# Treg cell diversity and homeostatic control

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2020 · $512,512

## Abstract

CD4+ T regulatory (Treg) that express the transcription factor FoxP3 cells play key and non-redundant
roles in maintaining immunologic tolerance and controlling inflammation. Treg proportions within CD4+ T cells
are under tight homeostatic control. There is considerable diversity to Treg cells, with differential ability to
control certain immune functions, and even tissue-resident Tregs with extra-immunologic functions. Our recent
recent single-cell RNAseq work has charted these finely, showing that Treg subsets express different effector
molecules. Treg subset structure appears very similar in mouse and humans. We hypothesize that Treg
subsets in lymphoid tissues respond to different homeostatic cues, and that these different inputs condition
specific Treg functions. (1) Our single-cell transcriptomics data, which highlighted the diversity of Treg
subphenotypes in an unbiased fashion, identified some cell surface markers for identification and sorting, but
we will expand this base by performing a combined transcriptome/proteome analysis (scRNAseq combined
with surface protein quantitation by DNA-tagged antibodies - CITE-seq) to identify a panel of flow cytometry
markers that accurately distinguish Treg subphenotypes, in both mouse and human. This will relate this novel
landscape to prior knowledge on Treg subsets, provide biomarkers, and enable cell sorting for functional
experimentation. We will assess the function of Treg subsets in regulating other immunocytes (proliferation and
differentiation), assess their developmental origin and stability (thymic or peripheral, plasticity?), and how they
vary in autoimmune or tumor lesions. (2) IL2 is the canonical controller of Treg cellsbut other common-
cytokines (IL7, 15, 21 and TSLP) and IL33 also affect Tregs. Our results show a strikingly divergent distribution
of cytokine receptors among Treg subsets, suggesting that different growth factors support them. We will use
both gain- (in vivo cytokines administration) or loss-of-function (blocking antibodies) strategies to modify Treg
subset balance, read out by flow cytometry (STAT phosphorylation, Treg subset number and activation) and
scRNAseq. (3) Most members of the large TNF Receptor (TNFR) superfamily are over-expressed in Treg cells,
and some have been shown to affect Treg maintenance and survival, but a comprehensive picture is lacking.
Our data show differential representation of TNFR across Treg subsets. We hypothesize that TNFR molecules
network with cytokine receptors to provide specific trophic signals to distinct Treg subsets. We have developed
a powerful CRISPR-based in vivo screen, based on barcoding stem cells, to evaluate how sets of genes
partake in Treg homeostasis. We will analyze the role of the entire TNFR family on maintenance of different
Treg subsets, at baseline in different lymphoid tissues and after stimulation by trophic cytokines. We will also
test several TNFRs combinatorially, to search for redundancy, complementari...

## Key facts

- **NIH application ID:** 9943857
- **Project number:** 1R01AI150686-01
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** CHRISTOPHE O. BENOIST
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $512,512
- **Award type:** 1
- **Project period:** 2020-02-11 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9943857

## Citation

> US National Institutes of Health, RePORTER application 9943857, Treg cell diversity and homeostatic control (1R01AI150686-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9943857. Licensed CC0.

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