CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

years-old for <1y, 1-10y, 10-20y, and >20y cohorts, respectively. Time on ART was <1, 8(IQR 6-9), 15(IQR 13-19), and 28(IQR 25-31) years. Median nadir, CD4 and CD8 count were 219(IQR 153.8-379.8), 822(IQR 539-976), and 816(IQR 611 1227), respectively, for all cohorts. Median CD4/CD8 was 0.52(IQR 0.38-0.78), 0.76(IQR 0.53-1.20), 0.98(IQR 0.77-1.38), and 0.86(IQR 0.61-1.23), respectively. 2) CD4+ naive (TN) cells decreased with longer ART duration, while TCM and TEM increased. There were no differences in TEMRA between groups. 3) CD4 from <1y cohort showed significantly higher levels of LAG-3, PD1, and TIGIT than 10-20y and >20y cohorts, as well as CD57 and CD32, while KLRG1 was significantly higher in >20y. 4) HIV-1 reservoir was higher in <1y than cohorts with longer time on ART. 5) Proviral reactivation in CD4 (p24-gag+) was lower as time on ART progressed: 1.9-fold(p=0.0287) in 1-10y, 2.3-fold(p=0.0051), and 2.4-fold(p=0.0039), compared to <1y (Figure 1A). 6) Cytotoxic response against infected TZM cells was 2.4-fold (p=0.0417) and 2.4-fold (p=0.0396) higher in >20y compared to the <1y and 10-20y cohort (Figure 1B), which could be related to enhanced NKT subpopulations. Conclusions: PWH on long-term ART showed reduced capacity for proviral reactivation and lower expression of exhaustion markers in CD4+ T cells, with improved cytotoxic response against HIV-1-infected cells. Time on ART should be considered for HIV cure interventions to achieve higher efficacy.

lowest NF-kB activity. Interestingly, distinct patterns of plasma IL-1b, TNF-a and GCSF were strongly associated with cluster membership. Conclusions: Collectively, these results suggest that specific patterns of cytokine secretion and NF-kB signaling are major drivers of CD4 T cell transcriptomic diversity in PWH on ART. These findings demonstrate the power of dimensionality reduction and data visualization tools to define subgroups of PWH with distinct clinical or immunological characteristics which may serve to risk stratify PWH on ART for future HIV cure strategies. TLRS Ligands and IL-15 Induce Trained Immunity in CD4 Cells and Limit HIV Infection and Persistence Muhammad Bilal Latif, Enoch S. Muyanja, Mojahidul Islam, Jeffrey A. Tomalka, Hilmi Al-Shakhshir, George N. Palakis, Susan P. Ribeiro, Ashish A. Sharma, Rafick P. Sekaly, for the Pathology Advanced Translational Research Unit (PATRU) Emory University, Atlanta, GA, USA Background: Despite effective HIV suppression by ART, the virus persists in latent reservoirs. Our single-cell data from human and primate studies show that individuals with better HIV control and smaller reservoirs have higher interferon stimulated gene (ISG) expression in CD4+ T cells, linked to greater chromatin accessibility and transcriptional activation. ISG expression could be induced by Toll-like receptors (TLRs) and IL-15. Thus, we hypothesize that; the activation of intrinsic innate antiviral immunity (IIAVI) in memory CD4+ T cells upregulates IRFs, enhancing ISG transcription. This "trained immunity" state will confer a poised antiviral response and limit reactivation of latent HIV upon exposure. Methods: We established two co-culture systems: one using negative selection with immune cell markers and the other labeling cells with Cell Trace Violet (CTV). We evaluated HIV infection in these systems with Poly I:C (TLR3 agonist), R848 (TLR7/8 agonist), and IL-15 stimulation. Mechanisms of PRR and IL-15-mediated HIV protection were explored using high-dimensional flow cytometry. Results: Activation of TLR3, TLR7/8, and the IL-15 pathway in the presence of macrophages, dendritic cells, and NK cells (first co-culture system) efficiently triggered intrinsic pathways, leading to both autocrine and paracrine IFN responses, conferring protection against HIV infection in memory CD4+ T cells. In the second system, memory CD4+ T cells induced and transferred IFN mediated protection to neighboring bystander CD4+ T cells. We investigated the mechanisms of TLR3, TLR7/8, and IL-15-induced refractoriness, focusing on effector molecules of innate antiviral immunity i.e. pIRF3, pIRF7, IFNα, IFNγ, pSTAT1, and ISGs (e.g., IFIT1, APOBEC3G) using an optimized kinetic timeline. Results show TLR3 and TLR7/8 activation stimulates IRF3 and IRF7, inducing IFNs and ISGs, including HIV restriction factors in memory CD4+ T cells (Fig. 1A), promoting a poised innate antiviral immunity state or “trained immunity,” that contributes to HIV refractoriness (Fig. 1B). Similarly, IL-15 activation leads to IFN production, ISG induction, and HIV restriction (Fig. 1). Conclusions: Our study shows that TLRs and IL-15-induced IFN responses, along with ISG expression in memory CD4+ T cells, create an antiviral innate memory state that protects against HIV infection and persistence upon re-exposure. Phenotypic and Functional Heterogeneity Contribute to the Persistence of the Clonal CD4 Reservoir Isabella A. T. M. Ferreira 1 , Alberto Herrera 1 , Tan Thinh Huynh 1 , Emily Stone 1 , Noemi L. Linden 1 , Virender K. Pal 2 , Cintia Bittar 2 , Marie Canis 2 , Marina Caskey 2 , Paul Bieniasz 2 , Michel Nussenzweig 2 , R. Brad Jones 1 1 Weill Cornell Medicine, New York, NY, USA, 2 The Rockefeller University, New York, NY, USA Background: The HIV reservoir persists within clonally expanded CD4+ T cells carrying intact proviruses. We describe the isolation and long-term in vitro expansion of these ‘authentic reservoir clones’ (ARCs) and insights into functional characteristics that may underlie their persistence. Methods: Authentic reservoir clones (ARCs) were derived from CD4+ memory T cells (n=4) using multiple rounds of limiting dilutions with irradiated feeder cells, anti-CD3, anti-CD28 stimulations, ARVs, and bNAbs. p24 ELISA was used to screen for wells containing clones. Proviruses were characterized through integration site analysis, intact proviral DNA assay (IPDA), and near full-length sequencing with clonality also verified by TCR sequencing. Flow cytometry based functional assays were performed, assessing proliferation (n=2), latency reversal (n=2), and susceptibility to CTL-mediated killing (n=3). Transcriptomic The figure, table, or graphic for this abstract has been removed.

