CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

cells from PTCs, in both the cytolytic CD56dimCD16+ and cytokine-producing CD56brightCD16- NK cell subsets. scATAC-seq analysis revealed increased accessibility of NF-kappa-B transcription factor family binding motifs in NK cells from PTCs, particularly within the CD56brightCD16- NK cell subset. Given the critical role of NF-kappa-B signaling in NK cell effector functions, these findings suggest that CD56brightCD16- NK cells from PTCs may be primed for heightened immune responses, including increased IFN-gamma production. Conclusions: Upregulation of genes within the NF-kappa-B signaling pathway among PTCs suggests that highly functional NK cells could play a critical role in mediating ART-free control of HIV. Further investigation of how NF-kappa-B influences NK cell function and recognition of HIV-infected cells may inform the development of therapeutic strategies aimed at enhancing immune-mediated control of HIV. HLA-E Prevents Natural Killer Cell Inhibition of HIV-1 Replication in Peripheral Blood CD4+ T Cells Amanda M. Dudek, Alex T. H. Cocker, Fabian P. Suchy, Katie Han, Elena J. Sasu, Peter Parham, Matthew H. Porteus Stanford University, Stanford, CA, USA Background: The HIV-1 Nef has been shown to alter MHC-1 presentation on infected T cells, and infection of cell lines suggest upregulation of non-canonical HLA-E may be advantageous for Natural Killer (NK) cell evasion. Conflicting clinical reports leave the role of NK cells in infection control unclear. True mechanistic understanding of HIV replication and control in primary T cells has been challenging due to technical limitations efficiently genetically modifying and infecting true primary cells ex vivo. Methods: We recently developed a strategy for efficient knock-out (~99%) and knock-in (~80% of alleles) in purified human peripheral blood CD4+ T cells, and supports in vitro infection challenge with both CCR5-tropic and CXCR4-tropic HIV-1. We therefore generated HLA-E knock-out primary T cells, challenged them with NL4-3, and quantified spreading infection with or without donor matched NK cells from 5 healthy human blood donors. Results: HLA-E KO caused loss of cell surface HLA-E on T cells from all donors, without any alteration of CD4, CXCR4, or CCR5 protein levels. HLA-E knock-out cells from all 5 donors showed increased control of virus replication during co-culture, suggesting that NL4-3 uses HLA-E to inhibit NK-mediated killing of infected cells. Control knock-out cells demonstrated a slight drop (average ~3-fold) in infection during T:NK co-culture, while HLA-E knock-out cells demonstrated a 28-fold average drop in supernatant p24 concentration, with one donor showing as much as an 82-fold reduction in p24 concentration. The level of virus control directly correlated with the ratio of HLA-E cognate inhibitory/activating receptors (NKG2A/NKG2C) present on the NK cell surface in all donors except one donor containing a naturally occurring homozygous deletion in NKG2C. To identify additional pathways that facilitate HLA-mediated evasion in the absence of NKG2C, we did single-cell RNA seq to identify NK cell genes upregulated during infection co-culture after NKG2C knock-out in NK cells. Conclusions: These data highlight that the ratio of NKG2A/NKG2C on individual patient cells may influence the level of HIV HLA-E mediated control, and redundant pathways exist for recognition of HIV infection in the absence of NKG2C. These data may explain discrepancies in previous studies on the role of NKG2C on viral load. Importantly, these data highlight a role for HLA-E in HIV-1 immune evasion, and highlight the potential of novel gene editing and infection models in true primary immune cells. Differential CD8T/NK Cell-Mediated HIV-1 Control After TIGIT or KLRG1 Blockade and ART in hBLT Mice Ildefonso Sánchez-Cerrillo 1 , Ilya Tsukalov 1 , María Agudo-Lera 2 , Olga Popova 1 , Patricia Fuentes 3 , Lucio Jesús Garcia Fraile Fraile 1 , Ignacio de los Santos 1 , María Lazaro-Díez 4 , Vladimir Vrbanac 5 , María Luisa Toribio 3 , Francisco Sánchez Madrid 1 , Julia G. Prado 4 , Meritxell Genescà 6 , María Jose Buzon 6 , Enrique Martin Gayo 2 1 Hospital Universitario de La Princesa, Madrid, Spain, 2 Universidad Autónoma de Madrid, Madrid, Spain, 3 Centro de Biología Molecular Severo Ochoa, Madrid, Spain, 4 IrsiCaixa Institute for AIDS Research, Badalona, Spain, 5 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 6 Hospital Universitari Vall d'Hebron, Barcelona, Spain Background: The combination of antiretroviral therapy (ART) and checkpoint receptor blockade may be an attractive strategy and to enhance immune responses, reduce persistent reservoirs and promote control of viral replication in people living with HIV-1 (PWH). Expression of inhibitory receptors TIGIT and

