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Background CD4+ T cells specific to tumor antigens can directly recognize tumor cells in the minority of tumors that express class II MHC on tumor cells, but the role of tumor specific CD4 + T cells in MHC class II negative human tumors is less understood. It is also unclear whether CD4+ T cell responses to shared tumor antigens could differ from CD4+ T cells targeting patient-specific neoantigens.Methods CD4 + T cells were expanded from the tumor of a patient with MHC II- NY-ESO+ melanoma, and NY-ESO and neoantigen specific CD4 T cell clones were identified by T Cell receptor (TCR) sequencing and expression of TCR α/β chains in donor CD4+ T cells. The peptide avidity of NY-ESO specific TCR and neoantigen-specific TCR from the same patient were compared, and the phenotypes of NY-ESO specific and neoantigen specific CD4+ T cells in the primary tumor were compared by scRNAseq. The function of NY-ESO specific transgenic CD4+ T cells in facilitating antitumor immunity in the presence and absence of direct recognition of tumor cells was assessed in the MISTRG humanized mouse model that allows formation of a melanoma human tumor xenograft that contains human antigen presenting cells (APC, figure 1).Results We identified 10 TCRs specific to the cancer testes antigen NY-ESO, and 8 TCRs specific to 3 different patient-specific neoantigens. NY-ESO specific TCR showed equal or greater peptide avidity as neoantigen specific TCR and showed a similar cellular phenotype by scRNAseq. One TCR recognized antigen presented by the HLA-DP04 allele present in 65% of patients of European ancestry, and was able to both directly recognize NY-ESO + HLA-DP04+ melanoma tumor cells as well as HLA-DP04+ monocyte-derived dendritic cells incubated with tumor cell extract. Adoptive transfer of NY-ESO specific CD4+ T cells into MISTRG mice humanized with HLA-DP04 immune cells mediated regression of NY-ESO+ tumors independent of direct presentation of antigen on tumor cells (figure 2).Conclusions CD4 + T cells specific to NY-ESO had comparable avidity and cellular phenotype to neoantigen specific CD4+ T cells in a patient whose tumor cells expressed NY-ESO but not class II MHC. An NY-ESO specific TCR directly recognized both MHC II positive tumor cells and MHC II negative tumor cells through antigen uptake into APC in-vitro, and mediated regression of human melanoma in a humanized mouse model through interaction with human APC, suggesting NY-ESO specific CD4+ T cells could target MHC II negative tumors.Abstract 342 Figure 1The MISTRG model of CD4+ T cell therapy. MISTRG humanized mice having melanoma human tumor xenograft that contains human HLA-DP04 immune cells and being treated with adoptively transferred NYESO CD4 T cellsAbstract 342 Figure 2NY-ESO targeted CD4+ T cells control human melanoma indpendent of direct tumor recognition. Human CD4+ T cells expressing the NY-ESO specific TCR but not an irrelevant TCR can control both NY-ESO+ HLADP+ (Left) and NY-ESO+ HLADP- SKMEL37 tumors (right) in MISTRG humanized mice. (n=9) Error bars are SEM. ****p<0.0001