Tu153 - Comprehensive Analysis of the Immune Landscape in the Peripheral Blood of Advanced Melanoma Patients Reveals Chronic Activation of Differentiated Cytolytic Cell Subsets

Background: Many advanced melanoma patients are either not responsive or relapse after immune checkpoint therapy, hence, finding diagnostic or prognostic markers is essential. To identify a potential immune signature in the peripheral blood, we delineated the phenotypic and functional characteristics of the PBMCs from melanoma patients and compared them to healthy individuals.

Methods: We used 1 dataset from metastatic melanoma patients (n=72) and 2 datasets from healthy individuals (n=31) generated by different mass cytometry (CyTOF) analyses in the lab. To accurately compare patient and healthy cohorts, we first normalized the FCS files using Fluidigm EQ^™ calibration beads. Then we performed manual gating in Cytobank, excluding the markers that were not shared between the different panels.

Results: Most of the major immune population in the PBMCs were unchanged in the patients compared to the healthy donors. However, in CD8 T cells from patients, we observed a decrease of the naïve subset and an increase of the terminally differentiated effectors (TEMRA), along with higher frequency of activated cells. Moreover, we also observed an increase of CD56^dimCD57⁺ NK cells. These data indicate a higher differentiation of the cytotoxic subsets in the melanoma patients. Upon PMA/Ionomycin stimulation, higher proportion of CD8 TEMRA from patients expressed cytokines (IL-2, IFNg, TNFa, GM-CSF, MIP-1b) and the degranulation marker LAMP-1. We also detected higher expression of lytic granules (granzyme B, perforin) along with increased expression of PD-1. Altogether, these data provide insight regarding the immune landscape in melanoma patients which may affect their overall immune responsiveness.