# Harnessing activated CD4 T cells to define new mechanisms of protection in tuberculosis

> **NIH NIH R01** · UNIVERSITY OF MINNESOTA · 2024 · $590,248

## Abstract

CD4 T cells are essential for protection against tuberculosis (TB). But current approaches to TB therapy have
not harnessed their potential benefit, and the mechanisms they use to control Mycobacterium tuberculosis (Mtb)
have not been completely defined. At the peak of adaptive immunity to TB, millions of Mtb-specific CD4 T cells
traffic to the lungs, but very few of them re-encounter their cognate antigen to become activated. The ability to
clearly discriminate and isolate these few activated cells from the greater population of inactive CD4 T cells could
enable the discovery of important new markers, effector functions, and clonally expanded T cell receptor (TCR)
sequences closely associated with protective immunity. To study activated CD4 T cells from Mtb-infected lungs,
we used a new approach that combines: (i) a reporter mouse to identify cells actively receiving TCR stimulation
in vivo (Nur77-GFP); (ii) BSL3-contained fluorescence cell sorting to isolate them live from tissues; and (iii) single
cell RNA sequencing (scRNA-Seq) with CITE-Seq and TCR immune-profiling to interrogate their function and
antigen-specificity. In our preliminary work, we discovered that Nur77-GFPHI CD4 T cells express an array of
activation markers and costimulatory receptors including OX40. This population was enriched for T regulatory
cells and effector T cells with TCR clonotypes localizing to lung parenchyma. Using adoptive transfer, we found
that Nur77-GFPHI cells are more protective than their Nur77-GFPLO counterparts. To therapeutically harness
activated CD4 T cells in vivo, we treated Mtb-infected mice with a monoclonal antibody that agonizes OX40 and
found that this treatment reduced the lung bacterial burden, prolonged survival of infected mice by >100 days
and did not cause toxicity. Immunotherapy specifically targeting activated CD4 T cells is a novel approach to TB
treatment that has not been thoroughly investigated. In Aim 1 of this proposal, we will define the function of
activated CD4 T cells in the lungs of Mtb-infected mice using scRNA-Seq and CITE-Seq. With adoptive transfer
and scTCR-Seq, we will uncover how antigen specificity shapes the function and fate of different CD4 T cell TCR
clonotypes throughout chronic infection. We will use TCR sequences we generate to develop TCR retrogenic
mice that can identify protective Mtb antigens and test the hypothesis that antigen specificity shapes T cell
phenotype in TB In Aim 2, we will determine the mechanisms underlying activation marker immunotherapy
mediated CD4 T cell control of TB. We will test the hypothesis that OX40 agonist treatment provides protection
by selectively modulating the function of both activated T conventional and T regulatory cells. Finally, we will
determine how OX40 agonism impacts activated CD4 T cell survival through chronic infection. The studies
proposed here represent the first high-definition characterization of the small population of activated CD4 T cells
at the site of M...

## Key facts

- **NIH application ID:** 10877769
- **Project number:** 5R01AI173780-02
- **Recipient organization:** UNIVERSITY OF MINNESOTA
- **Principal Investigator:** Tyler Dallas Bold
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $590,248
- **Award type:** 5
- **Project period:** 2023-07-01 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10877769

## Citation

> US National Institutes of Health, RePORTER application 10877769, Harnessing activated CD4 T cells to define new mechanisms of protection in tuberculosis (5R01AI173780-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10877769. Licensed CC0.

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