The identification of activation pathways linked to antitumor T-cell polyfunctionality in long surviving patients is of great relevance in the new era of immunotherapy. We have recently reported that dacarbazine (DTIC) injected one day before peptide-vaccination plus IFN-α improves the antitumor lytic activity and enlarges the repertoire of Melan-A-specific T-cell clones, as compared with vaccination alone, impacting the overall survival of melanoma patients. To identify the mechanisms responsible for this improvement of the immune response, we have analyzed the endogenous and treatment-induced antigen (Ag)-specific response in a panel of Melan-A-specific CD8+ T-cell clones in terms of differentiation phenotype, inhibitory receptor profile, polyfunctionality and AKT activation. Here, we show that Melan-A-specific CD8+ T cells isolated from patients treated with chemoimmunotherapy possess a late differentiated phenotype as defined by the absence of CD28 and CD27 co-stimulatory molecules and high levels of LAG-3, TIM-3 and PD-1 inhibitory receptors. Nevertheless, they show higher proliferative potential and an improved antitumor polyfunctional effector profile in terms of co-production of TNF-α, IFNγ and Granzyme-B (GrB) compared with cells derived from patients treated with vaccination alone. Polyfunctionality is dependent on an active AKT signaling related to the engagement of the co-stimulatory molecule ICOS. We suggest that this phenotypic and functional signature is dictated by a fine-tuned balance between TCR triggering, AKT activation, co-stimulatory and inhibitory signals induced by chemoimmunotherapy and may be associated with antitumor T cells able to protect patients from tumor recurrence.

We have recently described that DNA-damage inducing drug DTIC, administered before peptide (Melan-A and gp100)-vaccination, improves anti-tumor CD8+ Melan-A-specific T-cell functionality, enlarges the Melan-A+ TCR repertoire and impacts the overall survival of melanoma patients. To identify whether the two Ags employed in the vaccination differently shape the anti-tumor response, herein we have carried out a detailed analysis of phenotype, anti-tumor functionality and TCR repertoire in treatment-driven gp100-specific CD8+ T cells, in the same patients previously analyzed for Melan-A. We found that T-cell clones isolated from patients treated with vaccination alone possessed an Early/intermediate differentiated phenotype, whereas T cells isolated after DTIC plus vaccination were late-differentiated. Sequencing analysis of the TCRBV chains of 29 treatment-driven gp100-specific CD8+ T-cell clones revealed an oligoclonal TCR repertoire irrespective of the treatment schedule. The high anti-tumor activity observed in T cells isolated after chemo-immunotherapy was associated with low PD-1 expression. Differently, T-cell clones isolated after peptide-vaccination alone expressed a high level of PD-1, along with LAG-3 and TIM-3, and were neither tumor-reactive nor polyfunctional. Blockade of PD-1 reversed gp100-specific CD8+ T-cell dysfunctionality, confirming the direct role of this co-inhibitory molecule in suppressing anti-tumor activity, differently from what we have previously observed for Melan-A+CD8+ T cells, expressing PD-1 but highly functional. These findings indicate that the functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen nature, T-cell immune-checkpoints phenotype, TCR repertoire diversity and anti-tumor T-cell quality and highlights the importance of integrating these parameters to develop effective immunotherapeutic strategies.

