Characterization of naïve immune cell subsets important in HIV/SIV pathogenesis as a baseline for RNASeq deconvolution

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The human immunodeficiency virus-1 (HIV-1) currently infects 35 million people globally. HIV preferentially infects CD4+ T cells, a critical component of the host immune system, causing their rapid depletion, which has a broad negative impact on host immunity. Chronic HIV infection also results in increased expression of inhibitory immune regulatory proteins, which is associated with impaired functionality of a wide range of immune cells. This clinical phenomenon, referred to as "immune exhaustion," precludes the slow and eventual failure of host immunity against co-infecting pathogens and is the hallmark of AIDS-related disease. In Aim 1 of our studies, we used whole blood from Indian-origin rhesus monkeys to distinguish 12 discrete immune cell subsets utilizing antibody staining and flow cytometric cell sorting. The segregated "naïve" (uninfected) cell subsets will be characterized by RNASeq gene expression analysis, and will be used as the baseline population for a bioinformatics "deconvolution" method comparing the same cell subsets from simian immunodeficiency virus (SIV)-infected cell populations. We successfully developed an antibody panel that distinguishes activated and resting CD4+ T cells, activated and resting CD8+ T cells, activated and resting B cells, activated and resting NK cells, plasmacytoid dendritic cells, myeloid dendritic cells, and monocytes. We also developed a separate isolation protocol for neutrophils using different densities of Percoll. Finally, we optimized cell sorting of CD4+ and CD8+ T cells in order to obtain sufficient amounts of high quality RNA for future RNASeq gene expression analysis. As an adjunct to our above work, in Aim 2 of our experiments we sought to quantify the expression of immune inhibitory proteins that are increased upon the various cell subsets during SIV infection. We set out to optimize an antibody panel targeting three proteins of interest, PD-L1, LAG-3, and TIM-3, for the study of whole blood from SIV-infected rhesus monkeys. This data will be used to compliment our RNASeq dataset developed in Aim 1. By utilizing a biotinylated antibody specific for PD-L1 along with fluorescently conjugated streptavidin, we were able to detect PD-L1-positive cells and allow for amplification of positive fluorescence. We also performed multiple evaluations of monoclonal antibodies specific to either LAG-3 or TIM-3 and determined the concentrations at which detection was best for LAG-3+ and TIM-3+ cells.

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