Relative and synergistic contributions of PD1 and LAG3 on CD4+ T conventional cells in cancer

Cell surface inhibitory receptors (IRs) such as programmed cell-death-1 (PD1) and lymphocyte activation gene 3 (LAG3) are upregulated on activated and exhausted T cells (TEX). CD8+ TEX develop from chronic antigenic stimulation and are defined by sustained multi-IR expression, impaired effector functions, poor proliferative potential, transcriptional reprogramming, epigenetic scarring, and metabolic dysfunction. Immune checkpoint blockade (ICB) immunotherapies target IRs such as PD1 aiming to reinvigorate CD8+ TEX function for improved tumor clearance; however, many patients fail to respond. Our laboratory was the first to demonstrate that PD1 and LAG3 dual blockade was superior to PD1 alone in murine tumor models. PD1 and LAG3 are now major therapeutic targets. However, little is known regarding mechanistic impact of anti-PD1/anti-LAG3 combinatorial synergy. We recently reported that PD1/LAG3 synergize to drive CD8+ TEX in murine melanoma, and we reported similar results clinically. However, the combined activity of PD1/LAG3 observed during global deletion and monoclonal antibody-based approaches is not as robust following CD8+ T cell-specific deletion, suggesting PD1/LAG3 effects other T cell types, such as CD4+ T cells. CD4+ T cells play crucial roles in cancer immunotherapy by providing help to CD8+ TIL and exerting direct anti-tumor cytotoxic functions. On the other hand, recent evidence suggests that CD4+ T cells can display an exhaustion-like profile, including expression of PD1 and LAG3, that correlates with worse outcomes. Although there is growing evidence for CD4+ TEX in cancer, little has been done to mechanistically dissect their phenotype, function, molecular profile, or regulatory mechanisms to better understand their significance. While the role of PD1/LAG3 in CD8+ TEX is well established, it remains unknown whether these IRs drive a similar state in CD4+ T cells within the tumor microenvironment and how this might impact anti-tumor responses. My central hypothesis is that PD1 and LAG3 exert differential yet synergistic effects on CD4+ T cells to drive their exhaustion and impaired anti-tumor responses. My overall goal is to mechanistically dissect the cell-intrinsic effects of CD4+ Tconv-specific PD1/LAG3 loss and the cell- extrinsic impact on CD8+ T cells to better understand CD4+ TEX and their impact on anti-tumor immunity. I will perform adoptive transfers of melanoma tumor antigen-specific CD4+ T cells that lack PD1 and/or LAG3, and assess phenotypic, functional, transcriptional, and epigenetic features of CD4+ tumor-infiltrating lymphocytes (TIL) to determine the cell-intrinsic effects from PD1/LAG3 loss. I will compare our findings with our published data on PD1/LAG3-deficient CD8+ T cells (Aim I). Then, I will assess the impact of CD4+ T cell-specific PD1/LAG3 loss on CD8+ TEX by performing phenotypic, functional, and transcriptomic analyses of CD8+ TIL following A.T. of PD1/LAG3-deficient CD4+ TIL (Aim II). Elucidating the cell-intrinsic and cell-extrinsic contributions of PD1 and LAG3 on CD4+ T cells will improve our mechanistic understanding of CD4+ TEX and their significance in anti-tumor immunity, which is crucial for the advancement of immunotherapies. Project Number: 1F32CA301603-01 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Nicole Mihalik | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $75,520 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F09C-Z (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11163800