CROI 2020 Abstract eBook
30 HIV-INFECTED MACROPHAGES EVADE NK CELL-MEDIATED KILLING WHILE DRIVING INFLAMMATION Kiera L. Clayton 1 , Heather Stuart 1 , Geetha H. Mylvaganam 1 , Alonso Villasmil Ocando 1 , Marcela Maus 2 , Bruce D. Walker 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Massachusetts General Hospital, Boston, MA, USA Background: The primary targets for HIV infection are CD4+ T cells, however macrophages also become infected and persist despite antiretroviral therapy, suggesting evasion of immune responses. Our previous work shows that while HIV-infected macrophages are recognized by cytolytic CD8+ T lymphocytes (CTL), killing is inefficient due to resistance to CTL-derived granzymes. This poor killing delays CTL detachment from its target, causing hypersecretion of CTL- derived cytokines that propagate inflammation, emphasizing the need for rapid killing and release of effector-target contacts to limit inflammation. Thus, we hypothesized that cells with greater cytolytic potential compared to CTL, such as NK cells, would be able to rapidly kill HIV-infected macrophages while limiting excessive inflammation. Methods: To test this hypothesis, innate interactions between NK cells and autologous HIV-infected macrophages or CD4+ T cells were assessed via flow cytometry-based recognition and killing assays. To characterize the potential for antibody-dependent cellular cytotoxicity (ADCC), HIV envelope expression on macrophages was characterized by flow cytometry, imaging flow cytometry, and confocal microscopy using HIV-specific antibodies, and HIV-specific CAR T cells were used to confirm envelope accessibility on target cells. Finally, ADCC responses against infected CD4+ T cells and macrophages were assessed via flow cytometry. Results: Despite similar levels of total recognition of HIV-infected CD4+ T cells and macrophages (degranulation and TNF-α production), NK responses to macrophages were significantly skewed towards non-cytolytic, cytokine production (p<0.0001), which was associated with poor elimination (p<0.0001). HIV antibody-based detection confirmed that envelope was transiently expressed on the macrophage cell surface, and recognition of infected macrophages by HIV-specific CAR T cells was comparable to that of CD4+ T cells, suggesting that HIV envelope is equally accessible on both cell types. ADCC enhanced NK cell responses to both cell types, however, total responses to macrophages were significantly lower compared to that of CD4+ T cells (p<0.001 for 3BNC117 and p<0.05 for PGT121). Conclusion: Together, these data suggest HIV-infected macrophages employ a unique mechanism to evade cytolytic recognition by NK cells while preserving pro-inflammatory cytokine responses, emphasizing the need to develop alternative strategies to eliminate infected macrophages. 31 HIV DNA DETECTED IN IMMUNE CELL SUBSETS IN CSF DURING ART Shelli Farhadian 1 , Joshua C. Cyktor 2 , Asma Naqvi 2 , Michael J. Corley 3 , Jennifer Chiarella 1 , Rachela Calvi 1 , Michelle Chintanaphol 1 , Geoffrey Lyon 1 , Diane Trotta 1 , John W. Mellors 2 , Serena S. Spudich 1 1 Yale University, New Haven, CT, USA, 2 University of Pittsburgh, Pittsburgh, PA, USA, 3 University of Hawaii, Honolulu, HI, USA Background: HIV-infected cells persist in the central nervous system (CNS) in at least half of people with HIV (PWH) on antiretroviral therapy (ART). We previously reported on a novel population of myeloid lineage microglia-like cells in cerebrospinal fluid (CSF) from PWH on ART; however the identity of CNS cells containing proviruses remains unknown. Methods: Fresh CSF and blood were collected from PWH (median 20yrs on ART, range 4-24yrs). Single cell CITE-seq was performed to validate CD204 as a marker for CSF microglia-like cells. CSF cells and peripheral blood mononuclear cells (PBMCs) were separated using fluorescence activated cell sorting into three subsets based on expression of: CD3+CD4+, CD3+CD8+, and CD3-CD20- CD204+. HIV DNA levels were determined in each subset using a sensitive qPCR assay targeting HIV integrase (iCAD). HIV DNA measurements were normalized for cell equivalents determined by CCR5 qPCR. Results: Six donors had plasma HIV RNA levels <20 copies/mL; one had 748 copies/mL. Two donors had HIV RNA detected in CSF despite plasma viral suppression, with 95 and 163 copies/mL HIV RNA detected in CSF. The median number of CSF cells obtained per donors was 35,327 (range 13,000-85,000) in 25mL of CSF. HIV DNA was detected in blood CD4+ T cells from 6/7 donors, and not detected in blood CD4+ T cells in one donor. In CSF, HIV DNA was detected in CD4+ T cells in 6/7 donors (of which 5 donors also had HIV DNA detected in blood CD4+ T cells). HIV DNA copies per 1 million cell equivalents was higher
