CD4+ T cell-mediated Immune Surveillance during Intestinal Tumor Initiation

Introduction/Rationale: While the immune system’s role in tumor development has been broadly studied in established tumors , how the immune system can detect and kill mutated cells at early pre-cancerous stages is largely unknown. The intestine provides a unique environment to study immune responses during tumor initiation since it has a rich resident immune system. Intestinal stem cells marked by Lgr5 have been well-established as the cell-of-origin of intestinal cancer. Here, we aim to study immune surveillance of these Lgr5+ intestinal stem cells upon acquisition of the earliest oncogenic mutations.

Methods: To study early immune surveillance of oncogenic intestinal stem cells, we used Lgr5-GFP-CreERT2 x floxed Apc mice, since inactivating mutations in the Adenomatous polyposis coli (APC) gene are the initiating events in intestinal tumorigenesis in patients. Two weeks after inducing mutations with tamoxifen we performed single cell RNA-sequencing, immunofluorescence, and flow cytometry to characterize the cellular ecosystems in the early adenoma niche.

Results: Our in-depth analysis revealed an increase in IFNg-producing T helper 1 CD4+ T cells, specifically located near mutated Lgr5+ stem cells. Blockade of both IFNg and CD4 T cells leads to a decrease in stem cell death, and accelerates tiumor progression, indicating these CD4+ T cells play an anti-tumor role via IFNg. Early transformed Lgr5+ stem cells express MHC-II, suggesting direct recognition by CD4 T cells. To address this interaction in an antigen specific manner, we generated transgenic mice with CD4 T cells expressing a GFP-specific TCR. Upon adoptive transfer, these GFP-specific CD4+ T cells infiltrate the niche of oncogenic Lgr5+ cells supporting a direct recognition.

Conclusion: Overall, these results suggest that CD4+ T cells are early responders to early transformation of intestinal stem cells and play a part in control of early tumor development via MHCII.