# Understanding HIV-1 persistence in cytotoxic CD4+ T lymphocytes at the single cell level

> **NIH NIH R01** · YALE UNIVERSITY · 2024 · $840,074

## Abstract

PROJECT SUMMARY
Despite effective antiretroviral therapy (ART), HIV-1 persists in the latent reservoir lifelong. Although most HIV-
1-infected cells die of viral cytopathic effects or immune clearance, HIV-1-infected cells, even those having active
HIV-1 expression, can survive, persist, and proliferate. We want to examine how HIV-1 establishes infection
during viremia and persists under viral suppression in people living with HIV-1. Understanding the immune cell
subsets, immune programs, and cell markers of HIV-1-infected cells will identify therapeutic targets. The
challenge is the heterogeneity and rarity of HIV-1-infected cells: in ART-treated, virally suppressed individuals,
only 1–100/106 (<0.01%) CD4+ T cells harbor inducible HIV-1. To this end, we profiled CD4+ T cells from the
Sabes cohort during viremia and after viral suppression using single-cell ECCITEseq, which captures single-cell
transcriptome, protein expression, HIV-1 RNA, and T cell receptor sequence (TCR) within the same single cell.
We have established advanced single-cell bioinformatic analysis pipelines and machine learning tools. This
approach enables multi-dimensional, high-resolution, single-cell profiling of immune cell subsets, immune
programs, cell markers, T cell clonal expansion, and HIV-1 RNA+ cells at the same time. Our single-cell
ECCITEseq found that HIV-1 RNA+ cells upregulated cytotoxic CD4+ T cell genes. Using flow cytometric
measurement of HIV-1 p24 protein and granzyme B expression, our RNA-seq based results were validated by
a protein-based orthogonal approach, revealing that HIV-1 persists by hiding in granzyme B+ cytotoxic effector
memory CD4+ T cells. Based on our results from viremic samples, we can examine the rare HIV-1 RNA+ cells
under suppressive ART over time. Using single-cell ECCITEseq, we found that despite suppressive ART, tumor
necrosis factor (TNF) responses persist. Furthermore, antigen and TNF responses drive the proliferation of T
cell clones. In addition, different antigen responses drive distinct T cell polarization and proliferation. Altogether,
we hypothesize that antigen stimulation and TNF responses can shape T cell polarization, cellular susceptibility
to HIV-1 infection, cellular survival, and proliferation of the infected cells, particularly granzyme B cytotoxic CD4+
T cells. Our goal is to understand why HIV-1-infected cytotoxic CD4+ T cells can preferentially survive, proliferate,
and persist, as opposed to other T cell subsets. Achieving this goal will identify immune programs that drive
HIV-1 persistence and identify cell markers for HIV-1-infected cells for more specific therapeutic targeting. Our
approach is to combine cutting-edge single-cell ECCITEseq and orthogonal validations to examine how HIV-1
RNA+ granzyme B+ CTLs survive and persist under viral suppression by profiling cell subsets, immune program,
markers, and proliferation dynamics for the rare HIV-1 RNA+ cells over time during viral suppression in vivo and
in vi...

## Key facts

- **NIH application ID:** 10840336
- **Project number:** 5R01AI174863-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Ya-Chi Ho
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $840,074
- **Award type:** 5
- **Project period:** 2023-05-11 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10840336

## Citation

> US National Institutes of Health, RePORTER application 10840336, Understanding HIV-1 persistence in cytotoxic CD4+ T lymphocytes at the single cell level (5R01AI174863-02). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10840336. Licensed CC0.

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