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BACKGROUND: Ageing leads to altered immune responses, resulting in higher susceptibility to certain infections in the elderly. Immune ageing is a heterogeneous process also associated with inflammaging, a low-grade chronic inflammation. Altered cytotoxic T cell responses and cytokine storm have previously been described in severe COVID-19 cases, however the parameters responsible for such immune response failures are not well known. The aim of our study was to characterize CD8+ T cells and cytokines associated with ageing, in a cohort of patients aged over 70 years stratified by COVID-19 severity. RESULTS: One hundred and four patients were included in the study. We found that, in older people, COVID-19 severity was associated with (i) higher level of GM-CSF, CXCL10 (IP-10), VEGF, IL-1ß, CCL2 (MCP-1) and the neutrophil to lymphocyte ratio (NLR), (ii) increased terminally differentiated CD8+T cells, and (ii) decreased early precursors CD8+ T stem cell-like memory cells (TSCM) and CD27+CD28+. The cytokines mentioned above were found at higher concentrations in the COVID-19+ older cohort compared to a younger cohort in which they were not associated with disease severity. CONCLUSIONS: Our results highlight the particular importance of the myeloid lineage in COVID-19 severity among older people. As GM-CSF and CXCL10 were not associated with COVID-19 severity in younger patients, they may represent disease severity specific markers of ageing and should be considered in older people care.
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BACKGROUND: Restoring plasma arginine levels through enteral administration of L-citrulline in critically ill patients may improve outcomes. We aimed to evaluate whether enteral L-citrulline administration reduced organ dysfunction based on the Sequential Organ Failure Assessment (SOFA) score and affected selected immune parameters in mechanically ventilated medical intensive care unit (ICU) patients. METHODS: A randomized, double-blind, multicenter clinical trial of enteral administration of L-citrulline versus placebo for critically ill adult patients under invasive mechanical ventilation without sepsis or septic shock was conducted in four ICUs in France between September 2016 and February 2019. Patients were randomly assigned to receive enteral L-citrulline (5 g) every 12 h for 5 days or isonitrogenous, isocaloric placebo. The primary outcome was the SOFA score on day 7. Secondary outcomes included SOFA score improvement (defined as a decrease in total SOFA score by 2 points or more between day 1 and day 7), secondary infection acquisition, ICU length of stay, plasma amino acid levels, and immune biomarkers on day 3 and day 7 (HLA-DR expression on monocytes and interleukin-6). RESULTS: Of 120 randomized patients (mean age, 60 ± 17 years; 44 [36.7%] women; ICU stay 10 days [IQR, 7-16]; incidence of secondary infections 25 patients (20.8%)), 60 were allocated to L-citrulline and 60 were allocated to placebo. Overall, there was no significant difference in organ dysfunction as assessed by the SOFA score on day 7 after enrollment (4 [IQR, 2-6] in the L-citrulline group vs. 4 [IQR, 2-7] in the placebo group; MannâWhitney U test, p = 0.9). Plasma arginine was significantly increased on day 3 in the treatment group, while immune parameters remained unaffected. CONCLUSION: Among mechanically ventilated ICU patients without sepsis or septic shock, enteral L-citrulline administration did not result in a significant difference in SOFA score on day 7 compared to placebo. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02864017 (date of registration: 11 August 2016).
