# T regulatory cell subsets at the microbial interface: determinism and function

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2021 · $423,750

## Abstract

Peaceful coexistence with the essential microbes that populate the mammalian gut requires a careful
balance between tolerance of commensals, maintenance of barrier function, and avoidance of damaging
inflammation. A key role is played by FoxP3+ T regulatory (Treg) cells, which help maintain immunologic
tolerance and control inflammation in many organismal contexts. We have recently described the induction by
several symbionts from the human gut of a unique population of FoxP3+ Treg cells that also express and
require functionally the transcription factor RORg. This is paradoxal, as RORg is the master regulator of pro-
inflammatory Th17 cells. Rorg+ Treg cells expand in response to gut symbionts, perhaps through local
differentiation, and are functionally involved in the control of intestinal inflammation. They co-exist in the colon
with more typical Helios+Gata3hi Tregs, likely of thymic origin. We propose to address several important
questions opened by these observations, to explore the control, origin and function of Rorg+ Tregs.
 1. How do FoxP3 and RORg synergize at the molecular level, and how does RORg function so
differently in Th17 cells vs Rorg+ Tregs? This will be addressed by analyzing transcription and chromatin
changes resulting from carefully controlled expression of FoxP3 and RORg in CD4+ T cells, modulated with
other transcription factors that are differentially represented in Th17 and Tregs, or with oxysterol ligands of
RORg. These inferences will be validated in vivo with conditional knockout mice, and their relevance to human
physiology will be assessed by transducing human CD4+ T cells in parallel, and by low-input RNAseq analysis
of Rorg+ Tregs we found in the human colon.
 2. What are the cellular origin and differentiation pathways of Rorg+ Tregs? The origin and inter-
relationships between colonic Treg subsets in the colon will be analyzed by transfer experiments, by lineage
tracing after Treg-specific tagging, and by using TCR sequences as barcodes to assess relationships between
Treg and Tconv populations in mice colonized by a single microbe
 3. Relative roles of RORg+ and Helios+ colonic Tregs assessed by inducing Treg-specific knockouts
in the key transcription that reciprocally control colonic Treg populations (Rorc, Gata3, Ikzf2). The effects of
these perturbations will be assessed on colon inflammation at baseline, the control of chemical or bacterially-
induced inflammation, intestinal barrier integrity, microbe-specific IgA repertoire and whether changes in Treg
populations influence the balance of microbial phyla and species.
 These connected explorations will provide a unique mechanistic and functional understanding of these
essential Treg populations, and will have profound implications on our understanding of the control of
inflammation at the host/symbiont interface.

## Key facts

- **NIH application ID:** 10129879
- **Project number:** 5R01AI125603-05
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** CHRISTOPHE O. BENOIST
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $423,750
- **Award type:** 5
- **Project period:** 2017-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10129879

## Citation

> US National Institutes of Health, RePORTER application 10129879, T regulatory cell subsets at the microbial interface: determinism and function (5R01AI125603-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10129879. Licensed CC0.

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