CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

467

Longitudinal Phenotypic and Transcriptional Profiling of Immune Cells in Early-Treated Acute HIV Jozefien De Clercq, Sofie Rutsaert, Jolien Vermeire, Linos Vandekerckhove, Sarah Gerlo HIV Cure Research Center, Ghent University, Ghent, Belgium Background: Despite early antiretroviral treatment (ART) initiation, persistent immunological alterations have been described in individuals treated during acute HIV infection (AHI). We performed longitudinal phenotypic and transcriptional profiling of peripheral blood mononuclear cells (PBMCs) after AHI to better understand the impact of early ART on cellular immune responses. Methods: We conducted 26-color flow cytometry on matched PBMC samples (n=39) during AHI (T0, n=36), after a median of 0.76 years on suppressive ART (UD, n=36), and after a median of 1.99 years on ART (UD+1, n=26). Age matched HIV-uninfected controls (HCs, n=21) were also included. Cells were clustered using FlowSOM. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) was performed on a subset of participants at T0 (n=7), UD+1 (n=7), and HCs (n=7), using unsupervised weighted-nearest neighbor (WNN) clustering based on RNA and protein data to accurately define cell identities. Results: Flow cytometric analysis revealed 30 PBMC metaclusters of CD4 T cells, CD8 T cells, γδ T cells, MAIT cells, NK cells, B cells, and monocytes. During AHI (T0), there was a proportional expansion of highly activated T cells and plasmablasts and contraction of naïve cells. Despite early ART initiation, the CD8 T cell proportion remained elevated during viral suppression, compared to HCs, with persistent activation and relative enrichment of CD8 TEMRA cells. CITE-seq confirmed the expansion of CD8 T cells with an exhausted and effector memory profile. Additionally, the proportion of non-classical monocytes exhibited a decrease during AHI that did not normalize with ART. Principal component analysis demonstrated that terminal differentiation of T cells and residual immune activation defined individuals treated during AHI compared to HCs. Flow cytometry particularly showed profound alterations in γδ T cells, including the rapid relative accumulation of γδ TEMRA T cells and persistent upregulation of activation markers. Differential gene expression analysis of γδ T cells revealed upregulation of genes involved in ER stress-induced apoptosis, T cell activation/transmigration, and IFN-γ secretion in early treated individuals (UD+1) compared to HCs. Conclusions: Early ART initiation during acute HIV infection (AHI) does not fully prevent phenotypic and transcriptional changes in PBMCs. These immunological alterations might be linked to ongoing antiviral responses or persistent inflammation, highlighting the need for further research. The figure, table, or graphic for this abstract has been removed. Markers of Immunological Control for Future Identification of HIV Post-Treatment Controllers Raniero Chimienti 1 , Elena Bruzzesi 2 , Gabriel Siracusano 1 , Camilla Volpi 1 , Matilde Certo 1 , Riccardo Lolatto 1 , Francesca Sironi 1 , Lucia Garbarino 3 , Michela Mazzocco 3 , Angelo Roberto Raccagni 2 , Raffaele Dell'Acqua 1 , Stefania Dispinseri 1 , Daniela Cesana 1 , Silvia Nozza 2 , Mauro Malnati 1 1 IRCCS San Raffaele Scientific Institute, Milan, Italy, 2 San Raffaele Vita-Salute University, Milan, Italy, 3 Galliera Hospital, Genoa, Italy Background: People with HIV (PWH) who initiate antiretroviral therapy (ART) during primary HIV infection (PHI) achieve a more favorable immunological profile. Aim of the study is to find predictive biomarkers of immunological control by comparing HLA-KIR matching among PWH who started ART in PHI who normalized the absolute count of CD4 and CD8 T cell count and the CD4/ CD8 ratio within 8-12 weeks [Rapid Responders (RR)] vs between 12-48 weeks [Classical Responder (CR)] vs at >48 weeks or never [Immunological Non Responders (INR)]. Methods: Monocentric cross sectional cohort study enrolling PWH who started ART during PHI with HIV-1 plasma viremia < 1 copies/ml since ≥ 2 years, and with CD4 count >350 in care at IRCCS San Raffaele, Milan, Italy. The study is registered in CTIS (2024-515419-24-00). Kirotyping has been assessed using quantitative RT-PCR assays. HLA Class I typing has been performed by NGS sequencing (GenDx). Quantification of intact and defective provirus copies was assessed by intact proviral DNA assay. Non-parametric tests were used for assessing statistical significance.

