CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

173 INCREASED BIRC2 AND BIRC3 TRANSCRIPTION ARE ASSOCIATED WITH INCREASED HIV PRODUCTION Joseph P. Casazza 1 , Quang N. Nguyen 1 , Avery N. Sukienik 1 , David R. Ambrozak 1 , Jianfei Hu1, Sam Darko 1 , Amy Ransier 1 , Farida Laboune 1 , Daniel Douek 1 , Richard A. Koup 1 1 Vaccine Research Center, NIAID, Bethesda, MD, USA Background: ACH-2 cells are A3.01 cells containing a single proviral HIV genome. Unstimulated ACH-2 cells produce low levels of HIV. Prior to stimulation, surface staining of HIV Env identifies two ACH-2 populations, one that stains for Env and p24 and one that does not. With TNF stimulation >90% of virus production is from the Envbrt population while an Envdim population arises from the Env- population. We compared the cellular transcriptome of Envbrt to Envdim and A3.01 cells before and after stimulation with TNF. Methods: ACH-2 cells were dual stained with PG9 and VRC07 to identify the envelope bright, dim and null populations and bulk sorted at 0, 3, 6, 9 and 24h post-stimulation. A3.01 cells were sorted similarly. Cells were lysed and then frozen. Total RNA was extracted, poly-adenylated RNA purified, fragmented and then reversed transcribed using random hexamers. Illumina ready libraries were generated and sequenced by paired-end HiSeq 4000 2x75 reads. Changes that were 2-fold different with a P<0.005 were defined as significant. Individual protein levels were determined using chemiluminescent antibody arrays. Results: ACH2 and A3.01 transcriptomes were similar post-stimulation in many ways. In both ACH2 and A3.01 cells RELA and NFKB1 message were elevated prior to, and did not change with TNF stimulation. Increased NFKB2 and NFKBIA in both A3.01 and ACH2 cells 3h after stimulation and increased RELB and CD82 suggested rapid activation and transport of NFkB to the nucleus and early NFkB driven phosphorylation and transport of AP-1 to the nucleus. There were also differences. HIV RNA read frequency was significantly higher in ACH2 bright than in ACH2 dim cells and absent in A3.01 cells at all times. These changes were associated with differences in NFkB associated gene transcription. Most strikingly BIRC3 transcripts, which plays an important role in NFkB activation, were significantly increased in Envbrt compared to A3.01 cells post-stimulation. This change was not observed in the Envdim population. Increases in BIRC-3 protein at 1 hour and phosphorylation of AP-1 at 30mwere confirmed by protein array. Conclusion: These data indicate low level transcription of HIV RNA prior to stimulation in Envbrt cells are associated with higher level BIRC3 after stimulation. Increased transcription of BIRC3 may contribute to increased HIV production in these cells. 174 THE ARYL HYDROCARBON RECEPTOR NEGATIVELY REGULATES HIV REPLICATION IN TH17/TH22 CELLS Debashree Chatterjee 1 , Yuwei Zhang 1 , Huicheng Chen 1 , Tomas Raul Wiche Salinas 1 , Yasmine Smail 1 , Jean-Pierre Routy 2 , Petronela Ancuta 3 1 Université de Montréal, Montreal, QC, Canada, 2 McGill University Health Centre, Glen site, Montreal, QC, Canada, 3 Centre Hospitalier de l'Université de Montréal, Montreal, QC, Canada Background ART fails to restore the depletion of Th17-polarized CCR6+CD4+ T-cells in PLWH. Novel Th17-targeted HIV remission/cure strategies are needed to restore Th17-mediated mucosal immunity. Autoimmunity studies demonstrated the existence of pathogenic and non-pathogenic Th17 cells and identified the aryl hydrocarbon receptor (AhR) as a marker of non-pathogenic Th17 cells. AhR is a ligand-dependent transcription factor that regulates the expression of several genes (IL-22, IL-10, integrin B7) and is involved in proteasomal degradation via its E3 ubiquitin ligase activity. We hypothesized that AhR negatively regulates HIV replication in non-pathogenic Th17 cells. Methods: PBMC of ART-treated PLWH (n=8; median CD4 counts: 598, plasma viral load < 40 HIV-RNA copies/ml) and uninfected controls (n=5) were used in this study. Total/CCR6+/CCR6- memory CD4+ T-cells were isolated by magnetic/ flow cytometry sorting. Cells of uninfected donors were stimulated via CD3/ CD28, exposed to HIV, and cultured 9 days. Viral outgrowth assay (VOA) was performed with cells of ART-treated PLWH. AhR silencing was performed using CRISPR/cas9, with efficacy evaluated by T7 endonuclease assay and Western blotting. AhR agonist (FICZ) and antagonist (CH223191) were used. Cell viability/ proliferation, HIV replication, cytokines, and gene expression were quantified by ELISA, flow cytometry and/or real-time PCR. Results: AhR mRNA/protein expression was induced by T-cell receptor triggering. CRISPR/cas9-mediated AhR silencing significantly inhibited IL-22, IL-17A, IL-10 and integrin beta7 expression (p<0.01) and increased viral

