RESUMEN
T cell immunoglobulin mucin domain-containing protein 3 (Tim-3) negatively regulates innate and adaptive immunity in cancer. To identify the mechanisms of Tim-3 in cancer immunity, we evaluated the effects of Tim-3 blockade in human and mouse melanoma. Here, we show that human programmed cell death 1-positive (PD-1+) Tim-3+CD8+ tumor-infiltrating lymphocytes (TILs) upregulate phosphatidylserine (PS), a receptor for Tim-3, and acquire cell surface myeloid markers from antigen-presenting cells (APCs) through transfer of membrane fragments called trogocytosis. Tim-3 blockade acted on Tim-3+ APCs in a PS-dependent fashion to disrupt the trogocytosis of activated tumor antigen-specific CD8+ T cells and PD-1+Tim-3+ CD8+ TILs isolated from patients with melanoma. Tim-3 and PD-1 blockades cooperated to disrupt trogocytosis of CD8+ TILs in 2 melanoma mouse models, decreasing tumor burden and prolonging survival. Deleting Tim-3 in dendritic cells but not in CD8+ T cells impeded the trogocytosis of CD8+ TILs in vivo. Trogocytosed CD8+ T cells presented tumor peptide-major histocompatibility complexes and became the target of fratricide T cell killing, which was reversed by Tim-3 blockade. Our findings have uncovered a mechanism Tim-3 uses to limit antitumor immunity.
Asunto(s)
Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Melanoma , Animales , Linfocitos T CD8-positivos , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Humanos , Linfocitos Infiltrantes de Tumor , Melanoma/patología , Ratones , Receptor de Muerte Celular Programada 1 , TrogocitosisRESUMEN
Ample evidence indicates that the gut microbiome is a tumor-extrinsic factor associated with antitumor response to anti-programmed cell death protein-1 (PD-1) therapy, but inconsistencies exist between published microbial signatures associated with clinical outcomes. To resolve this, we evaluated a new melanoma cohort, along with four published datasets. Time-to-event analysis showed that baseline microbiota composition was optimally associated with clinical outcome at approximately 1 year after initiation of treatment. Meta-analysis and other bioinformatic analyses of the combined data show that bacteria associated with favorable response are confined within the Actinobacteria phylum and the Lachnospiraceae/Ruminococcaceae families of Firmicutes. Conversely, Gram-negative bacteria were associated with an inflammatory host intestinal gene signature, increased blood neutrophil-to-lymphocyte ratio, and unfavorable outcome. Two microbial signatures, enriched for Lachnospiraceae spp. and Streptococcaceae spp., were associated with favorable and unfavorable clinical response, respectively, and with distinct immune-related adverse effects. Despite between-cohort heterogeneity, optimized all-minus-one supervised learning algorithms trained on batch-corrected microbiome data consistently predicted outcomes to programmed cell death protein-1 therapy in all cohorts. Gut microbial communities (microbiotypes) with nonuniform geographical distribution were associated with favorable and unfavorable outcomes, contributing to discrepancies between cohorts. Our findings shed new light on the complex interaction between the gut microbiome and response to cancer immunotherapy, providing a roadmap for future studies.
Asunto(s)
Microbioma Gastrointestinal , Melanoma , Microbiota , Bacterias/genética , Microbioma Gastrointestinal/genética , Humanos , Inmunoterapia/efectos adversos , Melanoma/tratamiento farmacológicoRESUMEN
Anti-programmed cell death protein 1 (PD-1) therapy provides long-term clinical benefits to patients with advanced melanoma. The composition of the gut microbiota correlates with anti-PD-1 efficacy in preclinical models and cancer patients. To investigate whether resistance to anti-PD-1 can be overcome by changing the gut microbiota, this clinical trial evaluated the safety and efficacy of responder-derived fecal microbiota transplantation (FMT) together with anti-PD-1 in patients with PD-1-refractory melanoma. This combination was well tolerated, provided clinical benefit in 6 of 15 patients, and induced rapid and durable microbiota perturbation. Responders exhibited increased abundance of taxa that were previously shown to be associated with response to anti-PD-1, increased CD8+ T cell activation, and decreased frequency of interleukin-8-expressing myeloid cells. Responders had distinct proteomic and metabolomic signatures, and transkingdom network analyses confirmed that the gut microbiome regulated these changes. Collectively, our findings show that FMT and anti-PD-1 changed the gut microbiome and reprogrammed the tumor microenvironment to overcome resistance to anti-PD-1 in a subset of PD-1 advanced melanoma.