RESUMEN
Antibody-based immunotherapy is a promising strategy for targeting chemoresistant leukemic cells. However, classical antibody-based approaches are restricted to targeting lineage-specific cell surface antigens. By targeting intracellular antigens, a large number of other leukemia-associated targets would become accessible. In this study, we evaluated a novel T-cell bispecific (TCB) antibody, generated by using CrossMAb and knob-into-holes technology, containing a bivalent T-cell receptor-like binding domain that recognizes the RMFPNAPYL peptide derived from the intracellular tumor antigen Wilms tumor protein (WT1) in the context of HLA-A*02. Binding to CD3ε recruits T cells irrespective of their T-cell receptor specificity. WT1-TCB elicited antibody-mediated T-cell cytotoxicity against AML cell lines in a WT1- and HLA-restricted manner. Specific lysis of primary acute myeloid leukemia (AML) cells was mediated in ex vivo long-term cocultures by using allogeneic (mean ± standard error of the mean [SEM] specific lysis, 67 ± 6% after 13-14 days; n = 18) or autologous, patient-derived T cells (mean ± SEM specific lysis, 54 ± 12% after 11-14 days; n = 8). WT1-TCB-treated T cells exhibited higher cytotoxicity against primary AML cells than an HLA-A*02 RMF-specific T-cell clone. Combining WT1-TCB with the immunomodulatory drug lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean ± SEM specific lysis on days 3-4, 45.4 ± 9.0% vs 70.8 ± 8.3%; P = .015; n = 9-10). In vivo, WT1-TCB-treated humanized mice bearing SKM-1 tumors exhibited a significant and dose-dependent reduction in tumor growth. In summary, we show that WT1-TCB facilitates potent in vitro, ex vivo, and in vivo killing of AML cell lines and primary AML cells; these results led to the initiation of a phase 1 trial in patients with relapsed/refractory AML (#NCT04580121).
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Péptidos/uso terapéutico , Proteínas WT1/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Antígeno HLA-A2/inmunología , Humanos , Leucemia Mieloide Aguda/inmunología , Ratones , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
Caveolin-1 (Cav-1) is a key organizer of membrane specializations and a scaffold protein that regulates signaling in multiple cell types. We found increased Cav-1 expression in human and murine T cells after allogeneic hematopoietic cell transplantation. Indeed, Cav-1(-/-)donor T cells caused less severe acute graft-versus-host disease (GVHD) and yielded higher numbers of regulatory T cells (Tregs) compared with controls. Depletion of Tregs from the graft abrogated this protective effect. Correspondingly, Treg frequencies increased when Cav-1(-/-)T cells were exposed to transforming growth factor-ß/T-cell receptor (TCR)/CD28 activation or alloantigen stimulation in vitro compared with wild-type T cells. Mechanistically, we found that the phosphorylation of Cav-1 is dispensable for the control of T-cell fate by using a nonphosphorylatable Cav-1 (Y14F/Y14F) point-mutation variant. Moreover, the close proximity of lymphocyte-specific protein tyrosine kinase (Lck) to the TCR induced by TCR-activation was reduced in Cav-1(-/-)T cells. Therefore, less TCR/Lck clustering results in suboptimal activation of the downstream signaling events, which correlates with the preferential development into a Treg phenotype. Overall, we report a novel role for Cav-1 in TCR/Lck spatial distribution upon TCR triggering, which controls T-cell fate toward a regulatory phenotype. This alteration translated into a significant increase in the frequency of Tregs and reduced GVHD in vivo.
Asunto(s)
Caveolina 1/metabolismo , Caveolina 1/fisiología , Regulación de la Expresión Génica , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/citología , Caveolina 1/genética , Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , Estudios Prospectivos , Transducción de Señal , Linfocitos T Reguladores/citología , Factor de Crecimiento Transformador beta/metabolismo , Trasplante HomólogoRESUMEN
The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3ß. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Endopeptidasas/inmunología , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Timocitos/inmunología , Ubiquitina Tiolesterasa/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Colitis/genética , Colitis/inmunología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Humanos , Células Jurkat , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-7/inmunología , Receptores de Interleucina-7/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timocitos/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Regulatory T (Treg) cells maintain immune homeostasis and prevent inflammatory and autoimmune responses. During development, thymocytes bearing a moderately self-reactive T cell receptor (TCR) can be selected to become Treg cells. Several observations suggest that also in the periphery mature Treg cells continuously receive self-reactive TCR signals. However, the importance of this inherent autoreactivity for Treg cell biology remains poorly defined. To address this open question, we genetically ablated the TCR of mature Treg cells in vivo. These experiments revealed that TCR-induced Treg lineage-defining Foxp3 expression and gene hypomethylation were uncoupled from TCR input in mature Treg cells. However, Treg cell homeostasis, cell-type-specific gene expression and suppressive function critically depend on continuous triggering of their TCR.
Asunto(s)
Autoinmunidad/inmunología , Factores de Transcripción Forkhead/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Reguladores/inmunología , Animales , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Metilación de ADN/inmunología , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/genética , Inflamación/inmunología , Factores Reguladores del Interferón/biosíntesis , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transducción de Señal/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Timocitos/citologíaRESUMEN
Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.