CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

Methods: MDMs and alveolar macrophages were infected with HIV in vitro, and lipid accumulation was determined both by microscopy using Oil Red staining and by flow cytometry using Bodipy staining. Lipid accumulation was also measured in macrophages isolated from bronchoalveolar lavage (BAL) fluid of patients with and without HIV using Oil Red staining. RNA sequencing was performed on the BAL fluid of patients with and without HIV. Results: In MDMs in vitro, HIV infection resulted in lipid accumulation, as determined both by Oil Red and Bodipy staining. Similarly, alveolar macrophages infected with HIV in vitro demonstrated lipid accumulation by Oil Red staining. Surprisingly, alveolar macrophages from treated HIV+ patients demonstrated no increase in Oil Red positivity compared to HIV- controls. Furthermore, RNA sequencing data from alveolar macrophages in a separate cohort of untreated HIV+ patients demonstrated increased, not decreased, expression of ABCA1 compared to HIV- controls. Conclusion: HIV infection induces lipid accumulation in vitro in MDMs and alveolar macrophages, but no lipid accumulation is observed in the BAL fluid of treated HIV positive patients in vivo. A possible explanation could include the differential expression of ABCA1 in the blood and alveolar space in patients with and without HIV.

Poster and Themed Discussion Abstracts

233 TIM-3 IS A MARKER OF PDC DYSFUNCTION IN HIV INFECTION Jordan A. Schwartz 1 , Kiera L. Clayton 1 , Shariq Mujib 1 , Hongliang Zhang 1 , Nur-ur Rahman 1 , Jun Liu 1 , Feng Yun Yue 1 , Erika Benko 2 , Colin Kovacs 1 , Mario Ostrowski 1 1 Univ of Toronto, Toronto, Canada, 2 Maple Leaf Med Clinic, Toronto, Canada Background: The plasmacytoid dendritic cell (pDC) is a specialized type I interferon producing cell of the innate immune system; critical in the control of HIV-1 infection. Previous studies have identified pDC dysfunction in HIV-infected donors, including a decreased capacity to produce IFN-α. Recently, studies have suggested that Tim-3, a T cell marker of activation/exhaustion, has diverse effects on a wide array of leukocytes. In this study, we evaluated the role of Tim-3 in pDC dysfunction during HIV-1 infection. Methods: Ex vivo pDCs from PBMCs of 26 HIV-infected donors were examined for Tim-3 and cytokine expression after TLR and virus stimulation. Tim-3 regulation on pDCs from HIV-uninfected donors stimulated in the presence of HIV-1, influenza, CpG, imiquimod or Sendai virus was also assessed. Tim-3 localization with signaling proteins essential for IFN-α production, TLR9, IRF7 and PI3K within pDCs was assessed using confocal microscopy. Results: Tim-3 was upregulated on pDCs in HIV infection and was not restored to pre-infection levels by cART. Tim-3+ pDCs from HIV-infected donors displayed profound defects in IFN-α and TNF-α production. Surprisingly, in vitro stimulation of enriched pDCs from HIV-uninfected donors with exogenous HIV virions did not significantly enhance Tim-3 expression. However, Tim-3 was rapidly induced by strongly activating pDC agonists such as imiquimod and Sendai virus. Tim-3 expression positively correlated with the maximum levels of IFN-α or TNF-α induced after stimulation. Tim-3 expressing pDCs showed disrupted submembrane distribution of TLR9 and intracellular Tim-3 co-localized with PI3K and IRF7 within lysosomes. Conclusion: Dysfunctional Tim-3 expressing pDCs are induced during HIV infection. Strongly activating stimuli were found to increase Tim-3 expression, suggesting that during HIV infection, chronic activation from opportunistic pathogens contributes to Tim-3 expression on pDCs. Tim-3 may interfere with IFN-α and TNF-α production from pDCs by displacing submembrane TLR9 and by enhancing degradation of IRF7 and PI3K. 234 IMMUNE CHECKPOINT MOLECULES IN HIV AND AGING Lesley R. de Armas , Suresh Pallikkuth, Sidney Lane, Rajendra Pahwa, Varghese George, Stefano Rinaldi, Margaret Fischl, Gordon Dickinson, Allan Rodriguez, Savita Pahwa Univ of Miami, Miami, FL, USA Background: Immune checkpoint (IC) molecules are increasingly becoming targets for therapeutic intervention to stimulate the immune system. The objective of the current study was to examine the relationship of IC expression on T lymphocytes with biological age in anti-retroviral treated (cART), virologically suppressed HIV-infected and – uninfected (HC) participants. Methods: Multi-parameter (15-color) flow cytometry was performed on PBMC isolated from 4 participant groups: HIV+ Young (<40yr) (Y+, n=24), HIV- Young (Y-, n=14), HIV+ Old (>60yr) (O+, n=21), and HIV- Old (O-, n=27) to evaluate IC expression (PD1, LAG3, TIM3, TIGIT, 2B4), transcription factors (EOMES, TBET), and markers of immune senescence (CD57, CD28). Total HIV DNA was measured in PBMC samples. Unpaired t test and correlation analyses were performed. Results: First, we evaluated age-specific effects of HIV infection and found CD57+CD28- (immunosenescent) CD4 T cells to be low in Y-, but increased in Y+ as well as O- and O+ groups. Similarly, TBET expression (CD4 CD8) was lowest in the Y- group. Next, we evaluated markers of aging in HC and HIV. IC molecules LAG3 and EOMES were increased in CD4 T cells from both O groups compared to respective Y groups, while TIGIT (CD4) and TIM3 (CD4 and CD8) were lower in O- compared to Y-. Expression of PD1, TIM3, TIGIT, and 2B4 on CD4 and CD8 remained stable when comparing O+ to Y+. Interestingly, IC triple positive CD4 cells by Boolean analysis (LAG3+TIGIT+PD1+; negative for all other IC) were increased in O+ compared to Y+ (p=0.001) but did not correlate with HIV DNA (p=0.45). In fact, only 13/512 combination gates positively correlated with HIV DNA (cutoff r>0.35, p<0.05) and all 13 contained TIM3+ cells. HIV DNA did not show significant correlation with age. Conclusion: Our results reveal complex relationships between IC expression on T lymphocytes with aging in cART-controlled HIV infection. Differences in immunosenescent cells between HIV and HC was most striking in Y and was lost in O groups, indicating its early onset and the dominant effect of age versus HIV in O groups. Increase in LAG3+ cells (+/- TIGIT, PD1) in O+ individuals is relevant as these cells harbor replication competent virus and may represent an obstacle to HIV cure in Aging persons. Association of TIM3 with HIV DNA may implicate it as a general marker of reduced immune control. Studies aimed at understanding IC expression and regulation will help guide therapeutic intervention strategies aimed at inhibiting their function.

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CROI 2017