CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

58261, an adenosine receptor antagonist, to investigate the impact of cAMP modulation on the establishment, maintenance, and reversal of HIV latency. Methods: Intracellular cAMP in primary CD4+ T cells and cell lines was quantified using a colorimetric enzyme-linked immunosorbent assay (ELISA). To determine the impact of forskolin and SCH-58261 on latency, HIV latently infected J-Lat 5A8 cells, J-Lat NFkB.RFP cells (reporting NFkB activation via RFP), and HC69.5 microglia were pretreated with varying drug doses followed by phytohaemagglutinin (PHA) or PMA/ionomycin treatment; GFP-positive cells (reflecting viral expression) were enumerated by flow cytometry. To determine the impact of cAMP modulation on initial establishment of latency, Jurkat cells were infected with a dual-reporter virus construct, Duo-Fluo HIV BFP/GFP, which enables enumeration and isolation of HIV latently-infected, productively infected, and uninfected cells by flow cytometry. Data were analyzed using unpaired t tests. Results: Forskolin treatment increased cAMP levels in J-Lat (p=0.0071) and primary CD4+ T cells (p=0.0071) (Fig.1A). Treatment with SCH-58261 reversed the phenotype (p=0.0011). Forskolin exerted a dose-dependent inhibitory impact on PHA- (p=0.0286) and PMA/Ionomycin- (p=0.0011) mediated latency reversal in J-Lat cells (Fig.1B), and NFkB activation was suppressed in parallel (p<0.0001). Forskolin suppressed HIV transcription in HC69.5 microglia (p=0.0046). Forskolin also inhibited the initial establishment of latency in Jurkat cells, increasing the ratio of productively-infected to latently-infected cells as compared to DMSO negative controls (p=0.0036). Conclusions: Elevated cAMP levels block HIV transcription and prevent viral reactivation in T cells and microglia, in an NFkB-dependent manner. cAMP induction also prevents initial establishment of HIV latency. cAMP modulators including forskolin should be explored in HIV cure strategies.

not predominantly associated with T follicular helper (Tfh) cell phenotypes during viremia (16.9% of HIV DNA+ cells; 13% of HIV RNA+ cells) or during ART treatment (8.1% of HIV DNA+ cells; 5.3% of HIV RNA+ cells). HIV DNA+ cells were instead found in two different memory populations: a non-activated and non-resident central memory T subset (34.7% and 15.6% of HIV+ DNA+ cells during viremia and ART, respectively) and a phenotypically resting but recently activated NR4A1/NR4A2 high subset (16.8% of HIV+ DNA+ cells; 18.1% of RNA+ cells) during ART. Conclusions: Our scDOGMAseq atlas of transcriptionally inactive and active HIV-1 infected CD4+ T cells from the lymphoid tissue highlights the predominance of non-Tfh cell subsets in the HIV tissue reservoir during ART. We find the HIV+ cells to be more concentrated in resting/recently activated cells that have not been previously described. These data altogether demonstrate that HIV cure strategies primarily targeting the B cell follicles during ART may miss out on a large portion of the HIV reservoir in lymphoid tissues. Lymph Node Immune Landscape Reveals the Role of Innate Immunity on HIV Reservoir in ART-PLWH Michail Orfanakis 1 , Spiros Georgakis 1 , Jolien Vermeire 2 , Susan P. Ribeiro 3 , Vincent C. Marconi 3 , Linos Vandekerckhove 2 , Khader Ghneim 3 , Ashish A. Sharma 3 , Rafick P. Sekaly 3 , Constantinos Petrovas 1 1 Lausanne University Hospital, Lausanne, Switzerland, 2 HIV Cure Research Center, Ghent University, Ghent, Belgium, 3 Emory University, Atlanta, GA, USA Background: The LN factors regulating HIV reservoir maintenance, particularly in antiretroviral treated people living with HIV (cART PLWH), are not defined. Presumably, the interplay between innate (e.g. monocytes, dendritic cells) and adaptive immune cells, particularly TFH cells, a main HIV reservoir component, is critical for this maintenance. We have applied complementary methodologies to delineate the LN landscaping and search for possible associations between in situ factors and HIV viral DNA content. Methods: LN tissue sections from viremic (n=12) and cART (n=9) PLWH were analyzed by multiplex immunofluorescence imaging (7- and 12-plex panels) and spatial transcriptomic (GeoMx) methodologies while LN-derived cells were investigated by multiparametric flow cytometry and scRNA-10X. The blood HIV DNA content was measured by the Rainbow platform that quantifies intact HIV DNA. Computational tools (Histocytometry, R and python algorithms) were used for data analysis. Results: Our imaging analysis revealed that i) contrary to viremic LNs, no association was found between TFH cell prevalence and HIV DNA in cART LNs, ii) the cell densities of follicular and extrafollicular total and GrzB+CD8T cells were negatively associated with HIV DNA, both in viremic and cART LNs, iii) follicular and extrafollicular prevalence of IL10+cells (similar contribution by CD163 hi CD68 lo and CD163 lo CD68 hi cells) were positively associated with HIV DNA and v) follicular and extrafollicular prevalence of IFNb+ cells (higher contribution by CD163 lo CD68 hi ) were negatively associated with HIV DNA, suggesting a negative role for HIV persistence. Flow cytometry analysis of single cell suspensions showed a similar phenotypic clustering profile (relative expression of cell subsets) of Tregs cells among the donors regardless of their HIV DNA. The in situ transcriptomic analysis showed a significant overexpression of genes associated with Bile Acid metabolism and induced signaling in cART vs viremic follicles/LNs. Conclusions: We provide a multilevel assessment of PLWH LN immune dynamics, particularly in follicular/germinal center areas. The data point to a network of soluble factors produced by innate immunity cells and Tregs (TGFb, IL10 and IFNb) (Figure 1) as potential mediators for viral reservoir regulation. Furthermore, Bile Acids that can trigger the production of TGFb and Interferons (shown by members of RID HIV) could represent a crucial triggering factor for the aforementioned network.

