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.

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,...

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.

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.

A phase II randomized clinical study is ongoing in our Institution to prove the clinical efficacy of dacarbazine (DTIC) one day before peptide-vaccination (Melan-A and NY-ESO-1) in the prevention of melanoma relapse in clinically disease-free HLA-A2 patients. In a previous pilot study, this combination therapy (using Melan-A and gp100 peptides) increased the number of tumor-reactive long-lasting effector-memory CD8+ lymphocytes. To identify the mechanisms enhancing the immune response, induced by DTIC combined with peptide-vaccination, we analyzed the endogenous and treatment-induced antigen specific CD8 T-cell response at the clonal level. We analyzed the sequence of the TCR α-chain of these clones and the molecular results were correlated with the expression of CD27/CD28 co-stimulatory molecule, AKT activation and anti-tumor lytic activity. The combination of chemo/immunotherapy elicited in Melan-A-specific, but not in gp100 clones, a renewal of high-avidity/tumor-reactive T-cell clones, with a broadening TCR diversity in long-surviving patients, suggesting that the selection of immune-resistant tumor variants may be circumvented by this combination, and thus prevent tumor recurrence in melanoma. In gp100 clones, AKT activation (pSer473-AKT) canonically correlates with CD28 and/or CD27 expression, independent of the treatment while in Melan-A clones, lacking CD27 and CD28 expression, non-canonical AKT activation was only observed after the combination therapy. The identification of the extracellular stimuli and signaling pathway responsible for DTIC-mediated activation of the AKT signaling are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4406. doi:1538-7445.AM2012-4406

Combination of chemo/immunotherapy to increase the effectiveness of the antitumor immune responses associated with a clinical efficacy is an attractive antitumor strategy. We have recently reported that the administration of dacarbazine (DTIC) one day before peptide (Melan-A and gp100)-vaccination in disease-free melanoma patients increases the number of peptide-specific effector-memory CD8+ lymphocytes. Notably, patients treated with DTIC before vaccination, showing a longer survival, display a progressive enhancement of TCR repertoire diversity of Melan-A+ CD8+ clones, accompanied by the maintenance of high-avidity, whereas patients treated with vaccine alone showed a reduction of TCR repertoire diversity and a decline of their tumor lytic activity. To study the phenotype and the functional lytic activity of CD8+ T-cell clones in association with their renewal rate we have analyzed the expression of CD28 and CD27 co-stimulatory molecules, in both Melan-A- and gp100-specific T-cell clones, isolated before and at different times after the treatments. We found newly recruited clones with high-lytic activity and highly-differentiated phenotype only after the combined therapy. Studies are in progress to define the role of replicative senescence in the continuous renewal induced by the combined therapy, by studying activity and regulation of telomerase in endogenous-induced or vaccine-driven clones, in correlation with their different phenotype and anti-tumor lytic capability.

The identification of activation pathways linked to anti-tumor T-cell polyfunctionality, associated with clinical benefit, is of great relevance in the new era of immunotherapy and combined chemo-immunotherapy. Recently we have reported that Melan-A-specific CD8 T cells isolated from long-term surviving patients treated with DTIC injected in a tight window before peptide-vaccination plus IFN-α; possess higher anti-tumor reactivity and an enlarged T-cell repertoire, compared to cells isolated after vaccination alone, suggesting that DTIC before peptide-vaccination may favor a protective anti-tumor specific immune response (1). To identify the mechanisms enhancing the immune response induced by the combined therapy, we analyzed the endogenous and treatment-induced antigen specific CD8 T-cell response in a panel of Melan-A- and gp100-specific clones from five patients. To this purpose, we analyzed the maturation/differentiation phenotype of these clones (CCR7 and CD45RA), the co-stimulatory (CD27, CD28 and ICOS) and inhibitory (TIM-3, LAG-3 and PD-1) profile, in parallel with the polyfunctionality (IFN-γ; TNF-α; and Granzyme B) and the activation of AKT (pSer473-AKT). AKT activation was correlated with the differentiation profile (in term of CD28 and/or CD27 expression) and was associated with poor polyfunctionality and low ICOS expression in Melan-A-specific T cells isolated from the endogenous response of both treatments and from the peptide-vaccination-driven response. Conversely, in three patients the combined treatment with DTIC before peptide vaccination elicited tumor-specific CD8+ T cells displaying the hallmarks of differentiated and highly activated effector T cells. These clones are highly efficient in tumor killing, possess polyfunctional activity, up-regulate inhibitory receptors (with PD-1 expressed at the highest level), retain proliferative capability and activate an AKT pathway not-related to the expression of CD27/CD28 molecules and partially dependent on ICOS engagement. Strikingly, T-cell polyfunctionality elicited by the combined therapy was strictly dependent on this AKT activation, as demonstrated by the blockade with selective inhibitors, which occurred only in Melan-A-specific CD8+ T cells and not in cells specific for gp100, suggesting that the nature of the peptide is crucial for the activation of this pathway. We suggest that this phenotypic and functional T-cell signature related to a highly effective tumor-specific response is fine-tuned between TCR activation, co-stimulatory and inhibitory signals critical to preserve the self-tolerance during immunotherapy treatment. Of clinical relevance, these three patients treated with chemo-immunotherapy are clinically disease-free after 9 years of follow-up. The study represents a critical contribution for the comprehension of the mechanisms underlying the advantages of combined chemo-immunotherapy and paves the way for the identification of new biomarkers of T-cell activation that may be employed as markers of immune responsiveness. Reference: 1. Palermo B, Del Bello D, Sottini A, Serana F, Ghidini C, Gualtieri N, Ferraresi V, Catricalà C, Belardelli F, Proietti E, Natali PG, Imberti L, Nisticò P. Dacarbazine treatment before peptide vaccination enlarges T-cell repertoire diversity of melan-a-specific, tumor-reactive CTL in melanoma patients. Cancer Res. 2010 Sep 15;70(18):7084-92. Citation Format: Ornella Franzese, Belinda Palermo, Cosmo Di Donna, Isabella Sperduti, Mariangela Panetta, Maria Laura Foddai, Angela Santoni, Paola Nisticò. Polyfunctional antitumor CD8 T cells obtained from a broad repertoire elicited by chemo-immunotherapy and preventing melanoma relapse depends on the activation of an AKT pathway sustained by ICOS. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A003.