CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

344 CD38+CXCR3+ TFH CELLS SERVE AS ACTIVE HIV RESERVOIR IN THE TOTAL TFH CELL POPULATION Alessandra Noto 1 , Madeleine Suffiotti 1 , Francesco Procopio 1 , Khalid Ohmiti 1 , Jean-Marc Corpataux 1 , Matthias Cavassini 1 , Raphael Gottardo 2 , Craig Fenwick 1 , Giuseppe Pantaleo 1 1 Lausanne University Hospital, Lausanne, Switzerland, 2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA Background: T follicular helper cells (Tfhs) are a phenotypically heterogeneous cell population generally defined by the expression of CXCR5 and PD-1. Tfhs serve as a major reservoir for HIV transcription and production in both viremic and long-term ART treated subjects. In the presents study, we have dissected the phenotypic and functional heterogeneity of Tfhs and the role of the different Tfhs populations in serving as HIV reservoir and their relationship with HIV-specific B cell responses. Methods: Lymph nodes (LN) biopsies were obtained from 17 HIV uninfected, 27 viremic untreated and 23 aviremic ART treated subjects. Definition of B and T cell populations and cytokines was performed by mass cytometry using a panel of 40 metal-conjugated antibodies. To dissect the heterogeneity of Tfh cells, we performed self-organizing map (FlowSOM) and consensus clustering. Cell-associated HIV-RNA was assessed in Tfh cells sorted on the basis of CD38 and CXCR3 expression (CD38+CXCR3+, CD38+CXCR3-, CD38-CXCR3+ and CD38-CXCR3-). Results: Unsupervised clustering identified 20 different populations of Tfh cells within CXCR5 high PD-1 high Tfh cells. CD38+CXCR3+ Tfhs significantly increased in viremics (41.18%) as compared to ART treated (17.8%) and HIV uninfected (6.9%) subjects (p<0.0001). Frequencies of CD38+CXCR3+ Tfh cells positively correlated with the percentage of total germinal center B cells (r=0.58, p<0.0001) and GC gp140+ B cells (r = 0.65, p<0.0003). CD38+CXCR3+ Tfhs expressed higher levels of expression of BCL-6, ICOS, CD57, CD40L, CCR5, HLA-DR, T-bet as compared to the CD38+CXCR3-, CD38-CXCR3+ and the CD38-CXCR3- and of production of the Tfh signature cytokine IL-21 (averages of 33%) as compared to the other three Tfh cell populations (p<0.0002). Of note, only the percentage of CD38+CXCR3+ Tfhs positively correlated with viremia (r=0.5, p=0.01) in untreated subjects. More importantly, CD38+CXCR3+ Tfhs were greatly enriched in cell-associated HIV-RNA as compared to CD38+CXCR3- (average 3.3 fold), CD38-CXCR3+ (average 6.24 fold) and CD38-CXCR3- (average 8.24 fold) Tfh cell populations (p<0.05). Conclusion: CD38+CXCR3+ Tfhs correspond to a population of phenotypically and functionally active Tfh cells. The higher levels of expression of CCR5 may render these cells more susceptible to HIV infection and it explains why CD38+CXCR3+ Tfh cells serve as the major active HIV reservoir within the total Tfh cell population. 345 RESIDENT MEMORY T CELLS ARE A CELLULAR RESERVOIR FOR HIV IN THE CERVICAL MUCOSA Jon Cantero 1 , Judith Grau-Expósito 1 , Carla Serra Peinado 1 , Daniela A. Rosero 1 , Laura Luque-Ballesteros 1 , Josep Castellví 2 , Tamara Sanhueza 3 , Julia G Prado 4 , Antoni Tarrats 3 , Carla Lecumberri 3 , Laura Mañalich-Barrachina 2 , Cristina Centeno-Mediavilla 2 , Vicenç Falcó 2 , María J. Buzón 1 , Meritxell Genescà 1 1 Vall d’Hebron Research Institute, Barcelona, Spain, 2 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain, 3 Hospital Germans Trias i Pujol, Barcelona, Spain, 4 IrsiCaixa Institute for AIDS Research, Badalona, Spain Background: Viral reservoirs, which represent the main obstacle to cure HIV, are early established in different tissues. Target cells in peripheral tissues where HIV is acquired, such as the female genital mucosa, may express CD69 as a hallmark of their resident memory T cell (TRM) phenotype. Some of the features of TRM reported for other tissues, including long-lived and self- renewal capacities, shape them as an ideal cellular reservoir for HIV. However, the contribution of this relatively novel subset of cells to the pathogenesis and persistence of HIV remains unknown. Methods: TRMwere phenotyped in fresh cervical tissues obtained from HIV-uninfected women undergoing hysterectomy for non-neoplastic reasons (n=6-9). Activation of CD103+/-CD4+TRM subsets were compared between healthy and antiretroviral therapy (ART)-suppressed HIV+ women (n=6). The cervical explant model of HIV infection was established to determine proviral DNA (vDNA) content by qPCR and productive infection by p24 antigen expression in TRM subsets (n=7). In addition, we determined vDNA in purified cell subsets derived from blood and cervix obtained from ART-suppressed HIV+ woman (n=6). Finally, we also assessed viral HIV-RNA in cervical tissues from