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Sepsis , Choque Séptico , Adulto , Humanos , Femenino , Persona de Mediana Edad , Anciano , Masculino , Puntuaciones en la Disfunción de Órganos , Choque Séptico/complicaciones , Citrulina/farmacología , Citrulina/uso terapéutico , Insuficiencia Multiorgánica/etiología , Enfermedad Crítica/terapia , Respiración Artificial/efectos adversos , Sepsis/tratamiento farmacológico , Sepsis/complicaciones , Unidades de Cuidados Intensivos , Suplementos Dietéticos , Arginina/uso terapéuticoRESUMEN
Bevacizumab (Bv) remains frequently prescribed in glioblastoma (GBM) patients, especially at recurrence. We conducted a prospective clinical trial with 29 recurrent GBM patients treated with Bv alone with a longitudinal follow-up of different circulating immune cells [complete blood count, myeloid-derived suppressor cells (MDSCs), classical, intermediate, non-classical and Tie2 monocytes, VEGFR1+ and regulatory T cells (Treg)]. We observed a significant increase for leucocytes, neutrophils, eosinophils and classical monocytes and a decrease for the fraction of Treg during the treatment. The best prognostic values for survival under Bv were obtained for basal neutrophils and Treg. Counts below 3.9 G/L for neutrophils and above 0.011 G/L for Treg were associated with an overall survival of 17.5 and 19.9 months, respectively, as compared with 5.4 and 5.6 months, respectively, for counts above and below these cutoffs (p = 0.004 and p < 0.001). No prognostic impact was observed for neutrophils in a retrospective cohort of 26 patients treated with nitrosoureas alone. In another retrospective validation cohort of 61 GBM patients treated at recurrence with a Bv-containing regimen, an interaction was observed between neutrophils and corticosteroid intake. The predictive value of neutrophils on survival under Bv was lost in patients treated with corticosteroids, when steroid-free patients with a low neutrophil count had a particularly long median survival of 3.4 years. These two simply accessible criteria (basal neutrophils and steroid intake) could be used to reserve this relatively costly treatment for patients likely to be the most responsive to Bv and prevent unnecessary side effects in others.
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Bevacizumab/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Neutrófilos/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Femenino , Glioblastoma/inmunología , Glioblastoma/patología , Humanos , Estimación de Kaplan-Meier , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Neutrófilos/inmunología , Neutrófilos/metabolismo , Estudios Prospectivos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Tumors with the help of the surrounding environment facilitate the immune suppression in patients, and immunotherapy can counteract this inhibition. Among immunotherapeutic strategies, the immunostimulatory cytokine IL-15 could represent a serious candidate for the reactivation of antitumor immunity. However, exogenous IL-15 may have a limited impact on patients with cancer due to its dependency on IL-15Rα frequently downregulated in cancer patients. In this work, we studied the antitumor activity of the IL-15 superagonist receptor-linker-IL-15 (RLI), designed to bypass the need of endogenous IL-15Rα. RLI consists of human IL-15 covalently linked to the human IL-15Rα sushi(+) domain. In a mouse model of colorectal carcinoma, RLI as a stand-alone treatment could limit tumor outgrowth only when initiated at an early time of tumor development. At a later time, RLI was not effective, coinciding with the strong accumulation of terminally exhausted programmed cell death-1 (PD-1)(high) T cell Ig mucin-3(+) CD8(+) T cells, suggesting that RLI was not able to reactivate terminally exhausted CD8(+) T cells. Combination with PD-1 blocking Ab showed synergistic activity with RLI, but not with IL-15. RLI could induce a greater accumulation of memory CD8(+) T cells and a stronger effector function in comparison with IL-15. Ex vivo stimulation of tumor-infiltrated lymphocytes from 16 patients with renal cell carcinoma demonstrated 56% of a strong tumor-infiltrated lymphocyte reactivation with the combination anti-PD-1/RLI compared with 43 and 6% with RLI or anti-PD-1, respectively. Altogether, this work provides evidence that the sushi-IL-15Rα/IL-15 fusion protein RLI enhances antitumor activity of anti-PD-1 treatment and is a promising approach to stimulate host immunity.
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Adenocarcinoma/terapia , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Linfocitos T CD8-positivos/efectos de los fármacos , Carcinoma de Células Renales/terapia , Neoplasias del Colon/terapia , Inmunoterapia/métodos , Interleucina-15/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/uso terapéutico , Adenocarcinoma/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Memoria Inmunológica/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacosRESUMEN
Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.
