RESUMO
CD4+ T cells can either enhance or inhibit tumour immunity. Although regulatory T cells have long been known to impede antitumour responses1-5, other CD4+ T cells have recently been implicated in inhibiting this response6,7. Yet, the nature and function of the latter remain unclear. Here, using vaccines containing MHC class I (MHC-I) neoantigens (neoAgs) and different doses of tumour-derived MHC-II neoAgs, we discovered that whereas the inclusion of vaccines with low doses of MHC-II-restricted peptides (LDVax) promoted tumour rejection, vaccines containing high doses of the same MHC-II neoAgs (HDVax) inhibited rejection. Characterization of the inhibitory cells induced by HDVax identified them as type 1 regulatory T (Tr1) cells expressing IL-10, granzyme B, perforin, CCL5 and LILRB4. Tumour-specific Tr1 cells suppressed tumour rejection induced by anti-PD1, LDVax or adoptively transferred tumour-specific effector T cells. Mechanistically, HDVax-induced Tr1 cells selectively killed MHC-II tumour antigen-presenting type 1 conventional dendritic cells (cDC1s), leading to low numbers of cDC1s in tumours. We then documented modalities to overcome this inhibition, specifically via anti-LILRB4 blockade, using a CD8-directed IL-2 mutein, or targeted loss of cDC2/monocytes. Collectively, these data show that cytotoxic Tr1 cells, which maintain peripheral tolerance, also inhibit antitumour responses and thereby function to impede immune control of cancer.
Assuntos
Antígenos de Neoplasias , Linfócitos T CD4-Positivos , Citotoxicidade Imunológica , Imunoterapia , Neoplasias , Linfócitos T Reguladores , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL5/metabolismo , Células Dendríticas/imunologia , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-10/metabolismo , Interleucina-10/imunologia , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Reguladores/imunologia , Receptores Imunológicos/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Tolerância Imunológica , Linfócitos T CD8-Positivos/imunologiaRESUMO
Follicular helper T cells (Tfh cells) are the major producers of interleukin-4 (IL-4) in secondary lymphoid organs where humoral immune responses develop. Il4 regulation in Tfh cells appears distinct from the classical T helper 2 (Th2) cell pathway, but the underlying molecular mechanisms remain largely unknown. We found that hypersensitivity site V (HS V; also known as CNS2), a 3' enhancer in the Il4 locus, is essential for IL-4 production by Tfh cells. Mice lacking HS V display marked defects in type 2 humoral immune responses, as evidenced by abrogated IgE and sharply reduced IgG1 production in vivo. In contrast, effector Th2 cells that are involved in tissue responses were far less dependent on HS V. HS V facilitated removal of repressive chromatin marks during Th2 and Tfh cell differentiation and increased accessibility of the Il4 promoter. Thus, Tfh and Th2 cells utilize distinct but overlapping molecular mechanisms to regulate Il4, a finding with important implications for understanding the molecular basis of allergic diseases.
Assuntos
Interleucina-4/biossíntese , Interleucina-4/genética , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Sítios de Ligação/genética , Sequência Conservada , Citocinas/genética , Elementos Facilitadores Genéticos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Imunidade Humoral/genética , Interleucina-4/deficiência , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas , Deleção de Sequência , Subpopulações de Linfócitos T/imunologia , Células Th2/imunologia , Transcrição GênicaRESUMO
Patients with relapsed or refractory acute myeloid leukemia (AML) have a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell leukemias and lymphomas and could prove highly efficacious in AML. However, a significant number of patients with AML may not receive treatment with an autologous product due to manufacturing failures associated with low lymphocyte counts or rapid disease progression while the therapeutic is being produced. We report the preclinical evaluation of an off-the-shelf CAR T cell therapy targeting Fms-related tyrosine kinase 3 (FLT3) for the treatment of AML. Single-chain variable fragments (scFvs) targeting various epitopes in the extracellular region of FLT3 were inserted into CAR constructs and tested for their ability to redirect T cell specificity and effector function to FLT3+ AML cells. A lead CAR, exhibiting minimal tonic signaling and robust activity in vitro and in vivo, was selected and then modified to incorporate a rituximab-responsive off-switch in cis. We found that allogeneic FLT3 CAR T cells, generated from healthy-donor T cells, eliminate primary AML blasts but are also active against mouse and human hematopoietic stem and progenitor cells, indicating risk of myelotoxicity. By employing a surrogate CAR with affinity to murine FLT3, we show that rituximab-mediated depletion of FLT3 CAR T cells after AML eradication enables bone marrow recovery without compromising leukemia remission. These results support clinical investigation of allogeneic FLT3 CAR T cells in AML and other FLT3+ hematologic malignancies.
