CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

402

The Soft Escape: HIV Alters Host Cell Cytoskeleton as a Mechanism of Resistance to CTL Killing Louise Leyre 1 , Farah Mustapha 2 , Esther Lee 1 , Alberto Herrera 1 , Paul Zumbo 1 , Doron Betel 1 , Michael Galiano 2 , Nada Wahman 2 , Morgan Huse 2 , R. Brad Jones 1 1 Weill Cornell Medicine, New York, NY, USA, 2 Memorial Sloan Kettering Cancer Center, New York, NY, USA Background: HIV latency enables viral persistence during ART, but CTL pressure continues to shape the HIV reservoir. CD4+ T-cells exhibit varying susceptibilities to CTL attack, potentially leading to the selection of CTL resistant HIV reservoir-harboring cells. Effective CTL-mediated killing requires the formation of an immune synapse through biophysical forces, with CTLs being more efficient at eliminating stiffer targets. HIV-Nef manipulates the host cell cytoskeleton, potentially influencing cell stiffness. Our research focuses on how cell-intrinsic heterogeneity in HIV-infected CD4+ T-cell stiffness affects susceptibility to CTLs, and the role of Nef in this process. Methods: Central memory CD4+ T cells (Tcm) were infected with WT-JRCSF, DNef-JRCSF or TW10esc-JRCSF (CTL escape mutant) and exposed to HIV-Gag TW10-specific CTL clones. Surviving HIV-Env+ cells were sorted and analyzed for membrane tension and stiffness using optical tweezers (OT) and atomic force microscopy (AFM). Flow cytometry assessed killing efficiency, while confocal microscopy imaged actin. Two additional mutants, NefDPAK2-JRCSF and NefDMHCI-JRCSF, were studied to differentiate Nef's effects on cytoskeleton versus MHC-I downregulation. Results: Without CTL, WT, DNef, and TW10esc-infected Tcm showed similar levels of membrane tension, but WT-infected cells exhibited significantly lower stiffness and F-actin levels compared to DNef. After CTL coculture, WT 'survivors' displayed lower membrane tension (83pN vs 129pN and 132pN; n=75, 2-donors) and stiffness (767Pa vs 1047Pa and 1233Pa; n=92, 2-donors) compared to TW10esc 'bystanders' and DNef survivors. NefDPAK2 (cytoskeleton interaction mutant) and NefDMHCI (MHC-I interaction mutant) -infected cells, were both much more susceptible to killing than WT. NefDMHCI survivors resembled WT survivors in low membrane tension (80pN vs 83pN; n=52, 2-donors) and stiffness (867Pa vs 767Pa; n=77, 2-donors), while NefDPAK2 survivors resembled Nef survivors (1154Pa vs 1233Pa; n=82, 2-donors), showing no evident selection. Conclusions: Our findings reveal a collaboration between host-cell biophysical heterogeneity and HIV-Nef in promoting immunevasion. Nef reduces infected CD4+ T-cell stiffness by altering the cytoskeleton, allowing the softest cells to evade CTL-mediated killing. Therapeutics targeting cell stiffness, either by inhibiting Nef's cytoskeletal effects or modulating host factors, may enhance elimination of CTL-resistant infected cells. CD8+ T-Cell Function During ART Is Associated With Provirus Suppression and Reduced Viral Rebound David R. Collins 1 , Mpho Olatotse 1 , Tyler Lilie 2 , Xianbao He 3 , Emanuele Mazzola 4 , Manish Sagar 3 , Bruce Walker 1 , Athe Tsibris 2 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Brigham and Women's Hospital, Boston, MA, USA, 3 Boston University, Boston, MA, USA, 4 Dana–Farber Cancer Institute, Boston, MA, USA Background: Antiretroviral therapy (ART) typically fails to restore the ability of HIV-specific memory CD8 + T cells to mount secondary cytotoxic effector responses upon antigen re-encounter, which may contribute to the poor control of rebound viremia observed in most individuals upon analytical treatment interruption (ATI). Dysfunctional HIV-specific CD8 + T cells occasionally become functionally rejuvenated after prolonged ART, but their impact on viral persistence remains unclear. We hypothesized that targeting of autologous provirus by highly functional CD8 + T cells may contribute to containment of HIV reservoirs during ART and control of rebound viremia during ATI. Methods: We functionally mapped HIV-1 epitope-specific CD8 + T cell responses in 60 participants treated with ART for a median of 20 years using paired immunospot and proliferation assays. HIV-specific CD8 + T cell frequency, phenotype, recognition of autologous proviral epitopes, and ability to generate secondary cytotoxic effectors were assessed using flow cytometric assays. Total and intact HIV-1 DNA and cell-associated RNA (caRNA) were quantified. ATI with weekly monitoring of viremia and biweekly monitoring of HIV-specific CD8 + T cell responses was performed in one participant. The figure, table, or graphic for this abstract has been removed.

