R01AI174863

Understanding HIV-1 persistence in cytotoxic CD4+ T lymphocytes at the single cell level - 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 ECCITE-seq, 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 ECCITE-seq 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 ECCITE-seq, 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 ECCITE-seq 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 vitro.

Overall, we will understand why HIV-1 preferentially persists in cytotoxic CD4+ T cells, identify upstream immune drivers promoting the survival and proliferation of the HIV-1-infected cytotoxic CD4+ T cells, and guide the development of therapeutic strategies specific for HIV-1-infected cells.

Yale Univ

NOT APPLICABLE

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

New Haven, Connecticut 065191418 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 294% from $856,949 to $3,377,171.