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Autoantígenos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Vacunas contra el Cáncer/administración & dosificación , Neoplasias/terapia , Receptores CCR4/antagonistas & inhibidores , Escape del Tumor/efectos de los fármacos , Animales , Antígenos de Neoplasias/inmunología , Antineoplásicos/administración & dosificación , Autoantígenos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Terapia Combinada , Modelos Animales de Enfermedad , Femenino , Humanos , Factores Inmunológicos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Especificidad del Receptor de Antígeno de Linfocitos T/efectos de los fármacos , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Escape del Tumor/inmunologíaRESUMEN
CXCR6 is a receptor for the chemokine CXCL16, which exists as a membrane or soluble form. CXCR6 is a marker for resident memory T (TRM) cells that plays a role in immunosurveillance through their interaction with epithelial cells. The interaction of CXCR6 with CXCL16 expressed at the membrane of certain subpopulations of intratumor dendritic cells (DC) called DC3, ideally positions these CXCR6+ T cells to receive a proliferation signal from IL-15 also presented by DC3. Mice deficient in cxcr6 or blocking the interaction of CXCR6 with its ligand, experience a poorer control of tumor proliferation by CD8+ T cells, but also by NKT cells especially in the liver. Intranasal vaccination induces CXCL16 production in the lungs and is associated with infiltration by TRM expressing CXCR6, which are then required for the efficacy of anti-tumor vaccination. Therapeutically, the addition of CXCR6 to specific CAR-T cells enhances their intratumoral accumulation and prolongs survival in animal models of pancreatic, ovarian and lung cancer. Finally, CXCR6 is part of immunological signatures that predict response to immunotherapy based on anti-PD-(L)1 in various cancers. In contrast, a protumoral role of CXCR6+T cells has also been reported mainly in Non-alcoholic steatohepatitis (NASH) due to a non-antigen specific mechanism. The targeting and amplification of antigen-specific TRM expressing CXCR6 and its potential use as a biomarker of response to immunotherapy opens new perspectives in cancer treatment.
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Células T Asesinas Naturales , Neoplasias , Ratones , Animales , Receptores CXCR6 , Linfocitos T CD8-positivos , Quimiocina CXCL16 , Neoplasias/terapiaRESUMEN
PURPOSE: CD70 is a costimulatory molecule known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). Clear-cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors; however, the clinical consequences of CD70 expression remain unclear. EXPERIMENTAL DESIGN: Tumor tissue from 25 patients with ccRCC was assessed for the expression of CD27 and CD70 in situ using multiplex immunofluorescence. CD27+ T-cell phenotypes in tumors were analyzed by flow cytometry and their gene expression profile were analyzed by single-cell RNA sequencing then confirmed with public data. Baseline sCD27 was measured in 81 patients with renal cell carcinoma (RCC) treated with immunotherapy (35 for training cohort and 46 for validation cohort). RESULTS: In the tumor microenvironment, CD27+ T cells interacted with CD70-expressing tumor cells. Compared with CD27- T cells, CD27+ T cells exhibited an apoptotic and dysfunctional signature. In patients with RCC, the intratumoral CD27-CD70 interaction was significantly correlated with the plasma sCD27 concentration. High sCD27 levels predicted poor overall survival in patients with RCC treated with anti-programmed cell death protein 1 in both the training and validation cohorts but not in patients treated with antiangiogenic therapy. CONCLUSIONS: In conclusion, we demonstrated that sCD27, a surrogate marker of T-cell dysfunction, is a predictive biomarker of resistance to immunotherapy in RCC. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be extended to other tumors.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Ligando CD27/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Inmunoterapia , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Microambiente TumoralRESUMEN
The modulation of subpopulations of pro-angiogenic monocytes (VEGFR-1+CD14 and Tie2+CD14) was analyzed in an ancillary study from the prospective PazopanIb versus Sunitinib patient preferenCE Study (PISCES) (NCT01064310), where metastatic renal cell carcinoma (mRCC) patients were treated with two anti-angiogenic drugs, either sunitinib or pazopanib. Blood samples from 86 patients were collected prospectively at baseline (T1), and at 10 weeks (T2) and 20 weeks (T3) after starting anti-angiogenic therapy. Various subpopulations of myeloid cells (monocytes, VEGFR-1+CD14 and Tie2+CD14 cells) decreased during treatment. When patients were divided into two subgroups with a decrease (defined as a >20% reduction from baseline value) (group 1) or not (group 2) at T3 for VEGFR-1+CD14 cells, group 1 patients presented a median PFS and OS of 24 months and 37 months, respectively, compared with a median PFS of 9 months (p = 0.032) and a median OS of 16 months (p = 0.033) in group 2 patients. The reduction in Tie2+CD14 at T3 predicted a benefit in OS at 18 months after therapy (p = 0.04). In conclusion, in this prospective clinical trial, a significant decrease in subpopulations of pro-angiogenic monocytes was associated with clinical response to anti-angiogenic drugs in patients with mRCC.
