CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

390

Understanding NLRP3 Inflammasome Activation at the Intersection of HIV and Aging Heidi Zapata, Shanshan Du, Archit Kumar, Bill Lee, Christopher Radcliffe, Lydia Aoun-Barakat, Sui Tsang, Brent Vanderwyk, Albert Shaw Yale University, New Haven, CT, USA Background: With long-term survival of people living with HIV (PLH) increased rates of age-related diseases have been observed. Persistent immune activation in PLH and age-related chronic inflammation are hypothesized to be drivers for the development of such diseases. The NLRP3 inflammasome is a multiprotein complex that forms in response to stimulation of pattern recognition receptors by pathogen associated molecular patterns (PAMPs), or by Damage Associated Molecular Patterns (DAMPs). This complex mediates the cleavage of caspase-1, IL-1b and IL-18 to their active forms. Activation of the inflammasome requires a priming step (signal 1) that induces the TLR/NF-kB pathway, and a second step (signal 2), triggered by a PAMP/DAMP and leading to formation of the complex. There is evidence that the development of age-related diseases is related to dysregulated activation of the NLRP3 inflammasome and pathological production of IL-1b. However, significant knowledge gaps in the understanding of human NLRP3 function remain at the interface of HIV and aging. Methods: We recruited young (21-40) and older (>60 years) HIV-positive and HIV-negative subjects (n=64). Blood was drawn and peripheral blood mononuclear cells were isolated for subsequent experiments. Results: HIV-positive older subjects have increased inflammasome activation at baseline as demonstrated by higher levels of caspase-1, and higher levels of cleaved IL-1b, and IL-18 (flow cytometry). With LPS stimulation (signal one) we see decreased levels of cleaved IL-1b production via western blot in HIV-positive older adults as compared to HIV-negative older adults and HIV-positive young adults. When LPS stimulation (signal one) is examined via flow cytometry we see increased retention of cleaved IL-1b in the monocytes of HIV-negative young adults compared to HIV-negative and HIV-positive older adults. With signal two we see that HIV-positive older adults have decreased retention of cleaved IL-1b compared to HIV-negative young adults—suggesting differences in Gasdermin signaling. When inflammasome specks are examined (confocal microscopy) we see that HIV-positive older adults have increased number of specks when compared to HIV-negative young adults. Conclusions: Overall, HIV-positive older adults have increased baseline activation of the inflammasome and subsequently a decreased ability to respond when exposed to PAMPs/DAMPs. The results noted allude to a dysregulation of Gasdermin signaling and inflammasome machinery in HIV older adults. Ex Vivo Isolation and Characterization of Live, Envelope Expressing, SHIV-Infected CD4 T Cells Joseph P. Casazza, Joana Dias, Bianca C. Ralph, David R. Ambrozak, Jianfei Hu, Amy Henry, Farida Laboune, Lucio Gama, Stephen D. Schmidt, Timothy S. Johnston, Giulia Fabozzi, Sabrina Helmold Hait, Daniel C. Douek, Richard A. Koup Vaccine Research Center, Bethesda, MD, USA Background: Fully mature SHIV envelope is transported to the CD4 T cell surface. Therefore, expressed envelope represents a means of identifying live SHIV-infected CD4 T cells. Methods: CD4 T cells from Fiebig-staged SHIVAD8-EO-infected rhesus macaques were stained using 2 fluorescently labelled broadly neutralizing antibodies previously shown to identify in vitro SHIVAD8-EO-infected rhesus macaque CD4 T cells. SHIV-infected cells from 2 rhesus macaques were index sorted between Fiebig stages 1 and 6. For each animal lymph nodes (LNs) were longitudinally sampled 3 times, and blood was longitudinally sampled 5 times. SHIV infection was confirmed by measurement of single cell SHIV gag-RNA by RT PCR. SmartSeq NGS libraries were prepared, then sequenced using an Illumina NovaSeq 6000. Bcl2fastq2, STAR, Picard and HTSeq were used to demultiplex and map reads, sequence and calculate read number and analyze differentially expression for each gene. Benjamini Hochberg correction was used for multiple comparison correction. Results: The frequency of SHIV-infected cells in both peripheral blood and LN CD4 T cells, intracellular SHIV RNA copy number and blood viral load all peaked between Fiebig stages 2 and 5, and then decreased in these macaques. During this time the frequency of SHIV infected cells increased in PD1 positive and CD28 dim CD4 T cells and then decreased. Intracellular SHIV RNA was inversely associated with CD3 and CD4 fluorescence intensity. Infection of germinal center CD4 T cells was discordant in these animals. One animal showed no