(p=1.3e-6) whereas in another those in the EM subset were the youngest (p=0.01). Conclusion: The latent HIV proviral landscape differs markedly between individuals, and sometimes between different CD4+ T-cell subsets within the same individual: eradication strategies may need to take this into consideration. Inference of proviral sequence ages in different HIV T-cell subsets can yield insight into latent HIV dynamics and persistence. 343 B CELL-T CELL DOUBLETS IN GALT ARE ENRICHED FOR TFH CELLS BUT NOT FOR HIV DNA John P. Thornhill 1 , Matthew Pace 2 , Genevieve E. Martin 2 , Carolina Herrera 1 , Jonathan Hoare 1 , Simon Peake 1 , Helen Brown 2 , Nneka Nwokolo 3 , Julie Fox 4 , Sarah Fidler 1 , John Frater 2 , for the the CHERUB Investigators’ 1 Imperial College London, London, UK, 2 University of Oxford, Oxford, UK, 3 Chelsea and Westminster Hospital, London, UK, 4 Guy’s and St Thomas’ NHS Foundation Trust, London, UK Background: Gut-associated lymphoid tissue (GALT) is a key HIV reservoir site and may play a role in HIV persistence on ART. T Follicular helper (TFH) cells and CD32+CD4+ T cells have been proposed to be enriched for HIV DNA. Here, we show that CD32+CD4+ T cells in GALT are B cell-T cell (B:T) doublets and that sCD40 (a soluble marker shed after B:T cell interaction through CD40/CD154 signaling) but not CD32 is associated with HIV DNA in GALT. Methods: GALT from the terminal ileum (TI), rectum (R) & tonsil tissue (n=1) was obtained from consenting individuals treated during primary HIV infection (PHI). HIV DNA was quantified in GALT biopsies by qPCR. Concurrent plasma samples were used to measure IL-4, IL-5, IL-6, IL10, IL-15, MCP-1, MIP-1α, MIP-1β, IP-10, sCD163, CD40 & CD40L by Luminex (n=23). CD32 expression on GALT CD4 T cells was measured by flow cytometry (n=19) and imaging cytometry assessed CD19, CD3, CD4, ICOS, HLA-DR & CD32 expression in healthy control GALT and HIV+ tonsil. Associations between HIV DNA & CD32 were tested by Spearman’s correlation. LASSO regression analyses were used to test for associations between GALT HIV DNA & plasma variables. Results: 23 PHI individuals were studied; median (IQR) HIV DNA was significantly higher in TI compared to R [2.82 (2.58-3.05) vs 2.73 (2.42-2.96) log 10 CPM gut T cells, p=0.03]. CD32 expression on GALT CD4 T cells was not associated with HIV DNA. Imaging cytometry analysis showed that CD32 expression on CD4 T cells in GALT (n=1) & HIV+ tonsil (n=1) was consistent with B:T cell doublets with CD32 expression primarily from B cells, while associated CD4+ T cells expressed ICOS. Plasma (n=23) sCD40 (TI: r=0.36 P=0.04, R: r=0.34 P=0.05) and sCD14 (TI: r=0.39 P=0.04, R: r=0.44 P=0.01) were the variables most strongly association with HIV DNA. Conclusion: These data show that CD32 expression on CD4 T cells in GALT and tonsil when gated as singlets using standard methodology is due to B cell-TFH cell doublets, with CD32 expression primarily on B cells. The enrichment for TFH cells within these doublets raises the issue of whether they are artefactual or physiological. Plasma sCD40, a marker of the B:T cell interaction, & sCD14, a marker of bacterial translocation, were the factors most associated with HIV DNA, while CD32 expression was not. This suggests that the B:T cell interaction & microbial translocation in GALT may be supporting HIV persistence while CD32 is a surrogate marker of this interaction.

Poster Abstracts

CROI 2019 125

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