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Inhibidores de la Angiogénesis/uso terapéutico , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Monocitos/patología , Neovascularización Patológica/tratamiento farmacológico , Inhibidores de la Angiogénesis/farmacología , Animales , Antígenos CD/metabolismo , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/patología , Modelos Animales de Enfermedad , Humanos , Neoplasias Renales/sangre , Neoplasias Renales/patología , Ratones Endogámicos BALB C , Células Mieloides/efectos de los fármacos , Células Mieloides/patología , Metástasis de la Neoplasia , Sunitinib/farmacología , Sunitinib/uso terapéutico , Resultado del Tratamiento , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: In multiple oncological settings, expression of the coinhibitory ligand PD-L1 by malignant cells and tumor infiltration by immune cells expressing coinhibitory receptors such as PD-1, CTLA4, LAG-3, or TIM-3 conveys prognostic or predictive information. Conversely, the impact of these features of the tumor microenvironment on disease outcome among high-grade serous carcinoma (HGSC) patients remains controversial. EXPERIMENTAL DESIGN: We harnessed a retrospective cohort of 80 chemotherapy-naïve HGSC patients to investigate PD-L1 expression and tumor infiltration by CD8+ T cells, CD20+ B cells, DC-LAMP+ dendritic cells as well as by PD-1+, CTLA4+, LAG-3+, and TIM-3+ cells in relation with prognosis and function orientation of the tumor microenvironment. IHC data were complemented with transcriptomic and functional studies on a second prospective cohort of freshly resected HGSC samples. In silico analysis of publicly available RNA expression data from 308 HGSC samples was used as a confirmatory approach. RESULTS: High levels of PD-L1 and high densities of PD-1+ cells in the microenvironment of HGSCs were strongly associated with an immune contexture characterized by a robust TH1 polarization and cytotoxic orientation that enabled superior clinical benefits. Moreover, PD-1+TIM-3+CD8+ T cells presented all features of functional exhaustion and correlated with poor disease outcome. However, although PD-L1 levels and tumor infiltration by TIM-3+ cells improved patient stratification based on the intratumoral abundance of CD8+ T cells, the amount of PD-1+ cells failed to do so. CONCLUSIONS: Our data indicate that PD-L1 and TIM-3 constitute prognostically relevant biomarkers of active and suppressed immune responses against HGSC, respectively.
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Biomarcadores de Tumor/metabolismo , Linfocitos T CD8-positivos/inmunología , Carcinoma Epitelial de Ovario/inmunología , Cistadenocarcinoma Seroso/inmunología , Regulación Neoplásica de la Expresión Génica , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Adulto , Anciano , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/inmunología , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/patología , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patología , Femenino , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Humanos , Proteínas de Membrana de los Lisosomas/inmunología , Proteínas de Membrana de los Lisosomas/metabolismo , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Pronóstico , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
T cells harboring multiple co-inhibitory molecules lose their anti-tumoral functionality. PD-1 is a clinically approved target in cancer therapy, but its expression alone does not mean dysfunctionality. The expression of Tim-3 on numerous cell types (T cell, Treg, dendritic cell, myeloid cells) favors tumor escape to immune cells. Within many tumors, PD-1/Tim-3 coexpressing CD8-T cells lose their ability to secrete cytokines (IFNγ, IL-2, TNFα) and their intratumoral infiltration correlates with a bad prognosis. Tim-3 recently appeared as a potential biomarker of anti-PD-1 resistance. Combined blockade of PD-1 and Tim-3 axis demonstrated potent clinical efficacy in preclinical models and reinforced the rationale of using an anti-Tim-3 to override tumor resistance.