Table of contentsMELANOMA BRIDGE 2015KEYNOTE SPEAKER PRESENTATIONSMolecular and immuno-advancesK1 Immunologic and metabolic consequences of PI3K/AKT/mTOR activation in melanomaVashisht G. Y. Nanda, Weiyi Peng, Patrick Hwu, Michael A. DaviesK2 Non-mutational adaptive changes in melanoma cells exposed to BRAF and MEK inhibitors help the establishment of drug resistanceGennaro Ciliberto, Luigi Fattore, Debora Malpicci, Luigi Aurisicchio, Paolo Antonio Ascierto, Carlo M. Croce, Rita ManciniK3 Tumor-intrinsic beta-catenin signaling mediates tumor-immune avoidanceStefani Spranger, Thomas F. GajewskiK4 Intracellular tumor antigens as a source of targets of antibody-based immunotherapy of melanomaYangyang Wang, Soldano FerroneCombination therapiesK5 Harnessing radiotherapy to improve responses to immunotherapy in cancerClaire Vanpouille-Box, Erik Wennerberg, Karsten A. Pilones, Silvia C. Formenti, Sandra DemariaK6 Creating a T cell-inflamed tumor microenvironment overcomes resistance to checkpoint blockadeHaidong Tang, Yang Wang, Yang-Xin FuK7 Biomarkers for treatment decisions?Reinhard DummerK8 Combining oncolytic therapies in the era of checkpoint inhibitorsIgor PuzanovK9 Immune checkpoint blockade for melanoma: should we combine or sequence ipilimumab and PD-1 antibody therapy?Michael A. PostowNews in immunotherapyK10 An update on adjuvant and neoadjuvant therapy for melanomAhmad TarhiniK11 Targeting multiple inhibitory receptors in melanomaJoe-Marc Chauvin, Ornella Pagliano, Julien Fourcade, Zhaojun Sun, Hong Wang, Cindy Sanders, John M. Kirkwood, Tseng-hui Timothy Chen, Mark Maurer, Alan J. Korman, Hassane M. ZarourK12 Improving adoptive immune therapy using genetically engineered T cellsDavid F. StroncekTumor microenvironment and biomarkersK13 Myeloid cells and tumor exosomes: a crosstalk for assessing immunosuppression?Veronica Huber, Licia RivoltiniK14 Update on the SITC biomarker taskforce: progress and challengesMagdalena ThurinWorld-wide immunoscore task force: an updateK15 The immunoscore in colorectal cancer highlights the importance of digital scoring systems in surgical pathologyTilman Rau, Alessandro LugliK16 The immunoscore: toward an integrated immunomonitoring from the diagnosis to the follow up of cancer’s patientsFranck PagèsEconomic sustainability of melanoma treatments: regulatory, health technology assessment and market access issuesK17 Nivolumab, the regulatory experience in immunotherapyJorge Camarero, Arantxa SanchoK18 Evidence to optimize access for immunotherapiesClaudio JommiORAL PRESENTATIONSMolecular and immuno-advancesO1 Ipilimumab treatment results in CD4 T cell activation that is concomitant with a reduction in Tregs and MDSCsYago Pico de Coaña, Maria Wolodarski, Yuya Yoshimoto, Giusy Gentilcore, Isabel Poschke, Giuseppe V. Masucci, Johan Hansson, Rolf KiesslingO2 Evaluation of prognostic and therapeutic potential of COX-2 and PD-L1 in primary and metastatic melanomaGiosuè Scognamiglio, Francesco Sabbatino, Federica Zito Marino, Anna Maria Anniciello, Monica Cantile, Margherita Cerrone,...

The relationship between PD-1 expression and T-cell effector functions is still unclear, although crucial in the new era of immunotherapy targeting the PD-1/PDL-1 pathway. We have recently reported that Melan-A-specific CD8+ T-cells isolated from melanoma patients treated with combined chemo-immunotherapy (dacarbazine before peptide Melan-A/gp100-vaccination), show an enlarged T-cell repertoire, CD28− PD-1+AKT+ phenotype with high anti-tumor polyfunctionality sustained by ICOS (Franzese et al, OncoImmunology, in press). Herein, we have analyzed the gp100-specific CD8+ T-cell clones isolated from the same patients. Cells have been characterized for gp-100 specificity, and TCR beta-chain sequencing analysis evidenced that they possess an oligoclonal repertoire. The relationship among inhibitory receptors (PD-1, TIM-3 and LAG-3), co-stimulatory molecules (CD27 and CD28) and effector functions of T cells has been studied. Taking advantage from a battery of Melan-A- and gp100-specific T-cell clones, here we show that gp100-CD8+ T cells with a CD28+ PD-1+AKT+ phenotype are unable to kill gp100-expressing melanoma cells, differently from data obtained with Melan-A-specific clones showing a CD28− PD-1+ AKT+ phenotype, and able to lyse tumor cells with high efficiency. Studies are in progress to identify the biochemical pathways involved in the control of functionality of PD-1-expressing CD8+ T cells.