(median 7.1 fold, range 0.3-132) in CSF CD4+ T cells than in blood CD4+ T cells in 5/6 donors. No donor had HIV DNA detected in CSF CD8+ T cells. We isolated genomic DNA from CD204+ CSF cells in three participants and observed that one participant had HIV DNA detected in CD204+ CSF cells. This donor had plasma HIV RNA 748 copies/mL and CSF HIV RNA 87 copies/mL. HIV DNA levels in this participant were 4368 copies per 1 million CD204+ CSF cells, 2769 copies per 1 million in CSF CD4+ T cells, and 401 copies per 1 million blood CD4+ T cells. Conclusion: We detected HIV DNA in CD4+T and myeloid cells in CSF in a limited sample of PWH on ART. Normalized HIV DNA in CD4+ T cells from CSF was higher than in blood in most donors. Larger studies should assess whether the HIV DNA detected is in replication-competent proviruses, and whether other CNS immune cell types are HIV-infected. Patrick Luckett 1 , Anupama Melam 1 , Sarah A. Cooley 1 , Joshua Shimony 1 , Beau Ances 1 1 Washington University in St Louis, St Louis, MO, USA Background: Despite the use of combination antiretroviral therapy, many HIV associated conditions, such as HIV associated neurocognitive disorders and frailty still exist in people living with HIV (PLWH). A potential biomarker reflective of these conditions is resting state functional connectivity (rsFC). Changes in rsFC strength have been hypothesized to reflect a compensatory reaction due to damage caused by persistent inflammation and chronic immune activation. Within a large cohort of PLWH and HIV- controls we identified networks most affected over the life span of HIV infection using machine learning methods. Methods: A total of 538 rsFC scans from 318 PLWH (mean age 47.2y, 77%male, 31% Caucasian, mean duration of infection 12.8y +/-9.4, 84% viral load <200) collected from studies at Washington University School of Medicine (WUSM) and 2791 scans from 2133 HIV- controls (mean age 44.4y, 42%male, 69% Caucasian) collected from studies at WUSM and other sources were analyzed. Ages ranged from 20 to 70 years old (figure 1b). Ten rsFC networks were evaluated, and preprocessing was performed using in house methods. Correlation matrices were generated for all participants, and an average correlation matrix was computed for each year of age for both groups. A Relief feature selection algorithmwas used to identify the strongest predictive networks of HIV status. We then evaluated which networks showed significantly different trajectories with respect to aging among PLWH and controls. Results: The Relief algorithm identified the strongest predictors of HIV status as multiple connections between the somatomotor, cingulo-opercular, and dorsal attention networks. The strongest difference in average connectivity strength as a function of aging between PLWH and controls were between the dorsal attention and both the fronto-parietal (figure 1a) and cingulo- opercular networks (R2 = -.82, -.52, P<.01), as well as the ventral attention and somatomotor networks (R2 = -.38, P<.01). Conclusion: Our data suggest changes in rsFC occurs as a result of HIV infection, and are primarily associated with motor, control, and attention. Further, changes exhibit specific temporal patterns between networks which is independent of reorganization that occurs due to normal aging, and these changes begin around midlife. These results will allow for a more tailored treatment approach for PLWH, and should be considered when conducting clinical trials.
32 EFFECTS OF HIV AND AGING ON RESTING-STATE NETWORKS
Made with FlippingBook - professional solution for displaying marketing and sales documents online