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Resistencia a Antineoplásicos , Trasplante de Microbiota Fecal , Melanoma/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias Cutáneas/terapia , Linfocitos T CD8-positivos/inmunología , Microbioma Gastrointestinal , Humanos , Interleucina-8/inmunología , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Células Mieloides/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Mass spectrometric analysis identified the peptide recognized by a cytotoxic T lymphocyte (CTL) specific for the chemically induced BALB/c Meth A sarcoma as derived from a 17beta-hydroxysteroid dehydrogenase type 12 (Hsd17b12) pseudogene present in the BALB/c genome, but only expressed in Meth A sarcoma. The sequence of the peptide is TYDKIKTGL and corresponds to Hsd17b12(114-122) with threonine instead of isoleucine at codon 114 and is designated Hsd17b12(114T). Immunization of mice with an Hsd17b12(114T) peptide-pulsed dendritic cell-based vaccine or a non-viral plasmid construct expressing the Hsd17b12(114T) peptide protected the mice from lethal Meth A tumor challenge in tumor rejection assays. A Hsd17b12(114-122) peptide-pulsed vaccine was ineffective in inducing resistance in mice to Meth A sarcoma. These results confirm the immunogenicity of the identified tumor peptide, as well as demonstrate the efficacies of these vaccine vehicles. These findings suggest that the role of the human homolog of Hsd17b12, HSD17B12, as a potential human tumor antigen be explored.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad/inmunología , Péptidos/inmunología , Seudogenes/inmunología , 17-Hidroxiesteroide Deshidrogenasas/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Femenino , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Péptidos/genética , Péptidos/metabolismo , Sarcoma Experimental/inmunología , Sarcoma Experimental/patología , Sarcoma Experimental/prevención & control , Linfocitos T Citotóxicos/inmunologíaRESUMEN
PURPOSE: Natural killer (NK) cells play a critical role in tumor immunosurveillance. Multiple activating and inhibitory receptors (IR) regulate NK-cell-mediated tumor control. The IR T-cell immunoglobulin and ITIM domain (TIGIT) and its counter-receptor CD226 exert opposite effects on NK-cell-mediated tumor reactivity. EXPERIMENTAL DESIGN: We evaluated the frequency, phenotype, and functions of NK cells freshly isolated from healthy donors and patients with melanoma with multiparameter flow cytometry. We assessed TIGIT and CD226 cell surface expression and internalization upon binding to CD155. We evaluated the role of IL15 and TIGIT blockade in increasing NK-cell-mediated cytotoxicity in vitro and in two mouse models. RESULTS: NK cells are present at low frequencies in metastatic melanoma, are dysfunctional, and downregulate both TIGIT and CD226 expression. As compared with TIGIT- NK cells, TIGIT+ NK cells exhibit higher cytotoxic capacity and maturation, but paradoxically lower cytotoxicity against CD155+ MHC class I-deficient melanoma cells. Membrane bound CD155 triggers CD226 internalization and degradation, resulting in decreased NK-cell-mediated tumor reactivity. IL15 increases TIGIT and CD226 gene expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade, increases NK-cell-mediated melanoma cytotoxicity in vitro and decreases tumor metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred NK cells enhances the antimetastatic activity of IL15, while CD226 blockade decreases the effects of IL15 and TIGIT blockade. CONCLUSIONS: Our findings support the development of novel combinatorial immunotherapy with IL15 and TIGIT blockade to promote NK-cell-mediated destruction of MHC class I-deficient melanoma, which are refractory to CD8+ T-cell-mediated immunity.See related commentary by Pietra et al., p. 5274.
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Interleucina-15/farmacología , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Receptores Inmunológicos/genética , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Interleucina-15/genética , Células Asesinas Naturales/efectos de los fármacos , Melanoma/sangre , Melanoma/genética , Melanoma/patología , Ratones , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Virales/genéticaRESUMEN
CD4+ Tregs impede T cell responses to tumors. They express multiple inhibitory receptors that support their suppressive functions, including T cell Ig and ITIM domain (TIGIT). In melanoma patients, we show that Tregs exhibit increased TIGIT expression and decreased expression of its competing costimulatory receptor CD226 as compared with CD4+ effector T cells, resulting in an increased TIGIT/CD226 ratio. Tregs failed to upregulate CD226 upon T cell activation. TIGIT+ Tregs are highly suppressive, stable, and enriched in tumors. TIGIT and CD226 oppose each other to augment or disrupt, respectively, Treg suppression and stability. A high TIGIT/CD226 ratio in Tregs correlates with increased Treg frequencies in tumors and poor clinical outcome upon immune checkpoint blockade. Altogether, our findings show that a high TIGIT/CD226 ratio in Tregs regulates their suppressive function and stability in melanoma. They provide the rationale for novel immunotherapies to activate CD226 in Tregs together with TIGIT blockade to counteract Treg suppression in cancer patients.
