CD4+ T cell Infiltration during Intestinal Adenoma Initiation Shapes Tumor Evolution

Abstract

Colorectal cancers (CRC) are often “immunologically cold” and respond poorly to immunotherapy. However, these observations are generally made at advanced stages of disease, after the tumor and immune system have co-evolved. How the immune system can influence the evolution of tumor development at early stages remains poorly understood. Intestinal stem cells (ISCs), marked by Lgr5, play an important role in adenoma initiation and progression. These cells are the primary cell-of-origin for intestinal cancer, with loss of the gene adenomatous polyposis coli (APC) driving the outgrowth of early adenomas. These mutated Lgr5+ stem cells retain their stemness potential, maintaining the ability to both self-renew and give rise to diverse adenoma cell types. In the intestine, ISC behavior is tightly regulated by signals from their surrounding niche, and the immune system is known to influence ISC function in non-tumor settings. Thus, understanding how the immune system responds to early transformation of ISCs during adenoma formation and how this immune response influences early stem cell function is critical for defining the drivers of tumor evolution. To address these questions, we used Lgr5-eGFP-CreERT2 × floxed Apc mice, a genetic model of intestinal tumorigenesis driven by Apc loss in ISCs. Importantly, this system enables visualization of Lgr5⁺ stem cells via eGFP expression, allowing us to examine dynamic changes within the stem cell niche. We found that Th1-polarized CD4⁺ T cells are among the earliest immune responders to adenoma initiation and actively infiltrate the stem cell niche. To determine how these cells influence transformed ISCs, we performed RNA sequencing and immunofluorescence to profile ISC changes in response to CD4+ T cells. These studies revealed that Th1 CD4⁺ T cells promote differentiation of mutant Lgr5⁺ stem cells towards a fetal-like intestinal stem cell state. This ISC-CD4+ T cell interaction leads to expansion of a fetal-like intestinal stem cell population capable of excluding T cells, recapitulating the immune-excluded phenotype observed in more advanced CRC tumors. We next investigated the mechanisms underlying the induction of this fetal-like stem cell program. Although transformed ISCs exhibit an early IFNγ response and upregulate MHC class II (MHC-II), our data indicate that Th1 CD4⁺ T cells do not drive fetal-like differentiation through IFNγ signaling or MHC-II–mediated interactions with ISCs. To further interrogate CD4⁺ T cell– stem cell communication, we generated a transgenic mouse model expressing a CD4⁺ T cell receptor (TCR) specific for eGFP, termed the “CDForce” mouse. When combined with the Lgr5-GFP-CreERT2 × Apcfl/fl model, this system enables targeting of CD4⁺ T cells to the Lgr5+ intestinal stem cells during adenoma initiation. This approach will thus allow us to dissect the mechanisms by which CD4⁺ T cells induce the fetal intestinal stem cell program during early adenoma formation.