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Plasma Cytokines and NF-κB Signaling Drive Transcriptomic Diversity in CD4+ T Cells From PWH on ART Yingfan Wang 1 , German Gornalusse 2 , David Siegel 2 , Alton Barbehenn 2 , Rebecca Hoh 2 , Jeffrey Martin 2 , Frederick Hecht 2 , Christopher Pilcher 2 , David Murdoch 1 , David M. Margolis 3 , Cynthia Rudin 1 , Florian Hladik 4 , Steven G. Deeks 2 , Sulggi Lee 2 , Edward Browne 3 1 Duke University, Durham, NC, USA, 2 University of California San Francisco, San Francisco, CA, USA, 3 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 4 University of Washington, Seattle, WA, USA Background: Antiretroviral therapy (ART) is not a cure, and HIV rapidly rebounds upon ART interruption. Furthermore, people with HIV (PWH) on ART have elevated immune activation and inflammation which has been associated with increased risk of aging-associated diseases which may be driven by this viral persistence. Methods: To identify the potential mechanisms by which HIV persistence may drive ongoing inflammation and immune activation, we leveraged published data from 154 PWH treated during early (<6 months) and later (>2+ years) HIV infection from the UCSF SCOPE and Options cohorts. These data included host gene and HIV reservoir measures (HIV total DNA, HIV unspliced RNA) from peripheral CD4+ T cells, as well as plasma cytokine levels. Results: Using a novel dimensionality reduction tool (PaCMAP) that preserves both local and global data structure, we identified three distinct CD4+ T cell transcriptomic clusters. By analyzing differentially expressed genes between the clusters, we found that clustering was significantly associated with differential expression of genes that encode for members of the transcription factor NF-kB family (NF-ĸB1, NF-ĸB2, RelA, RelB and cREL), NF-kB inhibitor genes and downstream effector genes such as pro-inflammatory cytokines ( IL-1A , IL-1B ) or antiviral pathways ( IFNG ). Clustering was not associated with HIV reservoir size or HIV unspliced RNA but was associated with the timing of ART: early treated PWH (<6 months) were enriched in the cluster with the

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