Assuntos
Imunoterapia Adotiva , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Tirosina Quinase 3 Semelhante a fms/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/metabolismo , Modelos Animais de Doenças , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Leucemia Mieloide Aguda/diagnóstico , Camundongos , Receptores de Antígenos Quiméricos/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T/metabolismo , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3+ dendritic cells (DCs) and bone marrow FLT3+ stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Complexo CD3/antagonistas & inibidores , Hematopoese/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/uso terapêutico , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Complexo CD3/química , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/farmacologia , Imunofenotipagem , Leucemia Mieloide Aguda , Depleção Linfocítica , Macaca fascicularis , Camundongos , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/químicaRESUMO
Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613.
Assuntos
Diferenciação Celular , Macrófagos/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Placenta/citologia , Linfócitos T/citologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Isquemia/patologia , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Perfusão , Fenótipo , Gravidez , Linfócitos T/metabolismoRESUMO
The IL-23 pathway is genetically linked to autoimmune disease in humans and is required for pathogenic Th17 cell function in mice. However, because IL-23R-expressing mature Th17 cells are rare and poorly defined in mice at steady-state, little is known about IL-23 signaling. In this study, we show that the endogenous CCR6(+) memory T cell compartment present in peripheral lymphoid organs of unmanipulated mice expresses Il23r ex vivo, displays marked proinflammatory responses to IL-23 stimulation in vitro, and is capable of transferring experimental autoimmune encephalomyelitis. The prolyl-tRNA synthetase inhibitor halofuginone blocks IL-23-induced Stat3 phosphorylation and IL-23-dependent proinflammatory cytokine expression in endogenous CCR6(+) Th17 cells via activation of the amino acid starvation response (AAR) pathway. In vivo, halofuginone shows therapeutic efficacy in experimental autoimmune encephalomyelitis, reducing both established disease progression and local Th17 cell effector function within the CNS. Mechanistically, AAR activation impairs Stat3 responses downstream of multiple cytokine receptors via selective, posttranscriptional suppression of Stat3 protein levels. Thus, our study reveals latent pathogenic functions of endogenous Th17 cells that are regulated by both IL-23 and AAR pathways and identifies a novel regulatory pathway targeting Stat3 that may underlie selective immune regulation by the AAR.
Assuntos
Aminoácidos/deficiência , Encefalomielite Autoimune Experimental/imunologia , Ativação Linfocitária , Fator de Transcrição STAT3/imunologia , Células Th17/imunologia , Animais , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-23/genética , Interleucina-23/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fosforilação/genética , Fosforilação/imunologia , Piperidinas , Quinazolinonas , Receptores CCR6/genética , Receptores CCR6/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Th17/patologiaRESUMO
Tumor-specific CD8+ T cells are key effectors of antitumor immunity but are often rendered dysfunctional in the tumor microenvironment. Immune-checkpoint blockade can restore antitumor T-cell function in some patients; however, most do not respond to this therapy, often despite T-cell infiltration in their tumors. We here explored a CD8-targeted IL2 fusion molecule (CD8-IL2) to selectively reactivate intratumoral CD8+ T cells in patient-derived tumor fragments. Treatment with CD8-IL2 broadly armed intratumoral CD8+ T cells with enhanced effector capacity, thereby specifically enabling reinvigoration of the dysfunctional T-cell pool to elicit potent immune activity. Notably, the revival of dysfunctional T cells to mediate effector activity by CD8-IL2 depended on simultaneous antigen recognition and was quantitatively and qualitatively superior to that achieved by PD-1 blockade. Finally, CD8-IL2 was able to functionally reinvigorate T cells in tumors resistant to anti-PD-1, underscoring its potential as a novel treatment strategy for patients with cancer. Significance: Reinvigorating T cells is crucial for response to checkpoint blockade therapy. However, emerging evidence suggests that the PD-1/PD-L1 axis is not the sole impediment for activating T cells within tumors. Selectively targeting cytokines toward specific T-cell subsets might overcome these barriers and stimulate T cells within resistant tumors. See related article by Moynihan et al., p. 1206 (32).
Assuntos
Linfócitos T CD8-Positivos , Interleucina-2 , Neoplasias , Humanos , Linfócitos T CD8-Positivos/imunologia , Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Microambiente Tumoral/imunologia , Camundongos , Animais , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Antígenos CD8/metabolismoRESUMO
CD8+ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8+ T cell dysfunction showed that interleukin-2 (IL-2)-based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2's effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (Treg) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8+ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8+ T cells in the liver without substantially altering Treg or NK cell counts. These expanded CD8+ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen-positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8+ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8+ T cells without affecting NK or Treg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.
Assuntos
Hepatite B Crônica , Interleucina-2 , Humanos , Animais , Camundongos , Vírus da Hepatite B , Linfócitos T CD8-Positivos , Hepatite B Crônica/tratamento farmacológico , Antivirais/farmacologia , Antivirais/uso terapêuticoRESUMO
IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rßγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell-selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.