germinal center CD4 SHIV infection, in the other most infection occurred in the germinal center. Envelope staining did not strictly identify SHIV infected CD4 T cells. In some populations the frequency of SHIV RNA in envelope stained CD4 T cells was close to 40%. This allowed the comparison of SHIV-infected and non-infected CD4 T cells in 57 Env + SHIV RNA + and 80 Env - SHIV RNA - LN CD4 T cells from a single sample. The frequency of SHIV RNA was as high as 5% of the sequenced transcriptome. The frequency of HIV RNA reads increased with CD3 and CD4 down regulation. GSEA analysis suggests G2M arrest and decreased antiviral responses in SHIV-infected CD4 T cells. Conclusions: Ex vivo isolation of acutely infected SHIV CD4 T cells using surface markers is possible. This enabled the identification of specific subsets of CD4 T cells with greatly increased frequencies of infection, allowing further characterization and transcriptome analysis of SHIV-infected CD4 T cells. Lobule Zone-Dependent Accumulation of Vd1 and Vd2 gd-T Cells in the Livers of SIV+ Infant Macaques Nina Derby 1 , Brooke I. Johnson 1 , Kimberly A. Meyer 1 , Jeremy V. Smedley 2 , Don L. Sodora 1 1 Seattle Children's Research Institute, Seattle, WA, USA, 2 University of Louisiana at Lafayette, Lafayette, LA, USA Background: Gamma-delta (gd) T cells are enriched in liver and barrier tissues compared to the peripheral blood and aid in the response to gut-derived antigens and tissue homeostasis. They are depleted from the blood during HIV infection, yet what happens to them and the importance of the effect are not well characterized. We hypothesized that gd-T cells may be involved in liver dysfunction during HIV infection. Methods: Livers from uninfected infant rhesus macaques (SIV-, n=5) and infant macaques chronically infected with SIV (SIV+, n=5) were subjected to GeoMx digital spatial profiling (DSP) to evaluate SIV-associated transcriptional changes within distinct zones of the liver lobule, including changes that indicate enrichment of immune cell populations. Fixed tissue sections were immunostained for periportal (near portal tracts) and pericentral (near central veins) zones. Eighty segments were placed based on immunostaining, profiled using the whole transcriptome atlas and analyzed with linear mixed models, gene set enrichment analysis (GSEA), and mixed cell deconvolution with SpatialDecon algorithm. Results: DSP identified >100 up and downregulated genes in SIV+ vs SIV- infant macaque livers as well as effects specific to periportal vs. pericentral zones. Genes upregulated in SIV+ livers were associated with inflammation, the type I interferon response and oxidative stress. Deconvolution identified increases in cholangiocytes, liver sinusoidal endothelial cells, non-inflammatory macrophages, and gd-T cells. gd-T cells were the most significantly altered immune cell subset in SIV+ infants. Stress-responsive gd-T cells-1 were increased only periportally while phosphoantigen-responsive gd-T cells-2 were increased periportally and even more so pericentrally. The pericentral zone also experienced strong downregulation of biological oxidation genes (NES -3.68), including those within the glutathione S transferase (eg. GSTP1 (FDR 0.0001)) and cytochrome P450 (eg. CYP39A1 (FDR 1.28E-06)) pathways. Conclusions: gd-T cells are enriched in the livers of infant macaques infected with SIV and experiencing high viral loads. gd-T cells-1 and gd-T cells-2 are differentially affected by lobule zone, suggesting these subsets may respond to distinct stimuli or exert distinct effects in the SIV+ liver. Ongoing studies are investigating the relationship between accumulation of these cells and other zone-specific transcriptional changes. IL-15, Type I Interferon, Fatty and Amino Acid Pathways Link to SIV Reservoir and Rebound in Infants Tehillah Chinunga 1 , Giuliana X. Xavier 1 , Fernanda R. Bruno 1 , Felipe Ten-Caten 1 , Katherine Bricker-Holt 2 , Ann M. Chahroudi 1 , Susan P. Ribeiro 1 1 Emory University, Atlanta, GA, USA, 2 National Institutes of Health, Bethesda, MD, USA Background: Inflammation persists under antiretroviral therapy (ART) in HIV infection. Soluble mediators of inflammation play major roles in immune function, reservoir persistence and rebound post-analytical treatment interruption (ATI). Interactions between cytokines/chemokines and/or metabolites with reservoir size and viral rebound dynamics are not fully known. We evaluated associations between host and microbial-derived metabolites or cytokines/chemokines and reservoir size on ART and post-ATI viral rebound in a pediatric HIV model.

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CROI 2025