Transcriptomic Analysis of HIV-Infected Cells

Defining the attributes of rare CD4 T cells that harbor latent HIV under therapy with anti-retroviral agents has been challenging due to difficulties in identifying and isolating these cells. These cells represent an important barrier to cure, and characterizing them would aid the design and development of novel HIV cure strategies. In this study, we addressed this challenge using a custom microfluidic technology named Focused Interrogation of cells by Nucleic acid Detection and Sequencing (FIND-seq) that isolates HIV-infected cell transcriptomes, based on HIV DNA detection. We applied this method to HIV DNA+ memory CD4 T cells isolated from blood from five persons with HIV who were receiving anti-retroviral agents. After curating samples based on data quality, we proceeded with bioinformatics analysis from three persons. This analysis identified six transcriptomic pathways that were inhibited in HIV DNA+ cells compared to HIV DNA- cells. These included death receptor signaling, necroptosis signaling, and anti-proliferative G-alpha12/13 signaling. Furthermore, we identified two gene clusters, including 60 and 85 genes, that were significantly associated with HIV DNA+ cells identified by co-expression network analysis. These genes included negative regulators of HIV transcription that were higher in HIV DNA+ cells, positive regulators of HIV transcription that were lower in HIV DNA+ cells, and other genes involved in the negative regulation of mRNA translation, additional RNA processing functions, and the regulation of cellular state and fate. We further examined these signatures in transcriptomic data from CD4 T cell subsets sorted by flow cytometry from nine persons with HIV. We found a partial similarity between the smaller cluster of genes and the signature of CCR6- peripheral TFH cells. Raw sequencing data from all eleven human participant samples included in the study are available. These include samples from two participants who were studied only by FIND-seq, three participants who were studied by both FIND-seq and flow cytometry and six participants who were studied only by flow cytometry. ]]>
Inclusion Criteria:HIV-1 infected participants with any of the following criteria:Documented HIV viral load less than 2000 copies/ml WITHOUT taking antiretroviral therapy, orUndetectable HIV viral load with CD4 T-cells consistently less than 350 for the last 12 months while taking a stable antiretroviral regimen, orAntiretroviral naive and planning to start an antiretroviral regimen - any CD4 or HIV viral load acceptable, orLong-term Non Progressors: HIV-positive at least 10 years, no antiretroviral therapy for the past 10 years or more, any viral load acceptable, CD4-T cell count always above 500. Exclusion Criteria:Active opportunistic infection or systemic treatment for opportunistic infection within the last 4 months (oral candidiasis acceptable)Active treatment for cancerActive treatment for hepatitis C requiring interferon based therapyImmunosuppressive therapy taken within the last 4 monthsActive treatment for hepatitis C requiring interferon based therapyImmunosuppressive therapy taken within the last 4 months]]>