Unveiling the origin of tumor-reactive regulatory T cells in patients with mismatch repair proficient colon cancer

The critical preliminary data for this proposal relates to the nature of regulatory T cells (Treg) that infiltrate tumors in patient with colorectal cancer (CRC) and their relationship with tumor-reactive conventional CD4+ T cells (CD4+ Tconv). We discovered that tumor-infiltrating CD4+ Tconv expressing PD-1 and ICOS recognize tumor antigens. Subsequently, using bulk TCR sequencing on sorted T cell populations, we found TCR clonotypes that are shared between tumor-infiltrated PD-1+ICOS+ CD4+ Tconv and Tregs, suggesting a common origin. This observation was further supported by the presence of shared TCR clonotypes between tumor-infiltrating CD4 Tconv and Tregs in our single cell RNA sequencing and single cell TCR dataset from 5 CRC patients. Furthermore, we observed that in vitro expansion of tumor-infiltrating Tregs results in the significant loss of FOXP3 expression on a subset of cells, suggesting the presence of a population of unstable Tregs in the tumor. We hypothesize that soluble factors present in the tumor microenvironment (TME) can promote the conversion of tumor-reactive CD4+ Tconv into Tregs following TCR-mediated activation and that this Treg phenotype is unstable and can be reversed by effector cytokines. The specific aims of this study are to 1: Assess conversion of tumor-reactive CD4+ Tconv into tumor-induced Treg in CRC tumors; 2: Identify the factors that regulate CD4+ Tconv to Treg plasticity. Our study design incorporates the use of single cell RNA sequencing, single cell TCR sequencing and TotalSeq to apprehend the complexity of the CD4+ Tconv and Treg compartments in CRC tumors. These results will provide essential information regarding the origin of tumor- infiltrating Tregs and highlight the level of CD4 T cell plasticity in tumors. Analysis of the TCR repertoire of blood memory CD4+ Tconv and Treg will further define if CD4+ Tconv convert into Treg in the TME. To determine if this phenomenon is associated with tumor reactivity, we will clone the TCRs shared between CD4+ Tconv and Treg and introduced them into autologous CD4+ Tconv to determine whether they recognize tumor-specific somatic mutations identified by whole exome sequencing and RNA sequencing. To identify extracellular soluble factors that could participate in the CD4+ Tconv to Treg conversion, we will analyze the composition of the tumor secretome directly ex vivo using the Olink platform. We will determine the spatial distribution of the cells secreting those factors and their relationship with CD4+ Tconv and Tregs in the TME using sequential immunofluorescence and RNAscope. We will evaluate if the identified factors can promote CD4+ Tconv to Treg conversion in vitro and whether they synergize with the immunosuppressive cytokine TGF-β. Finally, we will determine the stability of tumor-induced Tregs and whether this Treg phenotype can be reverted by effector cytokines. The findings from this proposal will reveal if the conversion of tumor-reactive CD4+ T cells into Tregs occurs in CRC tumors. If our proposal is successful, it will provide an outstanding opportunity to both remove a negative signal and deliver a positive signal by preventing conversion (or reversing it), which may be superior to simple Treg depletion. Project Number: 1R21CA292306-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Thomas Duhen | Institution: PROVIDENCE HEALTH & SERVICES - OREGON, RENTON, WA | Award Amount: $437,113 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 BTC-F (80)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11374311