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Background Co-targeting of PD1 and LAG3 on T cells has shown superior efficacy vs. PD1 monotherapy and is now an FDA-approved combinatorial regimen in melanoma. We recently reported that PD1/LAG3 synergized to drive CD8 T cell exhaustion (T EX) in cancer, and features of the CD8 TEXprogram are well-established. However, recent evidence suggests that intratumor CD4 T conventional cells (Tconv) exhibit an exhausted-like profile that correlates with worse outcomes, although the defining features, significance, and regulatory mechanisms of CD4 TEX remain unclear. Further, little is known regarding mechanistic impact of PD1/LAG3 on CD4 Tconv. Given the dynamic ability of CD4 Tconv to exert direct cytotoxicity and also provide help, better understanding of CD4 TEX may be of particular importance for improving cancer immunotherapy. We hypothesize that PD1/LAG3 exert differential yet synergistic effects on CD4 Tconv to drive their exhaustion and impaired anti-tumor responses.Methods We utilized B16-F10 murine melanoma together with adoptive transfers of T cell receptor transgenic CD4 T cells specific to tyrosinase-related protein-1 (Trp1), a melanocyte differentiation antigen expressed by both normal melanocytes and melanoma, crossed to Pdcd1 -/- and/or Lag3 -/- mice. Trp1 CD4 T cells were adoptively transferred into tumor-bearing WT C57BL/6 mice. CD8 depletion antibodies were administered to assess direct vs. helper roles. WT tumor-bearing mice were also given anti-PD1/anti-LAG3 therapy and CD4 Tconv phenotype assessed.Results We found that tumor-infiltrating Trp1 CD4 T cells upregulated PD1, LAG3, TOX, and CD39, suggestive of T EX phenotype. Interestingly, as little as 10,000 Trp1Pdcd1 -/- CD4 T cells were sufficient to clear established tumors. In the absence of CD8 T cells, higher doses (up to 200,000) of Trp1Pdcd1 -/- CD4 T cells had a cytotoxic effect on tumor growth, whereas the effects were minimized when fewer cells were transferred, suggesting that CD8 T cells may also be required for Trp1 CD4 anti-tumor efficacy at suboptimal doses. Further, PD1/LAG3 therapeutic blockade appears to modulate CD4 Tconv phenotype.Conclusions Our preliminary data suggests that PD1 may play a crucial role in restraining antigen-specific CD4 responses. Further, tumor-specific CD4 T cells may leverage both direct cytotoxicity and CD8 help for anti-tumor effects. Ongoing work is focused on assessing effects of CD4 T cell-specific PD1/LAG3 loss on tumor growth and cytotoxic/helper functions using Trp1 Pdcd1 -/- /Lag3 -/- mice as well as CRISPR-mediated deletion. Future plans include quad-transfers to study cell-intrinsic effects of PD1/LAG3 across the same microenvironment. Collectively, these studies aim to enhance mechanistic understanding of CD4+ TEX and inform novel immunotherapeutic strategies.