CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Background: Successful pregnancy is reliant on the acceptance of a semi- allogeneic fetus, meaning the systemic regulation of the immune system to maintain tolerance is important. In complicated pregnancies changes in both frequency and activation of peripheral leukocytes have been found. HIV-1 positive women have increased incidence of preterm labour suggesting HIV-1 infection disrupts immunological interactions relevant to the regulation of immunological balance in pregnancy, though this has not been explored in depth. We aimed to identify central leukocyte populations in HIV-1 positive and negative pregnancy immune networks that were shared or discordant which may impact on systemic immune regulation. Methods: Freshly isolated peripheral blood mononuclear cells from uncomplicated ART treated HIV-1 positive pregnant (PP; n=21) and HIV-1 negative pregnant (NP; n=36) women were analysed using flow cytometry and ELISpot assays. Natural killer (NK) cells, monocytes (Mo), dendritic cells (DC), and both classical and non-classical T-cell subsets were identified, while IFNg, IL-2, IL-10 and granzyme B functional responses against Influenza, Epstein-Barr and Cytomegalovirus were quantified. Cytometer acquisition was optimised for longitudinal comparison between samples. Non-parametric correlation networks of the resulting 500+ parameter group datasets were generated and analysed to determine network centrality measures and compare group networks using R packages. The top 50 Strength (number of significant associations) and Betweenness (times passed through in shortest paths between all other interacting parameters) centrality measures were compared. Results: Mo PD-L1 expression was identified as highly central by both measures in both groups, suggesting this pathway of interaction shapes the pregnant immune system. CD40-L expression on CD4 and CD8 T-cell subsets and PD-L1 on CD8 T cells had high Strength scores in both groups, while NKG2A and CD11b expression on NK cells as well as CD56bright NK subset frequency had high Betweenness scores in NP and PP women. However, the PP group had more high Strength scoring T-cell parameters, predominantly CD38 expressing T cells, suggesting these activated T cells are more influential in the HIV-1 positive pregnancy network. Conclusion: Our work highlights shared immune components that may be key regulators of pregnancy tolerance and has identified parameters uniquely impacted by HIV-1 that may negatively influence the pregnancy immune network. 200 CXCR5+ NK CELLS IN THE LYMPH NODE ARE ASSOCIATED WITH CONTROL OF SHIV INFECTION Sheikh A. Rahman 1 , James M. Billingsley 2 , Chris C. Ibegbu 2 , Sadia J. Rahman 1 , R. Paul Johnson 1 , Steven E. Bosinger 2 , Rama R. Amara 2 , Vijayakumar Velu 2 1 Yerkes National Primate Research Center, Atlanta, GA, USA, 2 Emory University, Atlanta, GA, USA Background: Natural killer cells (NKs) play an essential role in antiviral immunity; however, their function in Lymph nodes (LN) during chronic HIV/SIV infection is not fully elucidated. LN follicles constitute major reservoir sites for HIV/SIV persistence. Cure strategies could benefit from the characterization of CXCR5+ NK cells able to access/eliminate HIV-reservoirs. Methods: Here we studied the phenotype, distribution and function of CXCR5+ NK cells in the LN of SHIV naïve and chronic SHIV1157ipd3N4-infected (>14 weeks PI) rhesus macaques (RM) and their association with plasma viral RNA levels. Flow cytometry was used for phenotypic analysis, function (IFN-g, TNF-a, CD107a) was assessed by intracellular staining and in vitro target cell killing experiments. Immunohistochemistry was performed to identify the location of NK cells in B cell follicles. Results: We found that prior to infection, a significant proportion of NK cells (~15%) expressed CXCR5. Following infection, the frequency of CXCR5+ NK cells was significantly higher in chronic SHIV-infected RM. Phenotypically CXCR5+ NK cells express higher levels of FCgRIIa and FCgRIIIa compared to CXCR5- NK cells, which might be important for ADCC function. The CXCR5+ NK cells demonstrated enhanced polyfunctionality with higher production of IFN-g, TNF-a and CD107a when stimulated with mitogen. Immunohistochemistry analysis confirmed the presence of NK cells in LN follicles. Transcriptional profiling (RNA-seq) of sorted CXCR5+ and CXCR5- NK cells from SHIV-infected RM revealed that CXCR5+ NK cells are activated and express increased levels of cytolytic markers (perforin, granzyme-B, granulysin and CD107a), suggesting that these cells have a higher capacity to kill. Gene set enrichment analysis of CXCR5+ cells additionally showed elevated transcripts associated with cell activation, TNF-alpha, interferon signaling and apoptosis. Importantly, the frequency of CXCR5+ NK cells

