Analysis of T and NK cells immune response in Ipilimumab treated Melanoma patients

BackgroundThe most promising immunotherapeutics tested in meta-static melanoma patients are the monoclonal antibodyblocking CTLA-4 (Ipilimumab), and those interferingwith PD-1 and PD-L1. However, the lack of knowledgeon predictive biomarkers that could assist the treatmentremains a limiting factor. We speculate that, along withadditional markers, the immunoscore [1] is fundamentalas prognostic and predictive marker for response toimmunotherapies in metastatic melanoma. Our previousdata demonstrate that NK cells control the melanomaprogression [2,3]. Therefore we have analysed bothT cells and NK cells subsets frequencies and receptorsrepertoire in the peripheral blood of Ipilimumab treatedpatients with Stage IV metastatic melanoma.Material and methodsPeripheral blood mononuclear cells (PBMCs) from 12different patients with stage IV metastatic melanomawere collected and analyzed. Each patient received 4infusions of Ipilimumab each 21 days. Before each infu-sion we collected patients’ blood and isolated PBMCs toanalyze the lymphocytes compartment.We stained for the following antibodies: CD56 PE, CD3Fitc, CD56 APC, CD4 PeCy7, CD8 APCCy7, CD152(CTLA4) PE, CD279 (PD-1) PE, CCR7 PeCy7, CD158a/h(KIR2DL1/S1) PE, CD158b (KIR2DL2/DL3) PE, CD158e(KIR3DL1) PE, CD16 APCCy7, CD57 PE, CD69 PE,CD314 (NKG2D) PE, CD226 (DNAM-1) PE, CD337(NKp30) PE, CD336 (NKp44) PE, CD335 (NKp46) PE(Miltenyi Biotech), CD192 (CCR2) AlexaFluor 647,CXCR2 (IL8RB) APC, 7-AADStaining Solution (BD Ita-lia), TIM3 PE (ebioscience), NKG2C PE (R&D Systems).The analysis was performed with FACS CANTO II.Statistical analysis was performed with Anova andStudent’s t-test.ResultsOur data indicate that, after the first Ipilimumab treat-ment, an inversion of CD4/CD8 ratio occurs with a conco-mitant increase in the CD56