and in vivo and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is essential for TGFβ signaling in CD4+ T effector cell differentiation and that it possesses functional capabilities distinct from SMAD3. Although SMAD2 is essential for the differentiation of Th17 cells, TGFβ signaling via SMAD2 is not required for the thymic programming of innate Tγδ17 cells. Among different γδ T cells, Vγ2+ (V2) γδ T cells are the major IL-17 producing subsets. We demonstrate that Sry-high mobility group (HMG) box TFs regulate the development of V2 Tγδ17 cells. We show that the HMG box TF, SOX13 functions in a positive loop for the intrathymic generation of V2 Tγδ17 cells. SOX13 regulates the programming of Tγδ17 cells by controlling the expression of B-lymphoid kinase (BLK) in developing immature V2 γδ T cells. BLK is an Src-family kinase expressed by all Tγδ17 cells. Furthermore, we show another HMG box TF, TCF1, the nuclear effector of canonical WNT signaling, is the primary negative regulator of IL-17 production by all γδ T cells. We propose that the antagonism of SOX13 and TCF1 determines the generation of IL-17 producing γδ T cells. We also show that extrinsic cues from αβ T cells do not affect the generation of IL-17 producing γδ T cells. Using OP9-DL1 culture system, we demonstrate that the progenitors of V2 Tγδ17 cells are the c-Kit+ early thymic precursors.

Immunologic self-tolerance is maintained by both central and peripheral mechanisms. Furthermore, regulation of mature lymphocyte responses is governed by inhibitory as well as stimulatory signals. TCR recognition of cognate peptide bound to MHC molecules provides the initial stimulus leading to T lymphocyte activation and determines the antigen specificity of any subsequent response. However, lymphocytes must discriminate between foreign and self antigens presented by self-MHC molecules to maintain self tolerance and avoid pathological autoimmunity. Consequently, TCR ligation alone is reported to result in abortive activation, T cell anergy, apoptosis, and tolerance. Under normal physiological conditions, costimulatory signals modify lymphocyte responsiveness to TCR ligation to prevent autoimmunity while enabling robust responses to foreign antigen. Members of the CD28/B7 superfamily provide the critical secondary signals essential for normal immune cell function. CD28 is an essential positive costimulatory molecule with critical functions in thymic development, lineage commitment, and regulation of peripheral lymphocyte responses to antigenic stimuli. CD28 ligation by APC-expressed B7 molecules alters proximal signaling events subsequent to MHC/TCR interactions, and initiates unique signaling pathways that alter mRNA stability and gene transcription. Furthermore, CD28 signaling is required for regulatory T cell development and function. Thus, CD28 has a central role in both potentiating lymphocyte activation mediated by TCR engagement and regulating peripheral tolerance. In contrast, Ctla-4 mediates an inhibitory signal upon binding B7 molecules on an antigen-presenting cell. Its importance in governing lymphocyte responses is manifested in the fatal lymphoproliferative disorder seen in Ctla-4-/- mice. The lymphocyte proliferation is polyclonal, antigen and CD28 dependent, and arises from defects in peripheral CD4+T cell regulation. The high percentage of peripheral T lymphocytes expressing activation markers is accompanied by lymphocyte infiltration into numerous non-lymphoid tissues and results in death by 3-4 weeks. While still controversial, Ctla-4 signaling has been reported to be essential for induction of peripheral T lymphocyte tolerance in vivo and in some model systems is proposed to regulate both T lymphocyte anergy induction and the immune suppressive effects of some regulatory T cells in the prevention of autoimmunity. Signaling pathways activated by TCR ligation and CD28 costimulation have been extensively characterized. In contrast, the mechanisms mediating Ctla-4 maintenance of tolerance remain largely unknown. Ctla-4 gene expression is tightly controlled during T cell development and activation, and its intracellular localization and expression on the cell surface is regulated by numerous pathways and intermediates. While a tailless Ctla-4 mutant is capable of inhibiting T cell activation, recent studies have shown that a ligand independent form of Ctla-4 is also capable of providing an inhibitory signal to T lymphocytes. In conjunction with the strictly controlled expression kinetics and the perfect amino acid homology between the intracellular domains of mouse and human Ctla-4, this data suggests that Ctla-4 may participate in the modulation or initiation of intracellular signaling pathways. Positive and negative costimulatory receptors on the T cell modify lymphocyte responses by altering both quantitative and qualitative aspects of the lymphocyte response including threshold of activation, cytokine secretion, and memory responses. Positive costimulation augments T cell responses, in part, by downregulating the expression of genes that actively maintain the quiescent phenotype. This study was initiated to determine the role of Ctla-4 ligation in modifying the global gene expression profile of stimulated T cells and to determine if the Ctla-4 mediated maintenance of T cell tolerance was achieved, in part, by altering the