CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

represented the 20% of the total cells expressing HIV transcripts. In lymph node tissues, we observed that a proportion of p24-expressing cells were also positive for the CD20 marker. Moreover, ex vivo infection of unstimulated PBMCs showed that viral replication induced the expression of CD20 (from 4.74% to 8.31% of CD20dim in CD4+ T cells). Notably, rituximab was able to target CD20dim CD4+T cells of ART-suppressed patients by the induction of apoptosis and antibody- mediated cell cytotoxicity, and the combination of rituximab and latency reversal agent Conclusion: CD20dim CD4+T cells harbored transcriptionally active HIV. Anti- CD20 treatment after induction of viral reactivation might represent a novel tool to target the active HIV-reservoir. 330 HIV-INFECTED CD4 T-CELL ISOLATION USING A MICROFLUIDIC MAGNETIC LEVITATION SYSTEM N. Gozde Durmus 1 , Kaushik Sridhar 1 , Erica Gibson 2 , Cassandra Thanh 2 , Rakhi Gupta 1 , Maya Ball-Burack 2 , Steven G. Deeks 2 , Utkan Demirci 1 , Timothy J. Henrich 2 1 Stanford University, Stanford, CA, USA, 2 University of California San Francisco, San Francisco, CA, USA Background: Strategies to identify and isolate HIV infected cells at various states of latency or transcriptional activation are urgently needed. We developed and implemented a novel microfluidic system that is able to sort untouched CD4+ T cells from HIV-infected individuals based on levitation heights within a magnetic field (i.e. magnetic density). We compared HIV DNA and unspliced RNA burden and immune phenotypes in cells from high and low density populations. Methods: Untouched CD4+ T cells from 15 ART-suppressed individuals were sorted based on magnetic density using our novel micirofluidic magnetic cell levitation and sorting system (Figure). A microfluidic chip-based platform incorporates collection channels for high-throughput isolation of cells levitating at different heights in a biocompatible paramagnetic medium for downstream characterization of HIV burden and immune phenotype. Results: Overall, CD4+ T cells from infected participants on ART levitated higher than cells from uninfected controls (229.6 vs 169 μM), but cell radii were similar. Two subpopulations of CD4+ T cells from ART-suppressed individuals were then isolated based on levitation height (the higher density populations contained 2.3-fold more cells). Markers of CD4+ T activation, immune checkpoint, and naive/memory phenotype (CD69, HLA-DR, CD38, PD1, CCR5, CD45RA, CCR7, CD4, CD3), as well as cell viability, were similar in both high and low density layers by flow cytometric analysis. Interestingly, HIV RNA levels from ART-suppressed individuals were significantly lower in the lower density subpopulation (0.81 log10 fewer copies/10^6 CD4+ T cells, P=0.007). Although there were no overall significant differences in HIV DNA levels between high and low density subpopulations, HIV DNA from three participant samples was highly enriched in lower density CD4+ T cells (>3 log10 copies/10^6 cells higher than in the higher density layer). characteristics that may be unrelated to expression of commonly tested surface protein markers, cellular activation state, and cell size. In addition, HIV DNA was observed almost exclusively in lower density CD4+ T cells in three samples, suggesting that isolating cells based on magnetic levitation has the potential to be refined and applied to various HIV reactivation, latency or eradication studies. Conclusion: We demonstrate that HIV infected CD4+ T cells of various transcriptionally active states have unique magnetic levitation/density