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Biomarcadores de Tumor , Receptor 2 Celular del Virus de la Hepatitis A/fisiología , Oncología Médica/tendencias , Terapia Molecular Dirigida/métodos , Animales , Biomarcadores de Tumor/fisiología , Células Dendríticas/fisiología , Receptor 2 Celular del Virus de la Hepatitis A/antagonistas & inhibidores , Humanos , Oncología Médica/métodos , Terapia Molecular Dirigida/tendencias , Linfocitos T Reguladores/fisiología , Escape del Tumor/genética , Escape del Tumor/inmunologíaRESUMEN
The efficacy of an antitumoral vaccine relies both on the choice of the antigen targeted and on its design. The tumor antigen survivin is an attractive target to develop therapeutic cancer vaccines because of its restricted over-expression and vital functions in most human tumors. Accordingly, several clinical trials targeting survivin in various cancer indications have been conducted. Most of them relied on short peptide-based vaccines and showed promising, but limited clinical results. In this study, we investigated the immunogenicity and therapeutic efficacy of a new long synthetic peptide (LSP)-based cancer vaccine targeting the tumor antigen survivin (SVX). This SVX vaccine is composed of three long synthetic peptides containing several CD4+ and CD8+ T-cell epitopes, which bind to various HLA class II and class I molecules. Studies in healthy individuals showed CD4+ and CD8+ T-cell immunogenicity of SVX peptides in human, irrespective of the individual's HLA types. Importantly, high frequencies of spontaneous T-cell precursors specific to SVX peptides were also detected in the blood of various cancer patients, demonstrating the absence of tolerance against these peptides. We then demonstrated SVX vaccine's high therapeutic efficacy against four different established murine tumor models, associated with its capacity to generate both specific cytotoxic CD8+ and multifunctional Th1 CD4+ T-cell responses. When tumors were eradicated, generated memory T-cell responses protected against rechallenge allowing long-term protection against relapses. Treatment with SVX vaccine was also found to reshape the tumor microenvironment by increasing the tumor infiltration of both CD4+ and CD8+ T cells but not Treg cells therefore tipping the balance toward a highly efficient immune response. These results highlight that this LSP-based SVX vaccine appears as a promising cancer vaccine and warrants its further clinical development.
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Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment. The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.
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Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/inmunología , Técnica del Anticuerpo Fluorescente/métodos , Receptor de Muerte Celular Programada 1/metabolismo , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Femenino , Humanos , Masculino , Estudios Retrospectivos , Microambiente TumoralRESUMEN
The large family of costimulatory molecules plays a crucial role in regulation of the immune response. These molecules modulate TCR signalling via phosphorylation cascades. Some of the coinhibitory members of this family, such as PD-1 and CTLA-4, already constitute approved targets in cancer therapy and, since 2011, have opened a new area of antitumour immunotherapy. Many antibodies targeting other inhibitory receptors (Tim-3, VISTA, Lag-3 and so on) or activating costimulatory molecules (OX40, GITR and so on) are under evaluation. These antibodies have multiple mechanisms of action. At the cellular level, these antibodies restore the activation signalling pathway and reprogram T cell metabolism. Tumour cells become resistant to apoptosis when an intracellular PD-L1 signalling is blocked. CD8+ T cells are considered to be the main effectors of the blockade of inhibitory receptors. Certain CD8+ T cell subsets, such as non-hyperexhausted (CD28+, T-bethigh, PD-1int), follicular-like (CXCR-5+) or resident memory CD8+ T cells, are more prone to be reactivated by anti-PD-1/PD-L1 monoclonal antibody (mAb). In the future, the challenge will be to rationally combine drugs able to make the tumour microenvironment more permissive to immunotherapy in order to potentiate its clinical activity.
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Inhibitory receptors expressed by T cells mediate tolerance to tumor antigens, with coexpression of these receptors exacerbating this dysfunctional state. Using the VectraR automated multiparametric immunofluorescence technique, we quantified intratumoral CD8+ T cells coexpressing the inhibitory receptors PD-1 and Tim-3 from patients with renal cell carcinoma (RCC). A second validation cohort measured the same parameters by cytometry. The percentage of tumor-infiltrating CD8+ T cells coexpressing PD-1 and Tim-3 correlated with an aggressive phenotype and a larger tumor size at diagnosis. Coexpression of PD-1 and Tim-3 above the median conferred a higher risk of relapse and a poorer 36-month overall survival. Notably, other CD8+T-cell subsets did not exert a similar effect on overall survival. Moreover, only the PD-1+Tim-3+ subset of CD8+ T cells exhibited impaired function after stimulation. Our findings establish intratumoral Tim-3+PD1+CD8+ T cells as critical mediators of an aggressive phenotype in RCC. Use of the Vectra tool may be useful to identify similarly critical prognostic and predictive biomarkers in other tumor types and their response to immunotherapy. Cancer Res; 77(5); 1075-82. ©2016 AACR.