DC (i.e., cis infection). Cell phenotype was assessed by flow cytometry and viral replication by HIVp24 production before and after stimulation with anti-CD3/ CD28 Ab or PMA/PHA. We quantified total HIV DNA in the TN and total CD4 T cell populations from 2 HIV nonprogressors (NPs). Results: After 12 days of incubation, there were low levels of p24 in the B cell-TN co-cultures (n=10), indicative of productive infection, but not in the DC-TN co-cultures. In contrast, both B cells and DC could efficiently HIV trans infect total CD4 T cells. As expected, TN were refractory to direct, cis infection with HIVBaL. Phenotypic analysis of the TN cells revealed that they maintained a CCR5neg phenotype. B cell-TN co-cultures exposed to anti-CD3/CD28 Ab or PHA/PMA resulted in high-levels of p24 production, whereas no virus expression was recovered from the DC-TN co-cultures. We previously demonstrated that APCs derived from NPs cannot trans infect CD4 T cells, which prompted us to quantify the HIV DNA reservoir in TN and total CD4 T cells isolated from 2 NPs. We detected HIV DNA in the total CD4 T cells but not in the TN of both NPs. Conclusion: B cells, but not DCs, efficiently trans infect CCR5neg TN cells with R5-tropic HIVBaL. No HIV DNA was detected in CD4 TN cells from NPs, consistent with the notion that APCs derived from NPs cannot trans infect CD4 T cells. B cell-mediated HIV trans infection of CD4 TN cells could be a key mode to establish early HIV reservoir. 172 TFR REDUCE HIV-1 INFECTED TFH IN VITRO IN AN IL-2 DEPENDENT MANNER Matthew T. Ollerton 1 , Elizabeth Connick 1 1 University of Arizona, Tucson, AZ, USA Background: Follicular CD4+ T cells (TFH) are highly permissive to HIV-1 infection and a major reservoir of HIV-1 in lymphoid tissues. Follicular regulatory CD4+ T cells (TFR) limit TFH numbers and function in vitro and in vivo. We hypothesized that TFR inhibit HIV-1 replication in TFH. Methods: TFH (CD3+CD8-CXCR5+CD25-) isolated from tonsils of individuals at low risk for HIV were spinoculated with the GFP reporter virus NLENG1, labeled with the proliferation dye VPD450, and co-cultured 1:1 with autologous TFR (CD3+CD8-CXCR5+CD25+) or TFH (control) for 5 days in Advanced R-10 media and 10 IU/ml IL-2. Percent GFP+VPD450+ TFH were determined by flow cytometry. Live VPD450+ TFH were isolated on a cell sorter, and total and integrated HIV DNA were quantified using QPCR. Cell counts were measured using counting beads. In some experiments, 0, 10, 30, or 100 IU/ml of IL-2, or blocking antibodies to TGF-beta, CD39, or IL-10 at 10 µg/ml were added. IL-2 supernatant concentrations were measured by ELISA. Statistical analyses were performed using non-parametric Wilcoxon matched-pairs test and Spearman’s correlation. Results: In comparison to control co-cultures, TFR reduced TFH numbers (p=0.023; n=14), %GFP+ TFH (p=0.001; n=14), total HIV DNA (p=0.016; n=7), and integrated HIV DNA (p=0.016; n=7). Blocking TGF-beta, CD39, and IL-10 did not reverse TFR inhibition of %GFP+ TFH. IL-2 increased TFH viability in a dose dependent manner (r= 0.946 p=<0.0001), but did not promote TFH proliferation. Compared to control co-cultures, %GFP+ TFH were reduced in TFR co-cultures with 10 IU/ml and 30 IU/ml IL-2, while no inhibition was detected in TFR co-cultures without IL-2 or with 100 IU/ml IL-2 (See Figure). TFH cell counts followed the same pattern. IL-2 supernatant concentrations were lower in TFR co-cultures compared to control co-cultures with 10 IU/ml (median, 1.0 vs 5.0 ng/ml; p= 0.031) and with 30 IU/ml (median, 5.3 vs 12.1 ng/ml; p= 0.156), but not with no IL-2 (median, 0 vs 0; p=0.999) or 100 IU/ml IL-2 (median, 67.39 vs 56.0 ng/ml; p=0.813). Conclusion: IL-2 promoted TFH viability and HIV-1 associated GFP expression in vitro. TFR reduced HIV-1 producing TFH at low, but not high or absent concentrations of IL-2. Consumption of IL-2 in B cell follicles may be one mechanism by which TFR reduce HIV-expressing TFH in vivo.

Poster Abstracts

58

CROI 2020