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.