Assuntos
Linfócitos T CD8-Positivos , Interleucina-2 , Animais , Linfócitos T CD8-Positivos/imunologia , Interleucina-2/farmacologia , Camundongos , Humanos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Feminino , Neoplasias/imunologia , Neoplasias/tratamento farmacológicoRESUMO
The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/terapia , Imunoterapia , Interleucina-15/uso terapêutico , Melanoma Experimental/terapia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Modelos Animais de Doenças , Humanos , Interleucina-15/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Monoclonal antibody therapies are an important approach for the treatment of hematologic malignancies, but typically show low single-agent activity. Bispecific antibodies, however, redirect immune cells to the tumor for subsequent lysis, and preclinical and accruing clinical data support single-agent efficacy of these agents in hematologic malignancies, presaging an exciting era in the development of novel bispecific formats. This review discusses recent developments in this area, highlighting the challenges in delivering effective immunotherapies for patients.
Assuntos
Anticorpos Biespecíficos/farmacologia , Neoplasias Hematológicas , Antineoplásicos Imunológicos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/imunologia , Humanos , Imunoterapia/métodosRESUMO
BACKGROUND: Decellularized, dehydrated human amniotic membrane (DDHAM) is an extracellular matrix devoid of cells, cell debris, and growth factors. This study examines the effect of cell attachment to the DDHAM and the induced cellular responses. MATERIALS AND METHODS: The cell types employed in this study were human dermal fibroblasts (HDF), human epithelial keratinocytes (HEK), and human dermal microvascular endothelial cells (HDMEC), all of which play critical roles in the wound healing process. Further, the DDHAM was compared to a dehydrated human amnion/chorion membrane (dHACM), which contains and releases biological entities including growth factors and cytokines. The HDF and HEK were cultured on the DDHAM and the dHACM, and cell imaging and proliferation assays were performed to evaluate cell attachment to and the ability to proliferate on the DDHAM relative to the dHACM. In addition, the effect of soluble factors released by the DDHAM and the dHACM on cell survival, attachment, and proliferation were examined. The authors also evaluated the effect of soluble factors produced by culturing cells on the DDHAM in in vitro functional assays, including cell survival and endothelial cell migration in a wound closure angiogenesis assay. RESULTS: The HDF and HEK cells readily attached to and proliferated on the DDHAM, while the dHACM did not support cell attachment and proliferation when cultured under the same conditions. Soluble factors secreted when HDF were cultured on the DDHAM enhanced both endothelial cell and keratinocyte survival and endothelial cell migration in a wound closure assay. CONCLUSIONS: Although DDHAM is only an extracellular matrix and serves primarily as a scaffold, it has sufficient cues to allow for cell attachment and proliferation. Further, the biological entities released as a consequence of cell attachment promote cell survival and migration.
Assuntos
Âmnio/química , Curativos Biológicos , Adesão Celular , Proliferação de Células , Neovascularização Fisiológica , Ferimentos e Lesões/patologia , Aloenxertos , Junções Célula-Matriz , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Resultado do Tratamento , CicatrizaçãoRESUMO
IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doença de Crohn/metabolismo , Regulação da Expressão Gênica/imunologia , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Doença de Crohn/imunologia , Citometria de Fluxo , Glucocorticoides/farmacologia , Humanos , Interferon gama/metabolismo , Análise em Microsséries , Células Th17/imunologiaRESUMO
Human memory T cells (T(M) cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. These T cell lineages are almost exclusively CCR6(+) and are important mediators of chronic inflammation and autoimmunity. However, little is known about the mechanisms controlling IL-17/IL-22 expression in memory Th17/Th22 subsets. We show that common γ chain (γc)-using cytokines, namely IL-2, IL-7, and IL-15, potently induce Th17-signature cytokine expression (Il17a, Il17f, Il22, and Il26) in CCR6(+), but not CCR6(-), T(M) cells, even in CCR6(+) cells lacking IL-17 expression ex vivo. Inhibition of phosphoinositide 3-kinase (PI-3K) or Akt signaling selectively prevents Th17 cytokine induction by γc-cytokines, as does ectopic expression of the transcription factors FOXO1 or KLF2, which are repressed by PI-3K signaling. These results indicate that Th17 cytokines are tuned by PI-3K signaling in CCR6(+) T(M) cells, which may contribute to chronic or autoimmune inflammation. Furthermore, these findings suggest that ex vivo analysis of IL-17 expression may greatly underestimate the frequency and pathogenic potential of the human Th17 compartment.