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Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/inmunología , Receptor 2 Celular del Virus de la Hepatitis A/biosíntesis , Neoplasias Renales/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Carcinoma de Células Renales/patología , Estudios de Cohortes , Receptor 2 Celular del Virus de la Hepatitis A/inmunología , Humanos , Inmunofenotipificación , Neoplasias Renales/patología , Estudios Retrospectivos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Anaplastic lymphoma kinase (ALK) inhibitors have been successfully developed for non-small cell lung carcinoma (NSCLC) displaying chromosomal rearrangements of the ALK gene, but unfortunately resistance invariably occurs. Blockade of the PD-1-PD-L1/2 inhibitory pathway constitutes a breakthrough for the treatment of NSCLC. Some predictive biomarkers of clinical response to this therapy are starting to emerge, such as PD-L1 expression by tumor/stromal cells and infiltration by CD8+ T cells expressing PD-1. To more effectively integrate all of these potential biomarkers of clinical response to immunotherapy, we have developed a multiparametric immunofluorescence technique with automated immune cell counting to comprehensively analyze the tumor microenvironment of ALK-positive adenocarcinoma (ADC). When analyzed as either a continuous or a dichotomous variable, the mean number of tumor cells expressing PD-L1 (p = 0.012) and the percentage of tumor cells expressing PD-L1 were higher in ALK-positive ADC than in EGFR-mutated ADC or WT (non-EGFR-mutated and non-KRAS-mutated) NSCLC. A very strong correlation between PD-L1 expression on tumor cells and intratumoral infiltration by CD8+ T cells was observed, suggesting that an adaptive mechanism may partly regulate this expression. A higher frequency of tumors combining positive PD-L1 expression and infiltration by intratumoral CD8+ T cells or PD-1+CD8+ T cells was also observed in ALK-positive lung cancer patients compared with EGFR-mutated (p = 0.03) or WT patients (p = 0.012). These results strongly suggest that a subgroup of ALK-positive lung cancer patients may constitute good candidates for anti-PD-1/-PD-L1 therapies.
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PURPOSE: E75, a peptide derived from the Her2/neu protein, is the most clinically advanced vaccine approach against breast cancer. In this study, we aimed to optimize the E75 vaccine using a delivery vector targeting dendritic cells, the B-subunit of Shiga toxin (STxB), and to assess the role of various parameters (Her2/neu expression, combination with trastuzumab) in the efficacy of this cancer vaccine in a relevant preclinical model. EXPERIMENTAL DESIGN: We compared the differential ability of the free E75 peptide or the STxB-E75 vaccine to elicit CD8(+) T cells, and the impact of the vaccine on murine HLA-A2 tumors expressing low or high levels of Her2/neu. RESULTS: STxB-E75 synergized with granulocyte macrophage colony-stimulating factors and CpG and proved to be more efficient than the free E75 peptide in the induction of multifunctional and high-avidity E75-specific anti-CD8(+) T cells resulting in a potent tumor protection in HLA-A2 transgenic mice. High expression of HER2/neu inhibited the expression of HLA-class I molecules, leading to a poor recognition of human or murine tumors by E75-specific cytotoxic CD8(+) T cells. In line with these results, STxB-E75 preferentially inhibited the growth of HLA-A2 tumors expressing low levels of Her2/neu. Coadministration of anti-Her2/neu mAb potentiated this effect. CONCLUSIONS: STxB-E75 vaccine is a potent candidate to be tested in patients with low Her2/neu-expressing tumors. It could also be indicated in patients expressing high levels of Her2/neu and low intratumoral T-cell infiltration to boost the recruitment of T cells-a key parameter in the efficacy of anti-Her2/neu mAb therapy. Clin Cancer Res; 22(16); 4133-44. ©2016 AACR.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Neoplasias/genética , Neoplasias/inmunología , Receptor ErbB-2/inmunología , Animales , Antígenos de Neoplasias/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Humanos , Melanoma Experimental , Ratones , Ratones Transgénicos , Neoplasias/patología , Neoplasias/terapia , Fragmentos de Péptidos/inmunología , Receptor ErbB-2/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Various arguments suggest that CD8+ T lymphocytes play a major role in the control of cytomegalovirus (CMV) infection. The detection of CMV-specific CD8+ T cells may therefore provide additional information about CMV virus detection to predict the risk of development of CMV disease, especially in immunodepressed transplant recipients. We compared and tested various experimental conditions to optimize an enzyme-linked immunospot assay (Elispot) assay for the detection of CMV-specific CD8+ T lymphocytes. The indirect Elispot assay with one