Background: The identification of activation pathways linked to anti-tumor T-cell polyfunctionality in patients responding to immunomodulatory agents is of great relevance in the new era of immunotherapy. An effective anti-tumor immune response is the result of a fine balance between TCR activation and co-stimulatory as well as inhibitory signals. The co-stimulatory molecule CD28 plays an essential role in T-cell activation, and with CD27 characterizes the different T-cell differentiation stages. Terminally differentiated T cells are CD28−CD27−, show an exhausted phenotype, low functionality along with up-regulation of co-inhibitory receptors including PD-1. However, beside its critical role in tumor-induced immune-suppression, PD-1 has recently been described as a marker of highly melanoma-reactive CD8+ T-cells. Accordingly, we have recently reported that Melan-A-specific CD8+ T-cells isolated from melanoma patients treated with combined chemo-immunotherapy [dacarbazine (DTIC) plus Melan-A/gp100 peptide vaccination], showed an enlarged TCR repertoire and high AKT-dependent anti-tumor polyfunctionality sustained by ICOS despite a late differentiated phenotype and high PD-1 expression. We hypothesize that this AKT-dependent anti-tumor polifunctionality may have contributed to protect patients from disease recurrence. Aim of this study is to identify the complex relationship between PD-1 expression and T-cell effector functions, taking advantage from a panel of antigen-specific (Melan-A and gp100) T-cell clones isolated from melanoma patients. Methods: We generated gp100-specific CD8+ T-cell clones from patients treated with peptide-vaccination alone or DTIC plus vaccination. We have analyzed the treatment-induced response in terms of TCR-βsequencing, differentiation phenotype, inhibitory receptor profile, polyfunctionality, cytotoxicity and AKT activation, by flow-cytometric, biochemical and functional analyses. Results: CD8+ gp-100-specific T cells isolated from patients treated with vaccination alone showed an early differentiated phenotype, while those isolated from patients treated with DTIC plus vaccination displayed mostly a late differentiated profile, as defined by the expression of CD28 and/or CD27. These clones possessed an oligoclonal TCR repertoire, irrespective of the treatment received by the patients, differently from results obtained for Melan-A-specific clones. In gp100-specific CD8+ T cells AKT pathway was activated according to their differentiation profile as defined by the expression of CD28 and/or CD27, irrespective of the treatment. High anti-tumor lytic activity and low PD-1 expression were observed in T-cell clones isolated after chemoimmunotherapy, while cells isolated after peptide vaccination alone expressed high level of the inhibitory molecule PD-1, either alone or along with LAG-1 and TIM-3 and were non tumor-reactive. This low anti-tumor polyfunctionality (in terms of TNF-α, IFN-γ and GrB) was increased after anti-PD-1 mAb blockade. Interestingly, differently from Melan A-specific T cells which showed high levels of PD-1 in the absence of CD28, these non-functional gp100-specific T-cell clones expressed PD-1 in the presence of CD28 co-stimulatory molecule. Conclusions: Our results, obtained in a panel of Melan-A and gp100-specific T-cell clones, show that while PD-1-positive-Melan-A specific T-cell clones have a polyfunctional effector profile in the absence of CD28 expression and in the presence of AKT activation sustained by ICOS, non-functional gp-100-specific PD-1-positive T cells express high level of CD28 molecule. The biochemical pathways involved in the control of functionality of PD-1-expressing CD8+ T cells are currently under investigation. Citation Format: Belinda Palermo, Ornella Franzese, Cosmo Di Donna, Mariangela Panetta, Isabella Sperduti, Antonella Soriani, Maria Laura Foddai, Angela Santoni, Paola Nisticò. The low antitumor functionality of PD1-positive gp100-specific CD8+ T cell clones isolated from melanoma patients correlates with the presence of CD28 co-stimulatory molecule [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A040.