CROI 2016 Abstract eBook

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Poster Abstracts

Results: After 12 days of culture, viral replication was totally inhibited in the presence of the antiretroviral drugs JM-2987 and raltegravir. Surprisingly, the addition of the pan-caspase inhibitor Q-VD-OPh resulted also in an almost complete inhibition (84%) of the viral replication. Furthermore, a massive cell depletion was observed in the absence of antiretroviral drugs (18%± 6% of cells remaining relative to uninfected controls) that was totally prevented by the addition of JM-2987 and raltegravir. Loss of cells was also prevented by Q-VD-OPh treatment (63%± 4% relative to uninfected control). HIV infection triggered high levels of both caspase-1 and 3/7 activity (34.7%± 4 and 20.4%± 3, respectively) that were inhibited to the uninfected control level by JM-2987 and raltegravir (15.6 %± 1 and 9%± 0.6, respectively) and partially inhibited by Q-VD-OPh (24.2 ± 3 and 15%± 1, respectively). After infection, increased caspase-3 was mainly detected in the CD3+CD8-CD4- population (productively infected cells) while increased caspase-1 was observed in both CD4+ and CD4- T cell subsets. Conclusions: HIV infection of histocultures of human lymphoid tissue triggered caspase-1 and 3/7 activity that was inhibited with antiretroviral drugs as well as with a pan- caspase inhibitor. Caspase inhibitor treatment prevented both viral replication as massive CD4 T cell depletion suggesting that, caspase activation contributes significantly to viral replication and HIV pathogenesis in lymphoid tissue. 242 Follicular Regulatory T Cells Are Highly Permissive to HIV-1 Ex Vivo Shannon Miller ; Elizabeth Connick Univ of Colorado, Denver, CO, USA Background: The majority of HIV replication occurs in follicular helper T cells (TFH) in secondary lymphoid follicles during asymptomatic disease. Follicular regulatory T cells (TFR) are a subset of TFH that control germinal center (GC) responses. Their role in HIV-1 replication is unknown. We hypothesized that TFR are highly susceptible to HIV and evaluated potential causative mechanisms. Methods: Cells were disaggregated from healthy human tonsils or HIV-uninfected human lymph nodes (LN) and spinoculated with R5-tropic GFP reporter virus or mock spinoculated, cultured for 2 days, and analyzed for GFP expression by flow cytometry. Due to downregulation of CD4 by HIV, cells were gated on CD3+CD8- cells and subsets within were defined as TFR (CXCR5+CD25+CD127-), GC TFR (CXCR5+PD1+CD25+CD127-), TFH (CXCR5+CD25-), and GC TFH (CXCR5+PD1+CD25-). In select experiments, tonsil TFR and TFH were sorted prior to spinoculation. CCR5 and Ki67 expression were determined at baseline prior to spinoculation in a subset of subjects. Statistical analyses were performed by Wilcoxon matched-pairs signed rank test using GraphPad Prism 6. Results: %GFP+ is higher in TFR compared to TFH (median, 4.8-fold higher; p=0.008) and in GC TFR compared to GC TFH (median, 4.0-fold higher; p=0.008; n=8). GFP MFI is not significantly different in TFR compared to TFH (medians, TFR=4599, TFH=6952; p=0.09, n=6), but is lower in GC TFR compared to GC TFH (medians, GC TFR=4114, GC TFH=6427; p=0.03, n=6). Spinoculated cultures of sorted tonsil TFH and TFR (n=4) confirm differences observed in unsorted cultures (median fold difference %GFP+ TFR:TFH = 3.5). In HIV-uninfected LN (n=2), %GFP+ is also higher in TFR compared to TFH (average, 4.7-fold higher) and GC TFR compared to GC TFH (average, 3.4-fold higher). In tonsil cells prior to spinoculation, %CCR5+ is higher in TFR (37%) compared to TFH (11%; p=0.03, n=6) and GC TFR (38%) compared to GC TFH (11%). %Ki67+ (n=3) is higher in TFR compared to TFH (medians, TFR=51%, TFH=14%) and in GC TFR compared to GC TFH (medians, GC TFR=63%, GC TFH=14%). Conclusions: TFR are highly permissive to R5-tropic HIV ex vivo , which may promote HIV infection and replication within secondary lymphoid follicles in vivo . Potential mechanisms underlying heightened permissiveness of TFR include elevated proliferation and CCR5 expression. 