RESUMO
Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.
Assuntos
Antígenos CD28/metabolismo , Antígenos CD40/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD28/genética , Antígenos CD40/genética , Proliferação de Células , Sobrevivência Celular , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Imunoterapia Adotiva/métodos , Leucemia/genética , Leucemia/imunologia , Leucemia/metabolismo , Leucemia/terapia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The phenylalanyl-glycyl-glycyl-alanyl-prolyl (FG-GAP) domain plays an important role in protein-protein interactions, including interaction of integrins with their ligands. Integrin-α FG-GAP repeat-containing protein 2 (Itfg2) is a highly conserved protein in vertebrates that carries two FG-GAP domains, but its role in mammalian physiology is unknown. In this article, we show that Itfg2 is an intracellular protein and it plays a critical role in B cell differentiation and development of autoimmunity. Itfg2-deficient mice displayed a phenotype consistent with retention of B cells in the spleen and had a lower concentration of IgG in the blood when compared with wild-type littermates. Itfg2-deficient splenocytes also showed a defect in cell migration in vitro. After immunization with a thymus-dependent Ag, the absence of Itfg2 caused a shift in B cell maturation from the germinal centers to the extrafollicular regions of the spleen and blocked deposition of Ag-specific plasma cells in the bone marrow. In support of hematopoietic cell intrinsic activity of Itfg2, bone marrow transplantation of Itfg2-deficient cells was sufficient to impair germinal center development in wild-type mice. Furthermore, Itfg2 deficiency exacerbated development of autoimmune disease in MRL/lpr lupus-prone mice. These results identify Itfg2 as a novel contributor to B cell differentiation and a negative regulator of the autoimmune response during lupus.
Assuntos
Doenças Autoimunes/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Cadeias alfa de Integrinas/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Sequência de Bases , Diferenciação Celular/imunologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Ordem dos Genes , Marcação de Genes , Genótipo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Imunoglobulinas/sangue , Cadeias alfa de Integrinas/química , Cadeias alfa de Integrinas/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Alinhamento de Sequência , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Sh2d3c is an adaptor protein that has been implicated in T cell activation and shown to associate with different components of the integrin signaling pathway ex vivo. However, the in vivo significance of Sh2d3c expression in the regulation of the immune response and/or hematopoietic cell lineage development is not known. In this study, we show that expression of Sh2d3c is more critical for development and function of marginal zone B (MZB) cells than for T cell maturation. Mice deficient in Sh2d3c expression (Sh2d3c(-/-)) had a reduced number of MZB cells, and the residual MZB cells failed to properly capture polysaccharide Ags. Activation-induced proliferation, cytokine production, and migration of Sh2d3c(-)(/)(-) splenic B cells were also significantly reduced in vitro compared with wild-type (Sh2d3c(+/+)) cells. In contrast, T cell development and function were largely normal in Sh2d3c(-/-) mice. The thymi of Sh2d3c(-/-) mice showed no maturational abnormalities, the number of splenic T cells was only modestly reduced, and the T cells responded normally to in vitro polyclonal activation. The observed B cell deficiency in the Sh2d3c(-/-) mice led to diminished humoral immune response against thymus-independent type 2, but not thymus-dependent Ags, which highlights the primary in vivo role of Sh2d3c in regulating B cell development and function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos T-Independentes/imunologia , Subpopulações de Linfócitos B/patologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Feminino , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Transdução de Sinais/genética , Baço/patologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting "low" cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.
Assuntos
Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/terapia , Fator 88 de Diferenciação Mieloide/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Antígenos CD19/imunologia , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Transdução de Sinais/imunologia , Células THP-1RESUMO
Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.