# Chromatin and transcriptional regulatory factors that initiate and stabilize memory CD8 T cell development

> **NIH NIH P01** · UNIVERSITY OF FLORIDA · 2022 · $669,976

## Abstract

PROJECT SUMMARY / ABSTRACT
Project 2 (Pipkin)
Activation of naive CD8 T cells during intracellular infections rapidly induces chromatin remodeling and
transcriptional reprogramming that results in the differentiation of memory (TMEM) CTLs that provide long-term
immunity. We discovered that the transcription factor (TF) Runx3 instigates chromatin accessibility of TMEM-
associated cis-regulatory regions in naive cells during initial T cell receptor (TCR) stimulation, and is essential
for the differentiation of both circulating and tissue resident TMEM CTLs. Runx3 activates transcriptional circuits
that establishes nascent CTLs, represses alternative cell fates and prevents terminal CTL differentiation. Using
mass spectrometry we identified all subunits of the NuRD/HDAC complex in association with Runx3. In addition,
we conducted a pooled, RNA interference (RNAi) -mediated, loss-of-function screen in CD8 T cells responding
to viral infection that targeted all mammalian chromatin regulator factors (CRFs, 312 genes). This revealed that
deficiency in multiple individual NuRD/HDAC complex subunits impaired memory precursor CTL differentiation,
similar to Runx3 loss-of-function. In addition, multiple subunits of the BAF-family of nucleosome remodelers and
its collaborator Chd7, which is mutated in a human immunodeficiency, were essential for initiating very early
aspects of CTL differentiation and driving terminal differentiation. To gain insight into early aspects of CTL
development, we used a single cell RNA-seq (scRNA-seq) approach and performed computational trajectory
analyses which identified common, and then branching developmental pathways from naive CD8s that lead
toward effector and memory cell fates. In part, we confirmed these pathways operationally using Blimp1-YFP
reporter alleles and adoptive transfer experiments. In the current application, we propose to build on these results
to elucidate how CRFs and TFs reprogram chromatin structure during naive cell activation and early
establishment of effector and memory-like developmental paths. Specifically, we will define how Runx3 and
NuRD/HDAC complexes remodel chromatin structure to establish initial TMEM transcriptional programs (Aim 1).
We will elucidate how hierarchical functions of Runx3, Ets1, Blimp1 and Bcl6 regulate the divergence of Blimp1hi
and Blimp1lo effector- and memory-like developmental paths, and use an in vivo conditional RNAi approach in
naive CD8s to screen all T cell-expressed TFs (1,751 genes) to identify their roles in this process during viral
infection (Aim 2). Finally, we will integrate these analyses with how BAF and Chd7 remodelers govern
nucleosome organization in cis-regulatory regions that control transcriptional reprograming during early CTL
differentiation (Aim 3). These studies integrate synergistically with analogous approaches addressing the roles
of CRFs and TFs in TFH differentiation and function (Crotty, Project 1), and how they function at later times to
...

## Key facts

- **NIH application ID:** 10488588
- **Project number:** 5P01AI145815-04
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Matthew Eugene Pipkin
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $669,976
- **Award type:** 5
- **Project period:** 2020-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10488588

## Citation

> US National Institutes of Health, RePORTER application 10488588, Chromatin and transcriptional regulatory factors that initiate and stabilize memory CD8 T cell development (5P01AI145815-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10488588. Licensed CC0.

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