CROI 2020 Abstract eBook
251 OPPOSING ASSOCIATIONS OF NK AND MZ B CELLS IN RECTAL EXPLANT MODEL OF HIV INFECTION S. Abigail Smith 1 , Praveen Kumar Amancha 1 , Phillip M. Murray 1 , Cassie Grimsley-Ackerley 1 , Rama R. Amara 2 , Colleen F. Kelley 1 1 Emory University, Atlanta, GA, USA, 2 Yerkes National Primate Research Center, Atlanta, GA, USA Background: Our understanding of innate immune cells in human rectal mucosal tissues (RM) and their contributions to promoting or restricting HIV transmission is limited. Studies focused on systemic responses or utilizing PBMC are not suitable proxies for mucosal responses. Here, we utilized the rectal explant model to elucidate associations between RM innate cell subsets and HIV-1 BaL replication ex vivo. Methods: Plasmacytoid dendritic cells (pDCs); CD1c+myeloid DCs; neutrophils; macrophages; natural killer cells (NK); Marginal Zone-like B cells (MZBs); gd T cells; and mucosal-associated invariant T cells were quantified in RM from 69 HIV-negative men aged 18-65 years by flow cytometry. Associations between these cell subsets and HIV replication (p24 production over days 3-18) in ex vivo RM explant challenge experiments from the same study participants were examined. Hierarchical Stochastic Neighbor Embedding (HSNE) analysis was used to compare MZB and NK from blood and RM. From the explant supernatants, longitudinal production of 22 cytokines were quantified via LegendPlex analysis. Results: In RM, pDCs were the least abundant innate cell subset (p<0.001), while MZB and NK cells were most abundant (p<0.01). There was an inverse correlation between the percentage of NK cells in RM and p24 production in parallel RM explants (r=-0.36, p=0.005); but there was a positive correlation between MZB cells and HIV replication (r=0.49, p<0.0001). No other innate subset was associated with p24 production. Comparison of RM and blood MZB and NK subsets illustrated quantifiable differences (Figure). Of the 22 cytokines quantified, IL17A, IFNg, IL10, IP10, GMCSF, Granzyme A (GzA), Granulysin, and Perforin, were positively correlated with HIV replication (p<0.01 for all). Detection of IL17A, IFNg, IL10, and GMCSF on day 3 positively correlated with later p24 production (p<0.01 for all). Conclusion: Our data demonstrate novel associations between MZB and NK cells and p24 production in RM, highlighting their potential importance in HIV replication, and that RM NK activity is likely mediated by GzA, Granulysin, and Perforin. Our data also underscore the critical importance of pro-inflammatory cytokines IL17 and IFNg early in mucosal HIV infection. Defining the innate cells subsets and their effector mechanisms that facilitate or hinder HIV infection in RM could identify new targets for biomedical interventions.
NK cell-mediated viral control in BCF and intestinal inflammation control via maintenance of IgA production. Methods: AGM and cynomolgus macaques (MAC) were infected with SIVagm. sab92018 and SIVmac251, respectively. We collected blood, mesenteric LN (mLN), jejunum, ileum and colon from each animal before infection, at day 9p.i. and during chronic infection. B, T and NK cell analyses were performed by fluorescence microscopy and/or flow cytometry. Immunoglobulins were isolated by affinity chromatography and quantified by ELISA. To determine IgA/ IgG ratios and gp140 antibody specificity, trimeric SIVagm and SIVmac gp140- foldon Env proteins and an IgA-specific probe were produced. Soluble markers of microbial translation and inflammation (i.e. sCD14) were quantified in plasma by ELISA. Results: NK cells migrated into BCF of mLN during acute SIVagm infection. CXCR5+NK cells showed a negative correlation (p=0.0004; r2=0.66) in LN with follicular Th1 cells, a population responsible of hypergammaglobulinemia. In AGM, intestinal IgA levels (jejunum, ileum and colon) were comparable between acutely infected (mean=0.51-0.45AU), chronically infected (mean=0.53- 0.39AU) and non-infected animals (mean=0.49-0.4AU). In acute SIVmac infection, intestinal IgA levels (mean=0.5-0.47AU) were similar to those of uninfected MAC (mean=0.54-0.42AU), but strongly decreased in chronically infected animals (mean=0.12-0.05AU). Similarly, IgA were decreased in BCF of chronically infected MAC. There was a negative correlation between sCD14 and IgA levels in MAC (p= 0.0037; r2=0.32) and not in AGM (p= 0.72; r2=0.012). Conclusion: Our data unraveled a negative correlation between gut IgA titres and inflammation in SIVmac infection, with a dramatic loss of intestinal IgA in SIV-infected MAC while IgA levels in chronic SIVagm infection remain stable. Hence, we propose that the viral control in BCF could help maintaining normal IgA responses and a better control of gut inflammation. 