# Intestinal regulation of ThPOK expression and CD4 helper T cell function

> **NIH NIH R01** · ROCKEFELLER UNIVERSITY · 2021 · $541,324

## Abstract

Regulatory T cells are the main cell population involved in the prevention or suppression of unwanted overt
inflammatory processes. At the intestinal surface, which is chronically exposed to stimuli from both microbes
and diet, peripheral Foxp3+ T cells (pTregs) are thought to prevent the development of inflammatory bowel
diseases and food allergies. Surprisingly, very few Tregs are found in the most exposed layer of the intestinal
mucosa: the single-cell layered epithelium. Yet, the understanding of immune regulatory processes at this layer
is of utmost importance for a better comprehension of how pathological inflammatory processes arise, and for
the development of novel intervention strategies. During the first four years of funding for this project we
described environmental cues, transcription factors, and functional consequences related to the adaptation of
CD4 T cells to the gut epithelium. We found that CD4 T cells belonging to different T helper lineages convert
into CD8αα+CD4+ intraepithelial lymphocytes (CD4-IELs) in a microbiota-, retinoic acid-, IFN-γ and TGF-β-
dependent manner. This unexpected T cell plasticity was also observed among total Tregs, which lose Foxp3
and convert to CD4-IELs upon migration to the epithelium, an effect attributed to the loss of the transcription
factor ThPOK. Importantly, our work also suggested that pTregs and CD4-IELs perform complementary roles
in the regulation of intestinal inflammation. Our preliminary repertoire analysis indicates strong TCR biases in
both pTregs and CD4-IELs in the epithelium. However, the role of TCR stimulation for epithelial CD4 T cell
residency and plasticity, the diversity of TCR repertoire and the nature of TCR ligands in epithelial CD4 T cell
populations remain to be determined. We hypothesize that intra-epithelial adaptation of anti-inflammatory T
cells is shaped by their TCR and by factors found in the epithelium, determining gut T cell functional
specialization. Based on extensive published and unpublished data, this proposal undertakes several
intertwined aspects of CD4 T cell adaptation to the gut epithelium, bringing together both conceptual and
technological innovations. Using a combination of intersectional genetics, single-cell repertoire analyses and in
vitro NFAT assays, we will define the TCR repertoire and ligands of epithelial CD4 T cells. Our unpublished
data suggest a coordinated response involving multiple transcription factors that regulate Treg stability, tissue
residency and CD4-IEL function. We will perform RNA-seq, ChIP-seq and ATAC-seq, combined with mouse
genetics, to define the molecular machinery involved in the transcriptional changes observed in CD4 T cells
that undergo epithelium-specific adaptation. Finally, we will define the role of continuous ThPOK and TCR
expression in Treg suppressive function and whether CD4 T cell “IEL differentiation” is important for immune
regulation in the epithelium both at steady state and in models of intestinal...

## Key facts

- **NIH application ID:** 10070102
- **Project number:** 5R01DK093674-09
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Daniel S Mucida
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $541,324
- **Award type:** 5
- **Project period:** 2013-01-01 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10070102

## Citation

> US National Institutes of Health, RePORTER application 10070102, Intestinal regulation of ThPOK expression and CD4 helper T cell function (5R01DK093674-09). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10070102. Licensed CC0.

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