Targeting Tr1 to enhance the efficacy of cancer vaccines

Background: Neoantigen cancer vaccines have shown promising clinical potential in preventing or delaying tumor recurrence in a fraction of people. The low efficacy of neoantigen cancer vaccines can be attributed to several reasons, including the low abundance of antigen-specific CD8 T cell responses induced by these therapies. Additionally, accumulating evidence suggests that suppressive cells such as Foxp3+ Tregs or Foxp3-negative type 1 regulatory CD4 T cells (Tr1) are profoundly immunosuppressive and function as a major obstacle to cancer vaccine-induced antitumor responses. Our recent publication in Nature demonstrated that the antitumor efficacy of neoantigen cancer vaccines depends on the peptide dose of MHC-II–restricted neoantigens included in the vaccine formulation. Whereas vaccines comprised of MHC-I restricted neoantigens plus low doses of MHC-II-restricted neoantigens (LDVax) promote tumor rejection, similar vaccines containing high doses of the same MHC-II neoantigen (HDVax) induce Tr1 cells and inhibit rejection. Notably, CD4 T cells with a Tr1-like signature have been detected in cancer patients and correlate with poor patients’ outcome. Objective/Hypothesis: Our preliminary studies indicate that neoantigen cancer vaccines containing high doses of altered peptide ligands (APLs) with low MHC-II binding affinity do not induce Tr1 cells and instead promote tumor rejection. Additionally, Tr1 induction also required antigen presentation by type 2 dendritic cells (cDC2) and the participation of signaling by EBI3 subunit of the inhibitory cytokine IL-35. Based on these findings, we hypothesize that DC2/monocyte-derived IL-35 synergize with strong interactions between peptide and MHC-II molecules and T cell receptors (pMHC: TCR) to drive Tr1 cell induction and thereby inhibit the antitumor efficacy of neoantigen cancer vaccines. Specific Aims: (1) Elucidate the role of the EBI3 cytokine subunit chain of IL-35 in the induction of Tr1 cells; and (2) Define the factors that regulate expression of IL-35 signaling receptors (IL-12Rβ2 and IL-27Rα) required for Tr1 cell induction. Study design: Using our well characterized tumor models, we will identify the cellular sources of IL-35 using IL-35 reporter mice and assess how IL-35 blockade alters the phenotype and effector function of Tr1 and CD8 T cells. We will test whether IL-35 neutralization enhances vaccine-driven tumor rejection by diminishing Tr1-mediated suppression and boosting CD8 T cell cytotoxicity. We anticipate that IL-35 blockade will reduce Tr1 differentiation, reprogram CD4 T cells toward a Th1 phenotype, and enhance CD8 T cell effector responses. In Aim 2, we will determine whether strong pMHC: TCR interaction favors the differentiation of tumor-specific CD4 T cells into Tr1 cells by upregulating IL-12Rβ2 and IL-27Rα receptors involved in the signaling of IL-35. We expect that stronger interaction in CD4 T cells correlates with Tr1 differentiation and suppression of antitumor immunity. Project Number: 1R21CA313364-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Hussein Sultan (+1 co-PI) | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $399,829 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 CDPT-Y (56)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11356944