253 ALTERED DNA METHYLATION OF CCR5 IN SIGMOID MUCOSAL TISSUES OF TRANSGENDER THAI WOMEN Michael J. Corley 1 , Alexandra Schuetz 2 , Alina P. Pang 1 , Carlo Sacdalan 3 , Suchada Sukhumvittaya 2 , Nitiya Chomchey 3 , Nisakorn Ratnaratorn 2 , Yuwadee Phuang-Ngern 2 , Jintanat Ananworanich 3 , Eugène Kroon 3 , Nittaya Phanuphak 3 , Sandhya Vasan 4 , Lishomwa C. Ndhlovu 1 , for the SEARCH013/RV304 study group 1 University of Hawaii, Honolulu, HI, USA, 2 Armed Forces Research Institute of Medical Sciences in Bangkok, Bangkok, Thailand, 3 Thai Red Cross AIDS Research Center, Bangkok, Thailand, 4 US Military HIV Research Program, Silver Spring, MD, USA Background: Transgender women (TGW) have a high global prevalence of HIV. Lifestyle factors including hormone use have been shown to alter epigenetic patterns and transcriptional regulation in tissues. Thus, host epigenetic modifications in TGWmay link exogenous factors and altered HIV-related cell gene regulation leading to increased risk of HIV transmission. Methods: In a pilot study of HIV uninfected Thai volunteers, cross-sectional sigmoid mucosa biopsies were obtained frommen who have sex with men (MSM, n=10), cisgender women not on hormonal contraception (CW, n=9), and TGWwho had undergone gender affirmation surgery and remained on hormonal therapy (n=3). DNA methylation was measured genome-wide using the HumanMethylationEPIC array on the biopsies. Immunophenotyping of peripheral and mucosal mononuclear cells was performed to assess T cell subsets for CCR5 expression by flow cytometry. Statistical analysis included t-tests, non-parametric tests, and false discovery rate. Results: Among TGW, mean age of hormone initiation was 14 yrs, and sex reassignment surgery 19 yrs. Median duration of hormone use in TGWwas 8.5 yrs, with 1/3 using estrogen/ progesterone, and 2/3 estrogen only. All TGW reported anal and neovaginal sex and median lifetime sexual partners was significantly higher in TGW compared to CW (P = 0.005). We observed the greatest differences in DNA methylation at 219,788 CpG sites showing absolute mean differences in methylation greater than 5% between TGW and MSM (Δβ-value > |0.05| and significant at FDR adjusted P < 0.05) in sigmoid biopsies. There were fewer differences between TGW and CW (188,833 methylation sites) and even fewer between CW and MSM (5,162 methylation sites). Of known HIV acquisition risk genes, methylation at CpGs at the promoter region of the CCR5 gene were significantly decreased in TGW compared to both CW (P < 0.05) and MSM (P < 0.05). Furthermore, mucosal CCR5+ CD4 T cell frequencies were higher in TGW compared to MSM (P = 0.002), but not different compared to CW (P = 0.419). Notably, methylation levels of CCR5 associated with the frequency of mucosal CCR5+ CD4 T cells (P = 0.016).
252 IgA PRESERVATION IN GUT IN SIVagm INFECTION IS ASSOCIATED WITH INFLAMMATION CONTROL Philippe Rascle 1 , Cyril Planchais 1 , Beatrice Jacquelin 1 , Marie Lazzerini 1 , Vanessa Contreras 2 , Hugo Mouquet 1 , Nicolas Huot 1 , Michaela Müller-Trutwin 1 1 Institut Pasteur, Paris, France, 2 INSERM, Le Kremlin-Bicetre, France Background: Lymph nodes (LN) and intestine are the major HIV reservoirs. During SIVagm infection in African Green Monkeys (AGM), NK cells express CXCR5 and migrate into B cell follicles (BCF) of peripheral LN (pLN) where they efficiently control SIVagm replication. In the intestine, SIVagm replicates at high levels but this does not lead to bacterial translocation and chronic inflammation. IgA are important for the control of bacterial translocation and inflammation in the gut. In this study, we aimed at investigating whether there is a link between
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