Results: Among 92 PWH enrolled, 19 (21%) were classified as RR, 19 (21%) were CR and 54 (58%) as INR. At diagnosis, 91 were male with a median (IQR) age of 35.6 (28.4-44.1) years, starting ART in 18 (10-13) days in 2015 (2012-2017), with no difference among groups. Interestingly, 17/19 RR patients maintained the 3 immunological parameters within normal range for the extent of the follow-up (≥ 4years), whereas among INR only 15/54 recovered CD4/CD8 ratio after > 48 weeks, maintaining it for ≥ 96 weeks. A significant fraction of RR shared at least one of the protective combinations of HLA-C2 high expressed alleles and their cognate KIR 2DS receptors; on the other hand, this match was strongly reduced amongst INR patients (Figure A). No significant difference was found in intact and defective proviral load among the 3 groups (Figure B); of note, an inverse correlation was found between the load of intact DNA and the maintenance of the CD4/CD8 ratio (p<0.0001). Conclusions: We identified specific HLA-KIR match related to immunological control which may pave the way to find post-treatment controllers and candidates for analytical treatment interruption studies. The figure, table, or graphic for this abstract has been removed. Impaired Antigen Presentation and TGFβ Signaling Fosters Th17 Differentiation in PLWH Under ART Valentino D'Onofrio 1 , Loïc Schrooyen 2 , Gonzalo Salgado-Montes de Oca 3 , Linos Vandekerckhove 2 , Ashish A. Sharma 4 , Rafick P. Sekaly 4 1 Ghent University, Ghent, Belgium, 2 HIV Cure Research Center, Ghent University, Ghent, Belgium, 3 National Institute of Respiratory Diseases, Mexico City, Mexico, 4 Emory University, Atlanta, GA, USA Background: Innate Lymphoid Cells type 3 (ILC3), abundant in the gut, sense the microbiome and present antigens to naive T cells, promoting regulatory T cell (Treg) differentiation over Th17 cells, fostering immune tolerance. Disruption of this balance is linked to inflammatory bowel disease. While HIV alters the Treg/Th17 ratio in the gut, it’s unclear whether it results from the impairment of the ILC3-T cell interaction in the gut of people living with HIV (PLWH). Methods: Single-cell RNA sequencing was performed on gut tissues from 4 compartments (duodenum, ileum, colon, rectum) of PLWH under ART (n=7) and PWoH (n=2) collected at CIENI (Mexico). To balance the number of participants, we merged the dataset of biopsies from 6 PWoH previously generated by the RID-HIV initiative (HCRC, Belgium) and publicly available gut data (Gut Reference survey). Using the Seurat (v5.0) and related packages in R, cells were processed and corrected for batch effects across datasets. Downstream analyses consisted of differential gene expression, gene set enrichment analysis (GSEA), and cell-cell communication. Results: There was a reduction in Treg/Th17 ratio and ILC3 frequencies, including RORC expression, in PLWH, suggesting an alteration of the ILC3-T cell interplay that could contribute to the well-established imbalance of the Treg/ Th17 ratio and heightened inflammation observed in PLWH. TGFβ expression, critical for Treg differentiation, was lower in PLWH but was the most important signaling pathway upregulated in these cells (Figure). ILC3-Th17 interactions were more frequent than ILC3-Treg in PLWH. Cytokine signaling between ILC3 and Th17 was mainly mediated by TGFβ, targeting JUNB, IRF7 and NFKB2 in Th17. On the other hand, IFN-II signaling, which upregulates MHCII that presents bacterial antigens to naive T cells and promote their differentiation to Tregs, was most important in PWoH. TGFβ alone is insufficient for Treg differentiation, and decreased IFN-II signaling could impair MHCII expression in ILC3 leading to insufficient antigen presentation. Indeed, GSEA showed downregulation of key ILC3 effector functions in PLWH, including antigen processing cross presentation. We cannot rule out the importance of other antigen presenting cells. Conclusions: Despite increased TGFβ signaling, these data suggest an impaired antigen presentation machinery that can foster Th17 differentiation. This likely contributes to intestinal inflammation and incomplete immune reconstitution in the gut despite ART.

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Poster Abstracts

468

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