transcription of quiescence genes necessary for the prevention of T cell activation subsequent to TCR and CD28 stimulation. Previous studies investigating the influence of Ctla-4 ligation on transcriptional profiles of activated lymphocytes detected only quantitative alterations in the transcriptional regulation initiated by CD28 signaling. In contrast, our data suggests that quantitative effects of Ctla-4 ligation that differentially influence pathways acting downstream of stimulatory receptors results in a stable and qualitatively unique phenotype detectable at the level of the transcriptome. Thus, the cumulative effect of Ctla-4 signaling is unique and not constrained to reversing alterations in expression initiated by CD28. In addition, Ctla-4 ligation can be shown to influence T lymphocyte responsiveness and the resulting global expression profile within 4 hours after stimulation and prior to detectable Ctla-4 surface expression. In a subpopulation of T cells, TCR stimulation activates pathways that result in commitment to activation with 2-6 hours. In contrast, CD28 signaling must be maintained for 12-16 hours to ensure maximal responses at the population level. The period of sensitivity to Ctla-4 inhibition of activation is more constrained and does not extend beyond 12 hours. Together, these data support a potential role for Ctla-4 in modification of the early transcriptional response and may explain various alterations in phenotype resulting from Ctla-4 ligation that have been reported in secondary responses. Identification of genes involved in lymphocyte activation, maintenance of selftolerance, and attenuation of immune responses opens the door to therapeutic manipulation of the pathways implicated. CD28 costimulation results in general amplification of TCR-initiated transcriptional responses, and specifically alters the expression profile of a subset of genes. In contrast, Ctla-4 ligation directly and specifically alters the expression of a select group of genes when ligated, and results in minimal suppression of the global CD28-mediated costimulatory transcriptional response. Ctla-4 regulated genes comprise a heterogeneous family, but include known quiescence factors, transcriptional regulators, and various determinants of cell cycle progression and senescence. The role of Ctla-4 in maintaining self-tolerance indicates that targeted manipulation of these gene products presents a novel therapeutic opportunity, and suggests that the mechanisms involved in Ctla-4-mediated maintenance of peripheral T cell tolerance and regulation of immune responsiveness is more nuanced than previously thought. In addition, this study provides the most comprehensive description of global gene expression during primary lymphocyte activation yet available. The integration of statistical and bioinfomatics analyses with large scale data mining tools identifies genes not previously characterized in lymphocytes and can direct future work by predicting potentially interacting gene products and pathways.

Murine infection by the Lyme disease spirochete, B. burgdorferi, results in the generation of pathogen-specific antibody that can provide protection against Lyme disease, but the cells involved in this response are poorly characterized. T cells are not required for generating a protective antibody response to B. burgdorferi infection, but their exact role in providing protection against tissue colonization had not been previously determined. We found that TCRβxδ;-/- mice were susceptible to high pathogen loads and decreased antibody titers, but inhibition of CD40L-dependent interactions resulted in partial protection suggesting that a portion of the help provided by T cells was not dependent on CD40L-CD40 interactions between T and B cells. RAG1-/- mice reconstituted with either un-fractionated or B1-enriched peritoneal cells from previously infected mice generated B. burgdorferi-specific antibody, and upon spirochetal challenge suffered significantly lower levels of pathogen load in the joint and heart. Peritoneal cells from previously infected TCRβxδ-/- mice or B2-enriched or B1-purified peritoneal cells conferred little to only moderate protection, suggesting T cells play an important role in protection against spirochetal infection the joint. Consistent with this, T cells from previously infected donor mice, when transferred with B1 or B2 cells into RAG1-/- mice, generated increased antibody titers and were capable of diminishing bacterial burden in the joint and heart. A previously identified class of protective antibody is directed against the spirochetal surface lipoprotein DbpA, and we found that DbpA is a prominent protein antigen recognized by RAG1-/- mice reconstituted with B1-enriched peritoneal cells. Additionally, we found that mice reconstituted with B1 cells also make antibody directed towards the spirochetal glycolipid antigen, BbGL-IIc, which is recognized by Vα14iNKT cells. Consistent with the idea that T cells are important in providing protection against spirochetal infection, RAG1-/- mice reconstituted with B1 and T cells generated a more robust response against DbpA and BbGL-IIc. These results support the hypothesis that T cells act with B1 cells in a CD40L-independent manner to promote the production of antibodies that play an important role in protection of the joint from spirochetal infection.