Assuntos
Citocinas/imunologia , Memória Imunológica/fisiologia , Fosfatidilinositol 3-Quinases/imunologia , Receptores CCR6 , Transdução de Sinais/imunologia , Células Th17/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doença Crônica , Citocinas/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/genéticaRESUMO
Members of the Runx family of transcription factors, Runx1-3, are essential regulators of the immune system: a deficiency in one of the members, Runx1, results in complete ablation of hematopoiesis, and all three Runx proteins play important, nonredundant roles in immune system development and function. Here, we review gene regulation by Runx proteins in T lymphocytes, with a focus on their recently emerging roles in the development and function of peripheral CD4+ and CD8+ T lineages.
Assuntos
Subunidades alfa de Fatores de Ligação ao Core/imunologia , Regulação da Expressão Gênica , Linfócitos T/imunologia , Animais , Diferenciação Celular , Linhagem da Célula , Humanos , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Regulação da Expressão Gênica , Proteínas com Domínio T/fisiologia , Linfócitos T Citotóxicos/metabolismo , Animais , Northern Blotting , Western Blotting , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Citotoxicidade Imunológica/imunologia , Granzimas/genética , Granzimas/metabolismo , Interferon gama/metabolismo , Interleucina-2/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Perforina/genética , Perforina/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A general theme in gene regulation is that transcription factors never function alone. Recent studies have emphasized this concept for regulatory T cells, a unique lineage of CD4+ T cells that exert active immune suppression and are essential to maintaining self-tolerance.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Fatores de Transcrição NFATC/fisiologia , Linfócitos T Reguladores/metabolismo , Transcrição Gênica , Animais , Humanos , Fator de Transcrição AP-1/fisiologiaRESUMO
Cell differentiation involves activation and silencing of lineage-specific genes. Here we show that the transcription factor Runx3 is induced in T helper type 1 (T(H)1) cells in a T-bet-dependent manner, and that both transcription factors T-bet and Runx3 are required for maximal production of interferon-gamma (IFN-gamma) and silencing of the gene encoding interleukin 4 (Il4) in T(H)1 cells. T-bet does not repress Il4 in Runx3-deficient T(H)2 cells, but coexpression of Runx3 and T-bet induces potent repression in those cells. Both T-bet and Runx3 bind to the Ifng promoter and the Il4 silencer, and deletion of the silencer decreases the sensitivity of Il4 to repression by either factor. Our data indicate that cytokine gene expression in T(H)1 cells may be controlled by a feed-forward regulatory circuit in which T-bet induces Runx3 and then 'partners' with Runx3 to direct lineage-specific gene activation and silencing.
Assuntos
Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Interferon gama/metabolismo , Interleucina-4/genética , Proteínas com Domínio T/metabolismo , Células Th1/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Subunidade alfa 3 de Fator de Ligação ao Core/deficiência , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Humanos , Interferon gama/genética , Interleucina-4/biossíntese , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Ativação TranscricionalRESUMO
Helper T cells coordinate immune responses through the production of cytokines. Th2 cells express the closely linked Il4, Il13, and Il5 cytokine genes, whereas these same genes are silenced in the Th1 lineage. The Th1/Th2 lineage choice has become a textbook example for the regulation of cell differentiation, and recent discoveries have further refined and expanded our understanding of how Th2 differentiation is initiated and reinforced by signals from antigen-presenting cells and cytokine-driven feedback loops. Epigenetic changes that stabilize the active or silent state of the Il4 locus in differentiating helper T cells have been a major focus of recent research. Overall, the field is progressing toward an integrated model of the signaling and transcription factor networks, cis-regulatory elements, epigenetic modifications, and RNA interference mechanisms that converge to determine the lineage fate and gene expression patterns of differentiating helper T cells.
Assuntos
Interleucina-4/genética , Células Th2/citologia , Células Th2/imunologia , Animais , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Citocinas/genética , Metilação de DNA , Epigênese Genética , Retroalimentação , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Histonas/metabolismo , Humanos , Memória Imunológica , Camundongos , Modelos Genéticos , Modelos Imunológicos , Receptores Notch/imunologia , Transdução de Sinais , Células Th1/citologia , Células Th1/imunologiaRESUMO
Helper T cell differentiation involves silencing as well as activation of gene expression. We have identified a conserved silencer of the gene encoding interleukin 4 (Il4) marked by DNase I hypersensitivity (HS IV) and permissive chromatin structure in all helper T cells. Deletion of HS IV increased Il4 and Il13 transcription by naive T cells and led to T helper type 2 skewing in vitro. HS IV controlled Il4 silencing during T helper type 1 differentiation, as HS IV-deficient T helper type 1 cells that expressed interferon-gamma also produced abundant interleukin 4 in vitro and in vivo. Despite mounting a vigorous interferon-gamma response, HS IV-deficient mice were more susceptible to Leishmania major infection than were wild-type littermate control mice, showing a critical function for Il4 silencing in T helper type 1-mediated immunity.