BACKGROUND: Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap. METHODS: We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and_CD8+ T_cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T_cell help in the antitumor effects of the anti-PD-1+LAG-3 combination. RESULTS: The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory_CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFN_ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T_cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFN_ release but, conversely, increased numbers of activated CD8+T cells and IFN_ release in anti-PD-1+CTLA-4 treated tumors. CONCLUSIONS: Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T_cell polarization, which in turn, impacted cytotoxic CD8+T_cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.