The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8 + T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8 + lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8 + T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigenspecific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

Melanoma survival increased with targeted-and immunotherapy agents, yet most patients ultimately progress and require salvage therapy. In our experience, some progressive disease patients on immune-checkpoint inhibitors (ICIs) demonstrate deep and sustained responses to chemotherapy. We hypothesized that ICIs improve the response to subsequent chemotherapy in metastatic melanoma. We conducted a retrospective study to compare the efficacy of chemotherapy given with prior immunotherapy, to its efficacy given without it. We measured progression free survival (PFS), overall survival, and response rate. Immune-monitoring was performed on sequential peripheral blood mononuclear cell samples taken from a chemotherapy-responsive patient. The chemotherapy post-immunotherapy group (CpI) included 11 patients, the chemotherapy without prior immunotherapy (CNPI) group included 24 patients. Median PFS was 5.2 months in the CpI vs. 2.5 months in the CNPI groups; HR 0.37 [95% Confidence interval (CI) 0.144-0.983], P = 0.046. Immune-monitoring showed an increased proportion of CD8 + cells, with elevated PD-1 and CD69 expression, while on chemotherapy, as compared with all-time points on ICIs, suggesting immune-activation. Immunotherapy potentiates the effect of chemotherapy in metastatic melanoma possibly through activation of CD8 + T cells.

Highlights d Unlike promoter-mediated PD-L1 induction by IFN-g, EGFR rapidly stabilizes PD-L1 mRNA d Once induced, PD-L1 enhances metastasis in vivo and chemotaxis toward EGF d PD-L1 physically binds with and enhances activation of phospholipase C-g1 by EGFR d PLC-g1 binds a PD-L1's cytoplasmic segment implicated in protection from cytotoxicity

Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor–T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-126–35–specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone’s IFN-γ and TNF-α production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vβ16 for clones 2E2 and 2G1 and Vβ14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vβ by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy.