# Defining the transcriptional, phenotypic, and functional heterogeneity of virus-specific CD4 T cells during chronic viral infection

> **NIH NIH K99** · VERSITI BLOOD HEALTH, INC. · 2021 · $112,127

## Abstract

CD4 “help” via soluble factors and cell-surface ligands is critical to sustain CD8 T cell responses and humoral
immunity during chronic viral infection. Recently, our lab has further identified a necessity for CD4-derived IL-21
in the formation of a previously unrecognized CX3CR1+CD8 T cell subset that exhibits potent cytolytic function.
However, despite their essential role, our understanding of how persistent exposure to viral load and
inflammation shapes helper T cell differentiation remains incompletely understood. Using scRNA-seq, our lab
has generated substantial preliminary data demonstrating that CD4 T cells responding to chronic LCMV infection
are more heterogenous than previously appreciated, with three transcriptionally distinct subsets dominating the
antiviral response: Cxcr6+Th1, Cxcr5+Tfh, and Slamf6+ memory-like cells. Notably, our data further indicate that
CD4 T cells responding to chronic, but not acute viral infection, preferentially redirect their differentiation towards
this memory-like subset. Taken together, we hypothesize that prolonged exposure to antigenic and inflammatory
signals (TGF-β, IL-10 etc.) modulate the transcriptional diversity of virus-specific CD4 T cells, and that CD4
differentiation during chronic viral infection is driven towards this memory-like subset to coordinate the following:
1) attenuate Th1-mediated immunopathology, 2) maintain a progenitor pool that can give rise to Th1 and Tfh
effector cells, and 3) facilitate a niche wherein memory-like CD4 T cells co-localize with progenitor CD8 T cells
to redirect their differentiation towards protective CX3CR1+CD8 T cells. In Aim 1, we will perform adoptive transfer
(AT) experiments to examine the proliferative potential, protective capacity, and lineage relationship between
the three major subsets of virus-specific CD4 T cells. Additionally, based on the gene regulatory network
formulated by SCENIC (an R package), we will use RNA interference models to test the contribution of key
transcription factors predicted to play a role in regulating CD4 differentiation during chronic infection. In Aim 2,
we will use genetic approaches and AT experiments to manipulate antigen-driven TCR-signaling in order to
assess how antigenic signals regulate CD4 differentiation during chronic infection. Moreover, we will block the
activity of immunoregulatory cytokines known to be overexpressed during chronic infection to determine their
impact on the fate commitment of virus-specific CD4 T cells. Lastly, in Aim 3 we will determine whether AT of IL-
21-producing memory-like CD4 T cells, either alone or in conjunction with PD-L1 blockade, can augment
progenitorCX3CR1hi CD8 T cell transition to improve T cell-mediated immunity. Knowledge gained from this
research will provide mechanistic insights into the functional adaption process CD4 T cells display in the face of
persistent infection and help identify novel strategies aimed at optimizing cellular or humoral-mediated antiviral
...

## Key facts

- **NIH application ID:** 10206009
- **Project number:** 5K99AI153537-02
- **Recipient organization:** VERSITI BLOOD HEALTH, INC.
- **Principal Investigator:** Ryan Zander
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $112,127
- **Award type:** 5
- **Project period:** 2020-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10206009

## Citation

> US National Institutes of Health, RePORTER application 10206009, Defining the transcriptional, phenotypic, and functional heterogeneity of virus-specific CD4 T cells during chronic viral infection (5K99AI153537-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10206009. Licensed CC0.

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