RESUMO
CRISPR/Cas9 gene-editing technology allows researchers to study protein function by specifically introducing double-stranded breaks in the gene of interest then analyze its subsequent loss in sensitive biological assays. To help characterize one of a series of highly potent, conditionally active, T cell engaging bispecific molecules called COBRA™, the human EpCAM gene was disrupted in HT29 cells using CRISPR/Cas9 and guide RNA targeting its Exon 2. Although a commercially available antibody indicated loss of cell-surface expression, the EpCAM targeting bispecific COBRA was still able to lyse these cells in a T cell dependent cellular cytotoxicity assay. RT-PCR sequence analysis of these cells showed a major alternative transcript generated after CRISPR/Cas9, with Exon 1 and 3 spliced together in-frame, skipping Exon 2 completely, to express a truncated cell-surface receptor recognized by the EpCAM-COBRA. Researchers who use CRISPR/Cas9 must be cognizant of this potential to express alternative versions of their proteins and use sensitive orthogonal detection methods to ensure complete gene disruption.
RESUMO
T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⺠T cells and CD8⺠tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⺠T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⺠TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1âºTIM-3⺠TA-specific CD8⺠T cells. PD-1âºTIGITâº, PD-1â»TIGITâº, and PD-1âºTIGITâ» CD8⺠TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⺠T cells and CD8⺠TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⺠T cells and CD8⺠TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⺠T cell responses in patients with advanced melanoma.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/fisiologia , Receptor de Morte Celular Programada 1/fisiologia , Receptores Imunológicos/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/genética , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citocinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Memória Imunológica , Imunofenotipagem , Subunidade beta de Receptor de Interleucina-2/biossíntese , Subunidade beta de Receptor de Interleucina-2/genética , Ativação Linfocitária , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/genética , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores Virais/biossíntese , Receptores Virais/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Regulação para CimaRESUMO
PURPOSE: The purpose of the present study was to evaluate granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapy, which is known to stimulate potent and long-lasting antigen-specific immune responses, in combination with PD-1 blockade, which has been shown to augment cellular immune responses. EXPERIMENTAL DESIGN: Survival studies were done in the B16 melanoma and CT26 colon carcinoma tumor models. Immune monitoring studies were done in the B16 model. GM-CSF-secreting tumor cell immunotherapy was administered s.c. and the anti-PD-1 antibody was administered i.p. RESULTS: The studies reported here show that combining PD-1 blockade with GM-CSF-secreting tumor cell immunotherapy prolonged the survival of tumor-bearing animals compared with animals treated with either therapy alone. Prolonged survival correlated with strong antigen-specific T-cell responses detected by tetramer staining and an in vivo CTL assay, higher secretion levels of proinflammatory cytokines by splenocytes, and the persistence of functional CD8+ T cells in the tumor microenvironment. Furthermore, in the biweekly multiple treatment setting, repeated antigen-specific T-cell expansion was only observed following administration of the cellular immunotherapy with the PD-1 blockade and not when the cellular immunotherapy or PD-1 blockade was used as monotherapy. CONCLUSION: The combination of PD-1 blockade with GM-CSF-secreting tumor cell immunotherapy leads to significantly improved antitumor responses by augmenting the tumor-reactive T-cell responses induced by the cellular immunotherapy. Readministration of the cellular immunotherapy with the anti-PD-1 antibody in subsequent immunotherapy cycles was required to reactivate these T-cell responses.