Virus infection is sensed by the innate immune system through germline encoded pattern recognition receptors (PRRs). Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) serve as PRRs that recognize different viral components. Microbial nucleic acids such as Ribonucleic acid (RNA) are important virus-derived pathogen-associated molecular patterns (PAMPs) to be recognized by PRRs. Virus recognition may occur at multiple stages of the viral life cycle. Replication intermediates such as single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) are detected by the RNA-sensing PRRs that initiate innate and adaptive immune responses. Triggering of the innate immune system is a critical event that can shape the adaptive immune response to virus infection. Better vaccination strategies that lead to improved T-cell and antibody responses are needed for protection against pathogens. We sought to delineate the RNA-sensing PRR pathways that are activated during infection with an RNA virus, the signaling mediators involved and the influence on subsequent virus-specific adaptive immune responses. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vitro, we performed direct co-stimulation experiments on CD4+ T-cells of high purity. We utilized synthetic RNA-like immune response modifiers (IRMs) R-848 (MyD88-dependent) and poly I:C (MyD88-independent) as RNA PAMPs to determine the direct effects of RNA-sensing PRR activation on CD4+ T-cells. RNA PAMPs can act directly on CD4+ T-cells and modulate their function and phenotype. Maximal direct co-stimulatory effects were observed in CD4+ T-cells cultured with poly I:C compared to R-848. The cytoplasmic dsRNA-dependent protein kinase R (PKR) was also involved in poly I:C-mediated signaling in CD4+ T-cells. We found differences in the RNA-sensing PRRs activated by R-848 between mouse and human CD4+ T-cells. We observed minimal direct co-stimulatory effects by R-848 in mouse CD4+ T-cells. In contrast, augmentation of Th1 responses by R-848 was observed in human CD4+ T-cells. TLR8 activation in human CD4+ T-cells may explain the observed differences. We next explored the signaling pathways activated by RNA PAMPs in conventional dendritic cells (cDCs) and CD4+ T-cells that drive Th1 CD4 T-cell responses in isolated cDC/CD4 T-cell interactions. Allogeneic cDCs and CD4+ T-cells of high purity were cultured together with R-848 and poly I:C in MHC congenic mixed leukocyte reactions (MLRs). R-848 and poly I:C stimulation of type I IFN production and signaling was essential but not sufficient for driving CD4+ Th1 responses. The early production of IL-1α and IL-1β was equally critical. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vivo, we utilized a mouse model of heterosubtypic influenza A virus (IAV) infections. Using MyD88-/-, TLR7-/- and IL-1-deficient mice, we explored the role of MyD88-signaling in the generation of heterosubtypic memory CD4+ T-cell, CD8+ T-cell and antibody responses. We found that MyD88 signaling played an important role in anti-IAV spleen and lung CD4+ T-cell, spleen CD8+ T-cell and Th1 antibody immune responses. Anti-IAV lung heterosubtypic CD8+ T-cell responses were not dependent on MyD88 signaling. Our in vitro and in vivo results show the pivotal role of RNA-sensing PRR pathway activation in T-cell immune responses. Understanding the complexity of the PRR pathways involved during viral infections and defining the subsequent immune response would have important implications for the generation of more effective vaccine strategies.

Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections contributing to pathology following DENV infection. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein, NS126-34. We predicted higher frequencies of NS126-34-specific CD8+ T cells in PBMC from individuals undergoing secondary, rather than primary, DENV infection due to the expansion of memory CD8+T cells. We generated a tetramer against this epitope (B57-NS126-34TET) and used it to assess the frequencies and phenotype of antigen-specific T cells in samples from a clinical cohort of children with acute DENV infection established in Bangkok, Thailand. High tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS126-34-specific, other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV-specific CD8+ T cells. In vitro stimulation of CD8+ T cell lines, generated against three different DENV epitopes, indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides with substantial upregulation of CD71 detected to peptides which also elicited strong functional responses. CD71 may therefore represent a useful marker of antigenspecific T cell activation. During the course of our analysis we found substantial binding of B57-NS126-34 TET to CD8- cells. We demonstrated that the B57-NS126-34 TET bound KIR3DL1, an inhibitory receptor on natural killer (NK) cells. NK sensitive target cells presenting the NS126-34 peptide in the context of HLA-B57 were able to dampen functional responses of only KIR3DL1+ NK cells. Analysis of the activation of an NK enriched population in our Thai cohort revealed peak activation during the critical time phase in patients with severe dengue illness, dengue hemorrhagic fever, compared to people with mild illness. Our data identified CD71 as biologically useful marker to study DENV-specific CD8+ T cell responses and highlighted the role of viral peptides in modulating NK cell activation through KIR-MHC class I interactions during DENV infection.