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Antígenos de Diferenciación de Linfocitos T/metabolismo , Melanoma/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos , Citocinas , Humanos , Inmunofenotipificación , Inmunoterapia , Activación de Linfocitos , Melanoma/terapia , Linfocitos T Reguladores/inmunología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
PURPOSE: Objective responses are reported in 34% to 37% of patients with programmed death-1 (PD-1)-naïve advanced melanoma treated with PD-1 inhibitors. Pre-existing CD8+ T-cell infiltrate and interferon (IFN) gene signature correlate with response to PD-1 blockade. Here, we report a phase Ib/II study of pembrolizumab/pegylated (PEG)-IFN combination in PD-1-naïve advanced melanoma. PATIENTS AND METHODS: PEG-IFN (1, 2, and 3 µg/kg per week) was dose escalated using a modified toxicity probability interval design in three cohorts of four patients each, whereas pembrolizumab was dosed at 2 mg/kg every 3 weeks in the phase Ib portion. Thirty-one patients were enrolled in the phase II portion. Primary objectives were safety and incidence of dose-limiting toxicities. Secondary objectives included objective response rate, progression-free survival (PFS), and overall survival. RESULTS: Forty-three patients with stage IV melanoma were enrolled in the phase Ib and II portions of the study and included in the analysis. At the data cutoff date (December 31, 2017), median follow-up duration was 25 months (range, 1 to 38 months). All 43 patients experienced at least one adverse event; grade 3/4 treatment-related adverse events occurred in 21 of 43 patients (48.8%). Objective responses were seen at all three dose levels among 43 evaluable patients. The objective response rate was 60.5%, with 46.5% of patients exhibiting ongoing response. Median PFS was 11.0 months in all patients and unreached in responders, whereas median overall survival remained unreached in all patients. The 2-year PFS rate was 46%. CONCLUSION: Pembrolizumab/PEG-IFN demonstrated an acceptable toxicity profile with promising evidence of clinical efficacy in PD-1-naïve metastatic melanoma. These results support the rationale to further investigate this pembrolizumab/PEG-IFN combination in this disease.
RESUMEN
Immune checkpoint inhibitors show great promise as therapy for advanced melanoma, heightening the need to determine the most effective use of these agents. Here, we report that programmed death-1(high) (PD-1(high)) tumor antigen (TA)-specific CD8(+) T cells present at periphery and at tumor sites in patients with advanced melanoma upregulate IL10 receptor (IL10R) expression. Multiple subsets of peripheral blood mononucleocytes from melanoma patients produce IL10, which acts directly on IL10R(+) TA-specific CD8(+) T cells to limit their proliferation and survival. PD-1 blockade augments expression of IL10R by TA-specific CD8(+) T cells, thereby increasing their sensitivity to the immunosuppressive effects of endogenous IL10. Conversely, IL10 blockade strengthened the effects of PD-1 blockade in expanding TA-specific CD8(+) T cells and reinforcing their function. Collectively, our findings offer a rationale to block both IL10 and PD-1 to strengthen the counteraction of T-cell immunosuppression and to enhance the activity of TA-specific CD8(+) T cell in advanced melanoma patients.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-10/fisiología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/fisiología , Neoplasias Cutáneas/inmunología , Escape del Tumor , Anticuerpos/farmacología , Células Cultivadas , Humanos , Tolerancia Inmunológica , Interleucina-10/antagonistas & inhibidores , Interleucina-10/inmunología , Activación de Linfocitos , Melanoma/metabolismo , Melanoma/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-10/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/fisiología , Receptor de Muerte Celular Programada 1/fisiología , Receptores Inmunológicos/fisiología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/genética , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Citocinas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Humanos , Memoria Inmunológica , Inmunofenotipificación , Subunidad beta del Receptor de Interleucina-2/biosíntesis , Subunidad beta del Receptor de Interleucina-2/genética , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/patología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/biosíntesis , Receptor de Muerte Celular Programada 1/genética , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores Virales/biosíntesis , Receptores Virales/genética , Especificidad del Receptor de Antígeno de Linfocitos T , Regulación hacia ArribaRESUMEN
Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Vacunas contra el Cáncer/uso terapéutico , Proliferación Celular , Activación de Linfocitos/genética , Melanoma/terapia , Proteínas de la Membrana/fisiología , Receptor de Muerte Celular Programada 1/fisiología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/patología , Vacunas contra el Cáncer/inmunología , Adyuvante de Freund/uso terapéutico , Receptor 2 Celular del Virus de la Hepatitis A , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/metabolismo , Lípidos/uso terapéutico , Melanoma/genética , Melanoma/inmunología , Oligodesoxirribonucleótidos/uso terapéutico , Especificidad del Receptor de Antígeno de Linfocitos T , Resultado del TratamientoRESUMEN
Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.