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Poster Abstracts

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Predominance of Non-T Follicular Helper Cell Subsets in the Lymph Node Viral Reservoir During ART Vincent H. Wu 1 , Jayme Nordin 1 , M. Betina Pampena 1 , Perla Mariana Del Río Estrada 2 , Fernanda Torres Ruiz 2 , Mauricio González Navarro 2 , Yara Andrea Luna Villalobos 2 , Santiago Avila Rios 2 , Gustavo Reyes-Teran 3 , Michael Betts 1 1 University of Pennsylvania, Philadelphia, PA, USA, 2 Instituto Nacional de Enfermedades Infecciosas, Mexico City, Mexico, 3 Secretaría de Salud, Mexico City, Mexico Background: HIV-1 cure strategies require a comprehensive understanding of cellular phenotype of HIV tissue reservoirs for effective and selective targeting. While recent data have demonstrated the highly heterogeneous nature of peripheral blood CD4+ T cells subsets comprising the HIV reservoir of antiretroviral therapy (ART) treated people with HIV (PWH), CD4+ T follicular helper (Tfh) cells are generally considered as the predominant cellular reservoir of virus within lymphoid tissue. However, there are limited data supporting this during ART. Here, we re-examined this notion in lymph nodes of viremic and ART-treated PWH using unbiased multiomic analysis. Methods: To define the cellular signature of infected cells in the lymphoid tissue compartment, we profiled memory CD4+ T-cells from lymph node samples of PWH during viremia (n = 9) and ART treatment (n = 10) using single cell DOGMAseq (scDOGMAseq) for the simultaneous assessment of epigenetics, transcriptomics, and surface antigen levels. Results: Using the presence of transposed HIV-1 DNA during the epigenetic profiling and/or HIV RNA during transcriptomic profiling, we found HIV-1 infected cells (n = 1388 during viremia; n = 398 during ART) and determined their cellular phenotype. HIV-1 infected cells were found to be distributed between many CD4+ T-cell subsets, including central, effector, regulatory and resident memory subsets. Importantly, the reservoir during ART or viremia was

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