243 Dead Cells Tell No Tales: The Enigma of Discerning HIV-Infected Cell Death Nathan Cummins ; Sekar Natesampillai; Michele Smart; Scott H. Kaufmann; Andrew D. Badley Mayo Clinic, Rochester, MN, USA Background: Multiple mechanisms contribute to CD4 T cell death in HIV. It is unclear which predominate, and whether cell death occurs mostly in uninfected, non-productively infected, or productively infected cells. This traditionally has been studied by measuring co-expression of markers of HIV infection (viral protein or nucleic acid) and cell death. During cell death, host cell proteases and endonucleases are activated. We hypothesized that activation of these enzymes during cell death degrades common markers of infection and therefore precludes reliable detection of infected-cell death. Methods: Jurkat T cells transiently or stably expressing eGFP, and J-Lat 10.6 cells (which contain integrated HIV provirus encoding GFP) were used. Cells were treated with camptothecin (CPT) or DMSO control to induce apoptosis, and monitored for apparent GFP expression and markers of cell death over time by flow cytometry. Beta-actin gene expression was measured by RT-qPCR. Results: Jurkats transiently expressing eGFP treated with CPT had decreased apparent GFP expression over 48 hrs compared to DMSO treated cells, coincident with loss of membrane integrity measured by vital staining. CPT treated cells also had significantly decreased RT-qPCR signal for the housekeeping gene beta-actin (P<0.001) compared to control. Jurkat T cells stably expressing eGFP, sorted twice to ensure >95% baseline GFP expression, were treated in a similar manner. These CPT-treated cells also lost apparent GFP expression by flow cytometry coincident with binding Annexin V (37% loss), expression of active Caspase 3 (50% loss) and becoming TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) positive (80% loss), whereas DMSO treated cells maintained >90% GFP expression. 96% of CPT treated, dead Jurkat-eGFP at 48 hrs by vital staining were apparent GFP-negative. Similar effects were seen with J-Lat 10.6 cells reactivated with prostratin, and subsequently treated with CPT compared to DMSO control. Conclusions: Our data suggest that common markers of cellular HIV-infection, including protein and mRNA expression, diminish as cells progress through the death process. Therefore, determining the proportion of dying or dead cells that are HIV “positive” necessarily under-estimates this fraction. Novel markers of infected cell death are needed. This is important for HIV cure, for which novel strategies, i.e. “shock and kill”, require specificity to infected cells versus uninfected cells. 244 Multidimensional Profiling of HIV-Infected Human CD4 T Memory Stem Cells Lara Manganaro 1 ; Jeffrey Johnson 2 ; Ekta Sharma; Patrick Hong; Benhur Lee 3 ; Nevan Krogan 2 ;Viviana A. Simon 1 Icahn Sch of Med at Mount Sinai, New York, NY, USA; 2 Univ of California San Francisco, San Francisco, CA, USA; 3 Univ of California Los Angeles, Los Angeles, CA, USA Background: Human CD4 T cells constitute the long-lived HIV reservoir, which prevents the virus eradication in HIV infected patients treated with highly active antiretroviral therapy (HAART). Emerging evidence suggest that the HIV reservoir is formed early upon infection within the CD4 T memory cell population. A new powerful technology which combines Flow Cytometry and Mass Spectrometry (Mass Cytometry, CyTOF) allows immune profiling of human cells at unprecedented resolution (>30 parameters for single cell). We hypothesized that T memory cells supporting infection differ with respect to phenotype, cell cycle and proliferation. Methods: We compared the immuno-phenotype, cell cycle profiles and proliferation capacity of CD4 T cells stimulated with IL2 or IL15 and infected with replication competent NL4.3-HIV. A custom antibody cocktail was used to detect >20 surface and intracellular markers including p24 by CyTOF. We also conducted quantitative phospho-proteomics of CD4 T cells treated with IL2 and IL15 different cytokines in order to define signaling pathways specific for the given CD4 T cells stimulation. Results: The CyTOF analysis revealed that T memory stem cells (TSCM) display high proliferation capacity, which is specifically amplified by IL15. Moreover TSCM efficiently support HIV infection. The susceptibility to infection was further increased by IL15 stimulation with TSCM constituting up to 9% of the infected cell population. Cell cycle analysis showed a skewing of the infected cell populations towards the G2 phase. The phosphoproteomic analysis of CD4 T cells showed that 247 proteins were differentially phosphorylated in IL15 compared to IL2. The main processes activated by IL15 were RNA metabolism, transcription regulation, chromatin remodeling, signal transduction and cytoskeleton remodeling. Of note, we also identified up-regulation of SAMHD1 phosphorylation upon IL15 stimulation.

Poster Abstracts

95

CROI 2016