Background: PD-1 antibodyNivolumab was administered with/without a multi-peptide vaccine to 105 patients (pts) with unresectable melanoma that failed at least one regimen and were IPI naïve, or progressed after IPI, to assess its toxicity especially in those with prior dose limiting immune related adverse events (irAEs) to IPI, and to update survival data, characterize T cell gene expression and immune cell subsets. Methods: HLA A0201 IPI-naïve pts received nivolumab at 1, 3 or 10 mg/kg (34 pts); additional pts that failed prior IPI received nivolumab at 3 mg/kg: two cohorts of pts were A*0201 positive and had either grade 2 or less IPI-related irAE (10 pts), or grade 3-4 dose limiting irAE (20 pts); 41 pts had grade 2 or less irAE, were not HLA restricted and received nivolumab alone. Pre- and 12 week post-treatment peripheral blood was analyzed. Results: Median follow-up for all pts was 15 months (mos);median progression-free survival (PFS) was 4.2 mos, and estimated median overall survival (OS) was 16.7 mos (95% CI: 14.4, not reached) with 1 year OS of 65%. Median OS was similar for IPI-naïve or IPI-relapsed pts (15.9 vs. 18 mos, p=0.6), older or younger than 62 (p=0.44) and by dose (p=0.86). ORR, median PFS and OS were longer in males than females (all p<0.05). Of 20 pts with prior IPI-induced grades 3-4 irAEs, only 1 had a subsequent grade 3-4 irAE with nivolumab and it was different than seen with prior IPI, but treatment emergent grades 1-2 rash and injection reactions were common. Biomarker studies showed that circulating HLA-DR lo/CD14+/CD11b+ myeloid-derived suppressor cells (MDSC, p=0.028), and FCRL2+ CD8 T cells (p=0.009) were associated with progression. Decreased regulatory molecules BTLA and LAG3 on CD8+ T cells were associated with response (p=0.04). Principal component analysis of genes expressed by flow sorted CD8 T cells showed clustering by response (p = 6.01E-10) Conclusions: Median OS of 16.7 mos and PFS of 4.2 mos with nivolumab was observed in previously treated melanoma patients naïve to or failing IPI. Prior irAEs to IPI were not replicated with nivolumab. Novel biomarkers of outcome were found on circulating blood cells, including MDSC and CD8 T cell FCRL2 pre-treatment. Clinical trial information: NCT01176461.