ABSTRACT: Although checkpoint immunotherapy has primarily focused on CD8⁺ T cells, emerging evidence highlights an important role for cytotoxic CD4⁺ T cells in mediating therapeutic responses. However, research on the functional properties of cytotoxic CD4⁺ T cells in the context of immunotherapy is still at an early stage and remains insufficiently defined. Utilizing single-cell RNA-sequencing datasets obtained from metastatic melanoma patients treated with checkpoint inhibitors targeting PD-1 and/or CTLA-4, we performed transcriptomic profiling of conventional CD4⁺ T cells, excluding proliferative and regulatory (FOXP3⁺) subsets, and compared responders and non-responders as distinct groups. Importantly, our analysis identified distinct clusters that discriminate between responders and non-responders, with cytotoxic CD4⁺ T cells occupying a central position within these clusters. In responder-specific clusters, cytotoxic CD4⁺ T cells exhibited features of early activation, whereas clusters specific to non-responders were characterized by an exhausted phenotype. Notably, non-responder-specific clusters were positioned proximally to Treg-like clusters, suggesting a potential transition from cytotoxic to regulatory CD4⁺ T cell states in non-responders. Our findings reinforce the emerging concept that cytotoxic CD4⁺ T cells play a central role in mediating immunotherapy responses. These results provide a foundation for the development of predictive biomarkers and novel therapeutic strategies aimed at modulating CD4⁺ T cell differentiation.