Results: In most participants, HIV-specific CD8 + T cells were poorly responsive to antigenic stimulation; however, 11 epitope-specific responses (5%) from 10 participants (17%) exhibited exceptional cytotoxic recall against autologous proviral epitopes. During ART, these participants harbored 2.5-fold lower median HIV DNA ( p =0.05) and 8-fold lower median caRNA ( p =0.03) than those with poor functionality. In the sole ATI participant, viremia rebounded after 4 weeks followed by a rapid decline in viral load before ART re-initiation. This decline was concurrent with recall expansion of immunodominant HLA B*44-restricted Gag-specific T cells to more than 11% of total CD8 + T cells, a 59-fold increase in the proliferation marker Ki-67, and a 21-fold increase in co-expression of the cytotoxic effector proteins perforin and granzyme B among epitope-specific cells. Conclusions: Highly functional, non-escaped HIV-specific memory CD8 + T cells during prolonged ART are infrequently observed but may contribute to suppression of persistent HIV reservoirs and of recrudescent viremia. Strategies to elicit cellular immune memory capable of robust cytotoxic recall against autologous proviral epitopes may hold promise toward achieving durable ART-free HIV remission. TCR Convergence in HIV Controllers: Insights Into Natural Viral Suppression Alexandra Vujkovic 1 , Philipp A. Adams 2 , Kadrie Ramadan 1 , Nicky de Vrij 1 , Vincent Van Deuren 3 , Alexander Pasternak 4 , Maartje Vanfrankenhuijsen 1 , Wim Adriaensen 1 , Benson Ogunjimi 3 , Guido Vanham 3 , Kris Laukens 3 , Pieter Meysman 3 , Koen Vercauteren 1 , for the PhenoCure Study Group 1 Institute of Tropical Medicine, Antwerp, Belgium, 2 Amsterdam University Medical Centers, Amsterdam, Netherlands, 3 University of Antwerp, Antwerp, Belgium, 4 University of Amsterdam, Amsterdam, Netherlands Background: HIV controllers are rare individuals who suppress viral replication without antiretroviral therapy (ART). We hypothesized that this might partly be driven by T-cell receptor (TCR) convergence, a phenomenon where different DNA sequences encode identical amino acids. TCR convergence may indicate antigen-driven selection, reflecting a more focused immune response. This study applies a novel method to calculate TCR convergence and its potential role in HIV control. Methods: TCR RNA sequencing was performed on four subsets of CD8+ T-cells: naive, central memory (CM), effector memory (EM), and effector memory cells re-expressing CD45RA (TEMRA). Sample selection included 32 HIV-infected individuals (22 HIV controllers; 10 ART-treated) and 20 HIV-negative healthy donors (HDs). Sequencing reads from ~50,000 cells/subset were annotated using MiXCR. TCR diversity was expressed as Shannon diversity. TCR convergence scores were calculated by comparing TCR repertoires of HIV positive and HIV negative donors using an in-house method. Viral specificity was assessed using Immunewatch Detect (IMWD) and TCRs derived from a Gag activation assay in HIV controllers (n=14/22) and ART-treated individuals (n=7/10). Results: HIV controllers, just as HDs, had a higher percentage of naive CD8+ T-cells compared to ART-treated individuals (p<0.01). TCR diversity was highest in naive subsets and declined through CM, EM, and TEMRA subsets across all groups. HDs showed greater TCR diversity in the TEMRA subset compared to ART-treated individuals (p<0.01), with no significant differences between HDs and controllers. Controllers displayed HIV-specific TCRs with high convergence scores, mainly in the EM subset. Clusters of Gag-associated TCRs were present in the convergent memory repertoires of controllers at a higher rate than in ART-treated individuals or HDs (p<0.001). About 75% of highly convergent HIV specific TCRs in controllers were linked to the HIV Gag KRWIIMFLNK epitope, that has previously been associated with HIV control, validating the performance/ accuracy of our method to identify control-associated TCRs. Conclusions: HIV controllers exhibit distinct TCR specificity, primarily in the EM subset. Our TCR convergence-based method identified control-associated TCRs. The heightened convergent TCR specificity toward control-associated HIV epitopes suggests a more efficient, antigen-driven, and targeted immune response. This novel approach produced valuable insights into immune mechanisms underlying HIV control.

404

Poster Abstracts

403

94

CROI 2025