1 Vall d’Hebron Research Institute, Barcelona, Spain, 2 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain Background: Several pharmaceutical compounds as the Latency reversal agents (LRAs) can reactivate HIV expression from infected cells and might help to reduce the pool of latently infected CD4 T cells. However, the capacity of the LRAs to reactivate all infected CD4 T subpopulations is currently unknown. Methods: Viral reactivation assays were performed using freshly-isolated CD4+T cells obtained fromwhole blood donations of n=9 ART-suppressed patients. A total of 13 conditions, including controls, Ingenol (ING) and Bryostatin-1 (BRY) (PKC agonists), Romidepsin (RMD) and Panobinostat (PNB) (HDAC inhibitors) and JQ1 (Bromodomain inhibitor), and the combinations of LRAs from different families, were set up per patient. Intracellular HIV-RNA and p24 production were evaluated by the flow-RNA/FISH technology within different CD4+T cell subsets (Naïve-NA; Stem Cell Memory-SCM; Central Memory-CM, Effector memory-EM; Transitional Memory-TM and Terminally Differentiated-TD) after 22h in culture. Synergies and antagonisms between compounds were calculated using the Bliss independence model. Results: In general, a median of 10.74% of the whole HIV-reservoir in CD4+T cells induced HIV-RNA after viral reactivation but only 10.1% of these reactivated cells produced p24. The highest levels of HIV-RNA and the viral protein p24 were induced by ING, RMD and PNB followed by BRY and JQ1. Moreover, the combination of RMD+ING induced a reactivation of 3.50-fold compared to negative control. Regarding CD4+T subpopulations, most memory subsets were reactivated by Romidepsin and the highest susceptibility was observed in the TEM cells (FC=4.24). PNB was more efficient at reactivating TCM (FC=2.11) and TEM cells (FC=2.32), and ING was performing better in TCM (FC=4.05) and TTM (FC=5.27) but not in TEM cells. For combinations of LRAs, RMD+ING became the most effective condition in all tested CD4+T cell subsets, primarily in TTM (FC=6.72), but with no effect in TSCM. Contrarily, an antagonistic effect in terms of HIV transcription was observed when PNB+ING were added. Conclusion: Our results validate the use of the flow-RNA/FISH protocol to assess the potency of LRAs in different CD4+T cell subsets. Although important synergies are identified when RMD and ING are combined, existing LRAs present limited capacity to induce HIV transcription in the most important reservoir cells. This study emphasizes the need to generate new drugs with wide reactivation ability in all CD4+T cells for future clinical trials. 329 HIV-TRANSCRIPTION INDUCES CD20 EXPRESSION AND RENDERS CELLS SUSCEPTIBLE TO RITUXIMAB Carla Serra Peinado 1 , Judith Grau-Expósito 1 , Laura Luque-Ballesteros 1 , Antonio Astorga Gamaza 1 , Rein Willekens 2 , Jordi Navarro 2 , Adrià Curran 3 , Joaquin Burgos 3 , Esteban Ribera 3 , Josep Castellví 3 , Meritxell Genescà 4 , Vicenç Falcó 3 , María J. Buzón 4 1 Vall d’Hebron Research Institute, Barcelona, Spain, 2 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain, 3 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain, 4 Vall d’Hebron Research Institute, Barcelona, Spain Background: One of the main obstacles to cure HIV is the existence of persistent HIV reservoirs in ART-suppressed patients. Newmarkers with which to identify and target these reservoirs will definitely advance the search for an HIV cure. Here, we studied the expression of CD20 in HIV-infected cells and the use of this molecule to target the persistent viral reservoir. Methods: Phenotypic characterization of CD20 expression in different CD4+T cell subsets was measured by flow cytometry in n=10 ART-suppressed, n=13 viremic and n=6 healthy controls. Intracellular levels of HIV-RNA were measured by the novel FISH/RNA flow cytometry assay, and quantification of HIV-DNA in sorted CD20+ T cells by qPCR. Expression of CD20 and p24 in lymph nodes of HIV+ patients was measured by immunohistochemistry. Moreover, the dynamics of CD20 expression upon HIV infection was examined by ex vivo infection. Finally, the susceptibility of CD20+ T cells to therapy with rituximab (anti-CD20 monoclonal antibody) was examined after ex vivo viral reactivation of CD4+T cells from ART-suppressed patients. Results: We systematically found CD4+T lymphocytes in blood with dim expression of the CD20 receptor, in both healthy and HIV-infected individuals (2.47% in healthy, 1.82% in ART-suppressed and 2.11% in viremics). These cells had a memory phenotype (mainly central memory) and an increased activation state (values of 22 and 27.6% for CD20dim CD4+T cells, and 16.4 and 20.7% for CD20- CD4+ T cells in ART and viremics, respectively). In patients on ART, CD4+T cells expressing CD20 contained viral DNA and were enriched in HIV-RNA compared to cells not expressing this receptor (p=0.001). This population

Poster Abstracts

CROI 2019 120

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