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
Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell-specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1- but HLA-B*07:02+ nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma.
Assuntos
Imunoterapia Adotiva/métodos , Linfoma não Hodgkin/imunologia , Mieloma Múltiplo/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transativadores/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Engenharia Genética/métodos , Humanos , Camundongos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Kidney fibrosis is marked by an epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs). Here we find that, during renal fibrosis, TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of the EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several solute and solvent transporters in TECs. We also found that transgenic expression of either Twist1 (encoding twist family bHLH transcription factor 1, known as Twist) or Snai1 (encoding snail family zinc finger 1, known as Snail) expression is sufficient to promote prolonged TGF-ß1-induced G2 arrest of TECs, limiting the cells' potential for repair and regeneration. In mouse models of experimentally induced renal fibrosis, conditional deletion of Twist1 or Snai1 in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while also restoring cell proliferation, dedifferentiation-associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of the EMT program in TECs during chronic renal injury represents a potential anti-fibrosis therapy.
Assuntos
Pontos de Checagem do Ciclo Celular , Transição Epitelial-Mesenquimal , Rim/patologia , Animais , Aquaporina 1/genética , Células Cultivadas , Fibrose , Fase G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteína 1 Transportadora de Ânions Orgânicos/genéticaRESUMO
It is now clear that anti-CTLA-4 (α-CTLA-4) antibodies stimulate tumor immunity either by relieving inhibition of effector T-cell function or by depletion of regulatory T cells (Treg). Several recent reports, however, have suggested that these antibodies may deliver a "go" signal to effector T cells, thus interrupting T-cell receptor signaling and subsequent T-cell activation. We examined the behavior of melanoma-specific CD8+ pmel-1 T cells in the B16/BL6 mouse model using intravital microscopy. Pmel-1 velocities in progressively growing tumors were lower than their velocities in tumors given a therapeutic combination that included α-CTLA-4 antibodies, suggesting that successful immunotherapy correlates with greater T-cell motility. When α-CTLA-4 antibodies were injected during imaging, the velocities of pmel-1 T cells in tumor-draining lymph nodes also increased. Because α-CTLA-4 Fab fragments had the same effect as the intact antibody, the higher T-cell motility does not seem to be due to CTLA-4 inhibitory signaling but rather to the release of nonproductive stable interactions between tumor-infiltrating T cells and tumor targets or antigen-presenting cells subsequent to CTLA-4 blockade. This phenomenon resembles the recently described reversal of the antiviral T-cell motility paralysis by programmed death 1 (PD-1)-specific antibodies during T-cell exhaustion in persistent viral infections.
Assuntos
Antígeno CTLA-4/antagonistas & inibidores , Vacinas Anticâncer/uso terapêutico , Melanoma Experimental/terapia , Linfócitos T Citotóxicos/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Linfonodos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BLRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Fibroblastos/patologia , Fibrose/patologia , Neoplasias Pancreáticas/patologia , Animais , Carcinoma Ductal Pancreático/imunologia , Modelos Animais de Doenças , Fibroblastos/imunologia , Fibrose/imunologia , Humanos , Tolerância Imunológica , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/imunologia , Análise de SobrevidaRESUMO
SUMMARY: The generation of productive adaptive immune responses depends on the antigen-specific activation of T and B cells. The outcome of T-cell receptor engagement is influenced by signals from both positive and negative regulatory molecules that can either activate or inhibit T-cell function. CD28 and cytotoxic T-lymphocyte antigen-4 are the prototypical members of an immunoglobulin domain-containing protein family that play important roles in the control of T-cell responses against infection, cancer, and in autoimmune disease. Although the precise molecular details of their functions are still under active investigation, tumors and chronic pathogens seem to have exploited these pathways to achieve immune evasion. Furthermore, malfunction of the inhibitory arm of the immune response appears responsible for the development of multiple autoimmune pathologies. As a result, the negative regulators of T-cell activation have become attractive targets for therapeutic intervention in cancer, chronic infection, and autoimmune disease. The application of findings from basic research has provided insight into the manipulation of these pathways in the clinic and offers promising strategies for the treatment of disease.
Assuntos
Doenças Autoimunes/terapia , Imunoterapia , Infecções/terapia , Ativação Linfocitária , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD28/imunologia , Antígeno CTLA-4 , HumanosRESUMO
Despite the importance of programmed death-1 (PD-1) for T cell inhibition, little is known about its intracellular trafficking or requirements for localization to the immunological synapse. Here, we show that in activated T cells, PD-1 is present at the plasma membrane, near the Golgi and in the trans-Golgi network. Unlike CD28 and CTLA-4, PD-1 accumulation at the synapse is extensive only when T cells interact with dendritic cells (DCs) expressing high B7-DC levels. However, B7-H1 is also critically important, especially when the DCs have little B7-DC. Despite this preference, B7-H1(-/-) DCs elicit greater cytokine secretion than B7-DC(-/-) DCs during T cell restimulation, possibly because they also express less B7-DC. PD-1 and CD28 have similar kinetics of synaptic accumulation, suggesting that the process involves T cell receptor-triggered cytoskeletal reorganization followed by ligand binding.
Assuntos
Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Membrana Celular/imunologia , Genes Reporter , Complexo de Golgi/imunologia , Humanos , Ligantes , Camundongos , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T/imunologia , Receptores da Transferrina/imunologiaRESUMO
The reported affinity differences between CD28 and CTLA-4 binding to B7-1 and B7-2 may serve to selectively regulate CD28 and CTLA-4 function by differentially recruiting and/or stabilizing these molecules at the immunological synapse. Here we show that ligand binding is important for the accumulation of both CD28 and CTLA-4 at the synapse. While CD28 is recruited to the synapse in the absence of B7-1 and B7-2 binding, it is not effectively stabilized there, as its localization can be disrupted by CTLA-4. In the case of CTLA-4, ligand binding is critical for its concentration at the synapse. We also demonstrate that the affinity and avidity differences in ligand binding translate into selective recruitment of CD28 or CTLA-4 to the immunological synapse--B7-1 is the major ligand mediating CTLA-4 localization, while B7-2 is the main ligand for CD28 concentration at the synapse.