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Single-cell multi-omics understanding of HIV-induced immune dysfunction

- Finding a cure for HIV

Despite effective antiretroviral therapy, HIV persists as an integrated provirus. These HIV-infected cells are resistant to immune clearance and antiretroviral therapy. Upon treatment interruptions, viral rebound will resume. Therefore, all HIV-infected individuals need to take life-long antiretroviral therapy. Given the adverse effects, drug resistance and economical burden of lifelong antiretroviral therapy, a cure is needed to control the global HIV endemic.

The mission of the Ho lab is to understand HIV persistence and find a cure for HIV infections. The scientific goal is to understand how HIV persists in cells, particularly CD4 T lymphocytes, and whether epigenetic silencing can permanently and irreversibly silence HIV expression. We use a molecular virology approach to examine mechanisms of HIV persistence using blood samples from HIV-infected individuals.

We are currently recruiting postdocs working on a combination of HIV-induced immune dysfunction, single-cell genomics, and bioinformatics. See details here!

Despite effective antiretroviral therapy (ART), HIV persists in the latent reservoir as the major barrier to cure. Although ART effectively suppresses plasma viral load to clinically undetectable levels, once ART is interrupted, high levels of viremia will inevitably occur. Therefore, all 37 million people living with HIV need to take lifelong ART. This is because HIV hides in the memory CD4+ T cells and evades immune clearance. First, HIV stably integrates into chromosomes of infected cells. ART cannot remove these integrated proviruses. Second, HIV infects the long-lived memory CD4+ T cells and persists for decades. Third, HIV enters latency and becomes transcriptionally silent when memory CD4+ T cells return to a quiescent state. Latently infected cells do not express viral antigens and cannot be recognized and cleared by the immune system. Fourth, HIV-infected cells undergo clonal expansion through antigen stimulation, homeostatic cytokine-mediated proliferation, and integration-site driven proliferation. This means that HIV-infected cells not only survive but also increase over time. Finally, despite ART, millions of HIV-infected cells already established the latent reservoir within 3 days of infection. Since ART does not inhibit HIV LTR promoter activity, these infected cells express viral antigens through stochastic activation and continue to induce immune activation and immune exhaustion. The exhausted and dysfunctional immune effector cells cannot effectively eliminate HIV-infected cells. A cure is desperately needed. However, many questions remain unsolved: how does virally suppressed HIV infection induce chronic immune exhaustion? How do HIV-infected cells survive, proliferate, and persist over time? How can we specifically target HIV-infected CD4+ T cells without harming uninfected cells?

Our goal is to understand how HIV induces immune dysfunction over different stages of HIV infection over time, both at the systemic level characterizing all CD4+ T cells and the single-cell level in HIV-infected cells. We will focus on CD4+ T cells, the central orchestrator of adaptive immunity and the major target of HIV infection. We hypothesize that despite the heterogeneity of HIV-infected cells, HIV drives a distinct cellular program that promotes persistence. We propose that identifying and targeting HIV-driven immune dysregulation leads to cure. The challenge to answering this question is the heterogeneity, rarity, and lack of marker of HIV-infected cells. Our approach is to develop cutting-edge single-cell methods, to apply these methods to challenging clinical samples, and to validate with CRISPR-mediated up- and down-regulation of candidate cellular pathways.

Single-cell transcriptional landscapes reveal HIV-1–driven aberrant host gene transcription as a potential therapeutic target

HIV SortSeq identified the rare HIV-infected cells and a new way to treat HIV. See our "Single-cell transcriptional landscapes reveal HIV-1–driven aberrant host gene transcription as a potential therapeutic target" published on May 13, 2020 at Science Translational Medicine.
1. We developed HIV-1 SortSeq which uses HIV RNA expression as a surrogate to identify HIV-infected cells. This RNA-preserving method enables single-cell RNAseq on formaldehyde fixed samples.
2. HIV RNA positive cells are enriched in genes related to RNA processing and viral gene expression.
3. When HIV integrates into a host gene, HIV dominates over the host promoter and drives aberrant cancer gene expression by splicing from HIV into the host RNA.

Targeting RNA processing and T cell activation halts HIV-1-driven aberrant host-transcription

We identified drugs which may suppress HIV-induced immune activation and the expansion of the HIV latent reservoir. See our manuscript at in-press preview "Filgotinib suppresses HIV-1-driven gene transcription by inhibiting HIV-1 splicing and T cell activation" published on June 23, 2020 at Journal of Clinical Investigation.
1. We used a drug screen on a dual-reporter cell line to identify drugs which can suppress HIV expression without affecting cellular gene expression.
2. We identified 16 cellular pathways and 11 FDA-approved drugs which can suppress HIV expression.
3. Using transcriptome landscape analysis, we found that HIV-suppressing agents, such as the JAK inhibitor filgotinib, can inhibit HIV-driven cancer gene expression at the HIV integration site.
4. Using differential expression analysis (Ingenuity Pathway Analysis, IPA) and gene-set enrichment analysis (GSEA), we identified T cell activation and RNA processing-related genes that mediates HIV suppression.
5. Using intron retention analysis, we found a new function of filgotinib as a splice inhibitor.
6. Using limiting dilution culture of CD4+ T cells from HIV-infected individuals, we found that treating cells with HIV-suppressing agents can reduce the proliferation of HIV-infected cells ex vivo.

Single-cell multi-omics profiling of HIV-induced immune dysfunction and T cell clones harboring HIV

HIV persistence in clonally expanding CD4+ T cells is the major barrier to cure. Studying HIV-1-infected cells in clinical samples has been challenging, due to the rarity, heterogeneity, and lack of cellular markers for HIV-infected cells. Using paired blood samples during viremia and after suppressive ART from a randomized and interventional clinical trial (Sabes study), we interrogated how immediate versus delayed ART affected HIV-1-induced immune dysfunction and HIV persistence. We combined a single-cell multi-omic ECCITE-seq and cutting-edge machine learning methods to profile CD4+ T cells and identified 90 HIV RNA+ cells. To our knowledge, this is the largest number of HIV-infected cells examined by single-cell multi-omics and the first transcriptome analysis of HIV-1-infected single cells from infected individuals without ex vivo stimulation. By capturing surface protein expression, cellular transcriptome, HIV RNA, and T cell receptor sequencing within the same single cells, we identified the clonal expansion dynamics of T cell clones harboring HIV and the transcriptional program driving HIV persistence and T cell proliferation.

We found immune drivers that promote HIV persistence and cellular markers as potential therapeutic targets. First, we found that an ongoing TNF response is the major immune dysfunction in delayed versus immediate ART, shapes the transcriptional program of HIV RNA+ cells, and is an upstream regulator shaping T cell clonal expansion. Second, we found that cytotoxic CD4+ T lymphocytes, particularly those expressing GZMK (granzyme K) and GZMB (granzyme B), harbor HIV RNA+ cells and T cell clones harboring them. The transcriptionally distinct GZMK cytotoxic T cells were recently found to be important in anti-tumor immunity and in SARS-CoV-2 infection but under-appreciated in HIV infection. Third, we found that T cell clones harboring HIV-1 RNA+ cells are larger in clone size. Fourth, using machine learning algorithms, we identified markers for HIV RNA+ cells and T cell clones harboring them. Altogether, we found that HIV resides in cytotoxic T cells which are naturally proliferative and continue to be impacted by antigen stimulation and TNF responses from viremia through viral suppression.

CFAR Seminar June 2018 Ya-Chi Ho MD, PhD