correlated inversely with plasma SHIV viral RNA levels and exhibited a significant negative association with germinal center Tfh cells Conclusion: Chronic SHIV infection is characterized by accumulation of NK cells within LN follicles and suggest that CXCR5+ NK cells could play an important role in controlling SHIV infection. Cure strategies should focus on inducing these cells for sustained HIV remission 201LB THE ROLE OF CD101 IN HIV/SIV PATHOGENESIS AND MAINTENANCE OF THE VIRAL RESERVOIR Timothy Hoang 1 , Zachary Strongin 1 , Gregory K. Tharp 1 , Justin L. Harper 1 , Zhan Zhang 1 , Steven E. Bosinger 1 , Deanna Kulpa 1 , Mirko Paiardini 1 1 Emory University, Atlanta, GA, USA Background: HIV infection results in depletion of CD4+ T cells, induction of systemic inflammation, and exhaustion of antiviral responses, all features that are not normalized during ART and implicated in promotion of viral persistence. CD101 is a surface glycoprotein that has been linked to highly suppressive TRegs and defining “terminally exhausted” CD8+ T cells during chronic infection. Here, we sought to understand implications of CD101 expression on CD4+ T cells during SIV infection and mechanisms leading to SIV persistence. Methods: 28 rhesus macaques (RMs) were infected with SIVMac239 and started ART 42 d.p.i. Samples were collected longitudinally for flow cytometric and RNAseq analysis. Latency and Reversion Assay (LARA) was used for latency induction and integrated HIV was measured by qPCR. Results: At 14 d.p.i. CD101+ CD4+ T cells were preferentially depleted, as compared to other CD4+memory subsets (p<0.0001). CD101+ CD4+ T cells remained significantly lower than CD101- CD4+ T cells in blood and tissues up to 42 d.p.i. (p<0.001). Reconstitution of CD101+ CD4+ T cells was delayed compared to CD101- CD4+ T cells after ART. In SIV+ RMs on ART for >1 year, PD-1 and CTLA-4 were upregulated in CD101+ as compared to CD101- CD4+ T cells (p=0.0156 and p=0.0078, respectively). We also detected higher levels of cell cycling (p=0.0078) in the CD101+ CD4+ T cells, suggesting that these cells may persist through homeostatic proliferation and replenish the reservoir via clonal expansion. RNAseq showed that CD101+ CD4+ T cells were transcriptionally distinct and in a more terminally differentiated state, aligning with reports stating that CD101+PD-1+CD8+ T cells were “terminally differentiated”. Using LARA, we detected similar levels of integrated HIV-DNA in CD101+ and CD101- CD4+ T cells. Interestingly, and consistent with their higher co-expression of PD-1/CTLA-4 and transcriptomic profile, p24 gag expression within CD101+ CD4+ T cells was significantly lower at 7 d.p.i., suggesting that HIV-infected CD101+ CD4+ T cells progress to a latent state more readily. Conclusion: Altogether, these data identify CD101+ CD4+ T cells as a cell subset that (i) is preferentially depleted during early SIV infection, (ii) leads to the establishment of immune exhaustion, and (iii) preferentially enter latency. As such, CD101+ CD4+ T cells could be vital contributors to the HIV reservoir and targets of future therapeutic approaches. 202 IMMUNE CONTROL OF LIVE ATTENUATED-HIV INFECTION AND DISEASE IN BLTS-HUMANIZED MICE Shivkumar Biradar 1 , Moses Bility 1 , Robbie B. Mailliard 1 , for the Bility Lab Group 1 University of Pittsburgh, Pittsburgh, PA, USA Background: We recently demonstrated the robust development of human lymphoid and myeloid cells in immunodeficient mice, achieved through co-transplantation of bone marrow-derived human hematopoietic stem cells (hHSCs), liver, thymus, and spleen (BLTS). Importantly, unlike other earlier mouse models, BLTS-humanized (hBLTS) mice exhibit lymphoid tissue with proper development of B cell follicles, a major site of the latent HIV reservoir. Therefore, we hypothesize that hBLTS mice with complete human immune cell repertoire and lymphoid tissue architecture will provide an improved model for studying HIV immunity. Methods: To generate hBLTS mice, NSG mice were engrafted with autologous hHSCs via intravascular injection, and with human hematopoietic lymphoid tissues (fetal thymus, liver and spleen) via kidney capsule transplant. Reconstitution and characterization of human immune cells was determined by flow cytometry. Wild type and Nef-deleted HIV strains were used to infect the hBLTS mice. Blood samples were analyzed by flow cytometry and qRT-PCR to measure impact on the human immune cell populations and HIV viral load, respectively. Lymphoid tissue pathology was examined via immunohistochemistry.

Poster Abstracts

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CROI 2020