Characterizing the Immune Landscape in Rhesus Macaque Lymph Nodes During SIV Infection

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology

First Advisor

Ho, Ya-Chi

Abstract

HIV-1 infects cells expressing CD4, primarily memory CD4+ cells, and stably integrates into the host genome as a provirus. These infected cells can persist long-term. Antiretroviral therapy (ART) prevents new rounds of infection but is unable to eliminate infected cells, which can become latent and avoid detection and elimination by other immune cells. This reservoir of rare and heterogeneous latent HIV-1-infected cells can reactivate, almost inevitably leading to viral rebound if ART is interrupted. As a result, this reservoir is the major barrier to HIV-1 cure. Lymph nodes are important sites of the HIV-1 reservoir, key locations of immune response coordination, and tissues that experience structural change such as fibrosis and follicular hyperplasia during infection. In addition, lymph nodes are a source of rebound virus and show detectable viral replication before virus is detectable in peripheral blood. However, they have not been studied as extensively as blood as they are harder to obtain from people living with HIV-1 (PLWH). SIV infection of rhesus macaques recapitulates key aspects of HIV-1 infection including progression to AIDS and death and overcomes the difficulties of tissue availability and allows experimental control. SIVmac239 infection of rhesus macaques causes initial interferon (IFN) responses in acute infection that peak at one to two weeks after infection. IFN responses and markers of inflammation in peripheral blood and tissues decrease after this peak, but they remain elevated above baseline levels even with suppressive ART, mirroring what occurs in PLWH. Infected cells can be found in lymph nodes even under ART. Rebound occurs within weeks of analytical treatment interruption (ATI) even with treatment initiation within three days of infection. Because of all of these similarities to PLWH, the SIV-infected rhesus macaque model is a useful tool for understanding more about HIV-1 infection and rebound. In our first data chapter (Chapter 3), we perform single-nucleus RNA-sequencing of an SIVmac239-infected rhesus macaque lymph node. Single-cell datasets are useful resources for researchers using the same model organism and tissue, but minimal resources are available for rhesus macaques compared to humans and mice, especially outside of the brain. We identified nearly 11,000 cells across 18 cell subtypes in a lymph node from an SIVmac239-infected, early-treated, early ATI (four days) rhesus macaque. Compared to the few other datasets from macaque lymph nodes, we noted increased B cell and macrophage frequency and decreased CD4+ T cell frequency in our early ATI lymph node. Cells exist within tissues, interact with nearby cells, form structures, and create and respond to local microenvironments. In order to understand this spatial context, we applied a genome-wide unbiased spatial transcriptomics method (DBiT-seq) with 10 m resolution to lymph nodes from uninfected, ART-suppressed, early ATI (four to five days), and late ATI (twenty-six days). After determining an appropriate quality threshold, we retained over 48,000 spatial spots. Because these spots are not single cells, we deconvoluted them with SPOTlight to predict the cell type composition of each spot. Comparing these results across conditions revealed a significant increase in myeloid cell infiltration in late ATI. We identified SIV RNA+ spots by mapping spatial-RNA-seq reads to SIVmac239 and detected SIV RNA in 150 spots across the four late ATI slides and one early ATI slide. We defined neighbors to these SIV RNA+ spots and assessed differential gene expression for spots by SIV RNA status, by neighbor status, from slides containing SIV RNA, and slides from late ATI. SIV RNA+ spots and neighbors shared upregulated genes involved in T cell signaling, activation, proliferation, and survival. Spots further from SIV RNA had increased expression of genes related to antigen processing and presentation, cellular migration particularly of endothelial cells, and innate immune responses. Slides containing SIV RNA or from late ATI showed increased expression of myeloid cell markers, genes involved in immune infiltration, and a gene linked to fibrosis development. RNAscope detection and imaging confirmed SIV replication in late ATI lymph nodes and supported an increase in myeloid cells at this time point. Our work created single-cell and spatial transcriptomic datasets from SIV-infected rhesus macaques. This data could be a useful resource for future work with SIV, lymph nodes, and rhesus macaques in general, an important animal model. The myeloid cell infiltration in late ATI can be further investigated to define cells and their effects more clearly, especially at intermediate time points to our samples. These future spatially-resolved studies of lymph nodes as SIV escapes immune control and causes rebound would contribute toward the knowledge required to control the viral reservoir in PLWH without continued ART.

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