Probing the unique attributes of the naïve reservoir

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Resting CD4+ T cells form a reservoir capable of refueling HIV viremia when treatment is stopped. This is a major barrier to an HIV cure. Resting CD4+ T cells can be divided into naïve and memory subtypes. It was previously thought that naïve T cells formed a negligible portion of the reservoir, as the total amount of HIV DNA in the naïve pool is small. We have shown, however, that there is relatively more intact than defective HIV DNA in the naïve T cells compared to memory T cell subsets. In fact, in chronic progressors with X4 tropic virus, naïve are often the major contributor to the HIV reservoir. Moreover, the integration sites and proviral sequences of the HIV DNA in naïve cells are more diverse than in the memory subsets, indicating that the proviruses originated from many different infection events. Naïve T cells have a much longer lifespan than memory T cells, higher proliferative potential, and apparent resistance to clearance, which suggest that this naïve T cell reservoir is capable of repopulating the HIV reservoir. The formation of the naïve reservoir by CCR5-tropic viruses is still somewhat mysterious, as naïve cells in the blood do not usually express the CCR5 co-receptor, but preliminary data shows that naïve cells in lymphoid tissues show different expression patterns, including occasional expression of CCR5. Overall objective: We will quantify the contribution of naïve cells to the reservoir in donors that represent a spectrum of reservoir size (Aim 1). We exploit our exciting finding that elite controllers have nearly absent naïve T cell infection, while naïve infection serves as a prognostic indicator in chronic progressors. We will dissect the heterogeneous nature of naïve T cells, especially in lymphoid tissue, as has previously been done for memory T cells using RNA sequencing (Aim 2). We will elucidate important naïve biology and determine how naïve T cells become infected. Design and Methods: In Aim 1, led by Dr. O'Doherty, we quantify the contribution of naïve T cells to the entire HIV reservoir in both size and diversity using sophisticated sequencing techniques, unique bioinformatic approaches (Dr. Polson), and math modeling (Dr. Zurakowski). We will also study the ability of naïve T cells to replenish the reservoir and evade the CTL clearance. In Aim 2, led by Dr. Levy, we couple RNAseq with antibody barcoding to identify memory and naïve T cells with elevated levels of CCR5. We will probe the mechanism of naïve cell infection by infecting subset and bulk T cells from lymphoid tissue and blood. We will determine viral tropism phenotypically, and monitor for reversion to naïve phenotype ex vivo. These studies will also provide insights into the signaling pathways that distinguish naïve T cell subsets, and that determine their susceptibility to HIV. The premise of our proposal is largely based on our prior work that has shone a light on the role of naïve T cells and simultaneously provided insights into why these cells have previously been ignored. This proposal may lead to a greater focus on naïve T cell infection, as these cells are well-suited to hide proviruses. Focused attention on this entirely different cell type will be necessary to eradicate HIV.