Biology of Human Tumors Circulating CD4þ T Cells That Produce IL4 or IL17 When Stimulated by Melan-A but Not by NY-ESO-1 Have Negative Impacts on Survival of Patients with Stage IV Melanoma

Purpose:We initially observed that the presence of circulating NY-ESO-1– and/or Melan-A–specific T cells in patients with stage IV melanoma was significantly associated with prolonged survival. Here, we report the ways in which the phenotypes and functions of these T cells differentially affect survival in patients preselected for NY-ESO-1 and/or Melan-A reactivity. Experimental Design: We assayed functional antigen-reactive T cells recognizing NY-ESO-1 and/or Melan-A after in vitro stimulation using overlapping peptide pools. After restimulation, we assayed six cytokines simultaneously by intracellular cytokine staining. This allowed us to analyze the functional antigen response of both CD4þ and CD8þ T cells at the single-cell level. Results: We observed that NY-ESO-1 stimulated mainly CD4þ T cells, whereas Melan-A more often stimulated CD8þ T cells. NY-ESO-1 reactivity was not associated with an additional impact on survival, whether CD4þ T cells, CD8þ T cells, or both types of T cells were responding. In contrast, recognition of Melan-A by CD4þ T cells was associated with reduced survival in our cohort of patients preselected for NY-ESO-1 and/or Melan-A reactivity (that is, in patients with exceptionally long survival). We further observed a negative effect on survival in patients with CD4þ T cells producing IL4 and IL17 upon Melan-A stimulation. Their prognosis was comparable to patients without any