six-day in vitro sensitization step was found to be the most sensitive method to detect CMV-specific CD8+ T cells compared to direct Elispot with unfractionated peripheral blood mononuclear cells or purified CD8+ T cells. We showed that low doses of interleukin-2 during the in vitro culture enhanced the sensitivity of this test, and tetramer staining was performed to verify the high efficiency of this in vitro stimulation step. We directly loaded the specific CMV peptide during the Elispot assay and demonstrated that the use of T2 cells did not improve its sensitivity. Elispot for the detection of interferon-gamma appears to be more sensitive and reliable than measurement of tumor necrosis factor alpha or granzyme B. This technique was successfully applied to detect CMV-specific CD8+ T cells in human leukocyte antigen A2 (HLA-A2) and HLA-B7 healthy patients and in one lymphopenic post-transplant patient with positive CMV serology. This highly sensitive test may be a useful tool to assess T-cell immunity directed against CMV in immunodepressed patients.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Línea Celular , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/virología , Granzimas , Antígeno HLA-A2/inmunología , Humanos , Técnicas para Inmunoenzimas/métodos , Interferón gamma/metabolismo , Interleucina-2/farmacología , Trasplante de Riñón/inmunología , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Fragmentos de Péptidos/inmunología , Serina Endopeptidasas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The type of T cell polarization and simultaneous production of multiple cytokines have been correlated with vaccine efficacy. ELISpot is a T cell detection technique optimized for the measurement of a secreted cytokine at the single cell level. The FluoroSpot assay differs from ELISpot by the use of multiple fluorescent-labeled anticytokine detection antibodies, allowing optimal measurement of multiple cytokines. In the present study, we show that an IFNγ/IL-10 FluoroSpot assay is more sensitive than flow cytometry to detect Tr1 regulatory T cells, an immunosuppressive T cell population characterized by the production of IL-10 and IFNγ. As many tolerogenic vaccines are designed to induce these Tr1 cells, this FluoroSpot test could represent a standard method for the detection of these cells in the future. The use of an IFNγ/IL-2 FluoroSpot assay during influenza vaccine monitoring showed that the influenza-specific IL-2-producing T-cell response was the dominant response both before and after vaccine administration. This study therefore questions the rationale of using the single-color IFNγ ELISpot as the standard technique to monitor vaccine-specific T-cell response. Using this same test, a trend was also observed between baseline levels of IFNγ T cell response and T cell vaccine response. In addition, a lower IFNγ+IL-2+ T-cell response after vaccine was observed in the group of patients treated with TNFα inhibitors (P=0.08). This study therefore supports the use of the FluoroSpot assay due to its robustness, versatility and the complementary information that it provides compared with ELISpot or flow cytometry to monitor vaccine-specific T-cell responses.
Asunto(s)
Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Interferón gamma/análisis , Interleucina-10/análisis , Interleucina-2/análisis , Linfocitos T Reguladores/inmunología , Ensayo de Immunospot Ligado a Enzimas/métodos , Fluorescencia , Humanos , Immunoblotting/métodos , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/inmunología , OsteoprotegerinaRESUMEN
Although many human cancers are located in mucosal sites, most cancer vaccines are tested against subcutaneous tumors in preclinical models. We therefore wondered whether mucosa-specific homing instructions to the immune system might influence mucosal tumor outgrowth. We showed that the growth of orthotopic head and neck or lung cancers was inhibited when a cancer vaccine was delivered by the intranasal mucosal route but not the intramuscular route. This antitumor effect was dependent on CD8⺠T cells. Indeed, only intranasal vaccination elicited mucosal-specific CD8⺠T cells expressing the mucosal integrin CD49a. Blockade of CD49a decreased intratumoral CD8⺠T cell infiltration and the efficacy of cancer vaccine on mucosal tumor. We then showed that after intranasal vaccination, dendritic cells from lung parenchyma, but not those from spleen, induced the expression of CD49a on cocultured specific CD8⺠T cells. Tumor-infiltrating lymphocytes from human mucosal lung cancer also expressed CD49a, which supports the relevance and possible extrapolation of these results in humans. We thus identified a link between the route of vaccination and the induction of a mucosal homing program on induced CD8⺠T cells that controlled their trafficking. Immunization route directly affected the efficacy of the cancer vaccine to control mucosal tumors.