KLRG1 has been linked to dysfunctional natural killer (NK) and CD8+ T cells, limiting the capacity to eliminate HIV-infected CD4+ T cells from PWH in vitro . However, the potential benefits of specific targeting TIGIT and KLRG1 in HIV-1 infection have not been evaluated in vivo . Methods: Reconstituted humanized Bone, Marrow, Liver and Thymus (hBLT) mice (n=15) were intravenously infected with 5,000 TCID 50 HIV-1 JRCSF and n=5 mice remained uninfected. After 7 days, oral ART was administered to all infected animals for two weeks in combination with a weekly intraperitoneal injection of either human IgG1 isotypic control, aTIGIT or aKLRG1 mAbs (n=5 mice per group). Upon ART interruption (ATI), immunotherapy was maintained for two additional weeks. HIV-1 plasma viral load (pVL) was quantified at 3, 6, 10 and 13 days after ATI. NK and CD8+T cell subsets were analyzed longitudinally in blood and in the spleen by FACS. Results: HIV-1 pVL was more significantly reduced to undetectable levels after co-administration of ART with aTIGIT (83%; p<0.0001), aKLRG1 (67%; p=0.0147) than combination with Isotype Abs (50%). Further, mice treated with aTIGIT (median pVL 1.515x10 3 copies/mL) and aKLRG1 (median pVL 1.562x10 3 copies/mL ) mAbs showed control of viral rebound compared to mice receiving Isotypic control Ab (median pVL 7.18x10 3 copies/mL; p<0.0001 in both cases) at 6 days post-ATI. Moreover, histological detection of HIV-1 p24 in the spleen was significantly associated with rebound pVL (p=0.0076) at 10 days post-ATI and these infected cells were restricted to the white pulp in the aKLRG1 group (p=0.0286). Notably, aTIGIT treatment was associated with a increased splenic IFNg+TNFa+CD107a- CD8+ T cells (40% Isotype vs 80% aTIGIT, p<0.0001) and higher CD107a+ NKG2C+ CD57- NK cells associated with lower pVL (3% Isotype vs 17.10% aTIGIT; p=0.01) in these mice. In contrast, lower pVL in the aKLRG1 group was associated with increased Granzyme B+ CD8+ T cells (p=0.0083) and higher CD107a+ TNFa+ NK cells (p=0.04) in the spleen. Conclusions: Differential modulation of NK and T cell by ART and TIGIT or KLRG1 blockade may be relevant for future immunotherapies against HIV-1.

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Elucidating the Role of Natural Killer Cells in Antibody Breadth During HIV Infection Izumi de los Rios Kobara 1 , Uma Maheswari Mangalanathan 1 , Kishor Mandaliya 2 , Julie Overbaugh 3 , Catherine Blish 1 1 Stanford University, Stanford, CA, USA, 2 International Centre For Reproductive Health-Kenya, Mombasa, Kenya, 3 Fred Hutchinson Cancer Center, Seattle, WA, USA Background: Natural Killer (NK) cells are underappreciated regulators of the antibody response to infection. In mice, NK cells can kill T follicular helper cells which decreases somatic hypermutation and vaccine response. In the context of HIV, dysfunctional NK cells are associated with broadly neutralizing antibodies. Understanding the temporal immune factors that influence breadth is an important component of developing a successful HIV vaccine. Methods: For this project, we profiled subjects from a longitudinal cohort of HIV positive women in Mombasa, Kenya. This cohort was initiated in 1993 and HIV negative who enrolled were followed for seroconversion. Antiretroviral therapy was not available in Kenya at this time which gives us unique longitudinal samples of untreated HIV infection to understand factors that contribute to breadth development over the course of infection. We profiled 22 subjects with a range of neutralization breadth by scRNAseq and mass cytometry by time of flight (CyTOF). Results: In our cohort, gene modules related to NK cell cytotoxicity were enriched in broad neutralizers in early infection, but enriched in narrow

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CROI 2025 109