Single-cell Analysis of Glycomic and Proteomic Features of the HIV Reservoir

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PROJECT SUMMARY: It has emerged in recent years that multiple types of HIV reservoir cells can persist in the face of antiretroviral therapy (ART). HIV reservoir cells include those that are “latent” (transcriptionally- inactive) and those that are transcriptionally-active. The transcriptionally-active reservoir cells may be a major contributor to HIV rebound after ART interruption, as shown in analytic treatment interruption studies. In addition, some reservoir cells are able to produce viral proteins upon stimulation (i.e., inducible reservoir cells), and these reservoir cells may also be important for rebound as they are enriched for genome-intact and replication- competent HIV provirus. Unfortunately, the lack of a detailed understanding of the cell surface phenotypes of these HIV reservoir cells has precluded a full understanding of the biology of HIV persistence and hampered the development of a cure. Importantly, prior efforts to characterize reservoir phenotypes all focused on cell surface proteins and ignored cell surface glycans, despite the fact that glycans play a critical role in regulating multiple key cellular processes and immunological functions and have served as useful biomarkers in many diseases. We recently demonstrated that peripheral CD4+ T cells harboring high levels of fucosylated carbohydrates are enriched for HIV+ transcriptionally-active cells and deficient in HIV+ transcriptionally-inactive ones, and that this fucosylation is directly induced by HIV infection. These studies suggest that glycans may have utility as novel biomarkers of specific types of reservoir cells. In this study, we combine newly developed single-cell surface glycomic + proteomic technologies with bioinformatic analyses to analyze cells from an HIV latency model and ART-suppressed HIV+ individuals in order better understanding the makeup of these cells, with the ultimate goal of understand how they can persist in the face of ART and whether they express a unique profile of therapeutically targetable biomarkers. In Aim 1, we will test the hypothesis that cell-surface glycosylation patterns, including fucosylation, distinguish transcriptionally-active and inactive HIV reservoir cells in vitro and in vivo. These studies will take advantage of our recently established technique CyTOF-Lec, which pairs in-depth CyTOF phenotyping with surface glycan characterization at the single-cell level. In Aim 2, we will examine the glycan features of in vivo inducible reservoir cells. To do this, we will adapt our recently established and validated PP-SLIDE (predicted precursor single-cell linkage using distance estimation) approach to characterize the glycan + protein features of patient-derived reservoir cells inducible by ex vivo stimulation. Together, these analyses will allow us to test our central hypothesis that specific cell-surface glycosylation patterns, including fucosylation, can distinguish HIV reservoir cells from uninfected ones, and furthermore distinguish different types of reservoir cells (e.g., latent, transcriptionally-active, and inducible).