CROI 2016 Abstract eBook
Results: B/R treatment of primary, resting CD4 T cells resulted in a 12.3±3.0 fold increase in cell-associated HIV mRNA and a 30.4±10.7 fold increase in supernatant HIV mRNA. Despite the stimulation of the primary CD8 T cells, there was no significant difference in either cell-associated or supernatant HIV mRNA when B/R-treated CD4 T cells were incubated with CTL in 3 patients. However a 2-fold reduction in cell-associated mRNA was seen in the other 3 patients. Conclusions: While the combined treatment of latently infected CD4 T cells with romidepsin and bryostatin induces upregulation of HIV mRNA, primary CD8 T cells stimulated with Nef and Gag peptides in the presence of high levels of IL-2 were unable to eliminate the reactivated cells in the majority of chronic progresses on ART. However effective responses were seen in several patients. The results suggest that in some cases, effective priming of the cytotoxic T lymphocyte responses may lead to elimination of reactivated latently infected CD4+ T cells. 375 Dendritic Cell T-Cell Culture Facilitates HIV Latency in Proliferating CD4+ T Cells Nitasha A. Kumar 1 ; Renee M. van der Sluis 2 ;Talia Mota 2 ; Sharon R. Lewin 3 ; Paul U. Cameron 3 1 Emory Univ, Atlanta, GA, USA; 2 The Peter Doherty Inst, Univ of Melbourne, Melbourne, Australia; 3 Doherty Inst for Infection and Immunity, Univ of Melbourne, Melbourne, Australia Background: Latent infection of resting CD4 + T-cells is a major barrier to HIV eradication. We have previously shown that latency occurs in non-proliferating CD4 + T-cells following co-culture with myeloid dendritic cells (mDC) and monocytes, and have now examined whether latency can be established in proliferating CD4+ T-cells under similar conditions. Methods: Resting CD4 + T-cells stained with the proliferation dye eFluor670 were cultured alone or with syngeneic mDC, plasmacytoid DC (pDC) or monocytes (CD14 + ) in the presence of staphylococcal enterotoxin B (SEB). After 24hrs, cultures were infected with CCR5-tropic enhanced green fluorescent protein (EGFP)-reporter HIV. On day-5 post- infection, non-productively-infected, non-proliferating and proliferating T-cells were sorted and further cultured until day 12 with IL-7, T-20 (fusion inhibitor) and L8 (integrase inhibitor). On day 5 and 12 post-infection EGFP was quantified following activation with αCD3/αCD28+L8 as a marker of inducible latent infection. At day 5, expression of markers of activation (CD69, CD25), proliferation (Ki67), immune checkpoints (PD-1, Tim-3) and T cell subsets (CCR7, CD27) was determined. T-cell receptor Vβ chains that were SEB specific (17, 3) and non-specific (13.1) were measured on T-cells. Results: Inducible latent infection was detected in non-productively infected, proliferating T-cells co-cultured with mDC, pDC and monocytes following activation with αCD3/ αCD28+L8 (median(interquartile range), 403(53-1470), 40(1-213), 177(1-980) EGFP+ cells/10,000 viable cells respectively; n=15) and only in mDC and monocyte cultures using ALU-LTR nested PCR (1000 copies per 10 6 cells each; n=7). At day 12, inducible latent infection was observed in proliferating T-cells co-cultured with mDC and monocyte, but not pDC where cell viability was less than 20% (n=4). Proliferating CD4 + T-cells frommDC, pDC and monocyte co-cultures expressed high levels of CD25 (97, 86, 99% respectively, n=6), CD69 (39, 32, 46%; n=5), Tim-3 (23, 67, 48; n=4), lower levels of PD-1 (0.1, 1.9, 3%; n=4) and Ki67 (7, 18, 11%; n=7). The cells remained CCR7 + CD27 + until day 12. SEB non- specific Vβ was not enriched in either non-proliferating or proliferating T-cells, indicating that these cells do not differ in MHC-II interaction. Conclusions: Proliferating latently infected cells may be an important mecahism for HIV persistence and these cells should be included in studies of HIV persistence in HIV- infected individuals on ART. 376 Role of Dendritic Cell Polarization in the Induction of HIV-1 Latency Reversal Robbie B. Mailliard 1 ; Charles Rinaldo 1 ; Jan R. Kristoff 1 ; Margaret Carlson 1 ; Jennifer M. Zerbato 1 ; Nicolas Sluis-Cremer 1 ; Phalguni Gupta 2 1 Univ of Pittsburgh, Pittsburgh, PA, USA; 2 Univ of Pittsburgh, Grad Sch of PH, Pittsburgh, PA, USA Background: Dendritic cell (DC)-based strategies have been used to induce antigen specific CTL immunity in HIV during combination ART (cART), with reduction in viral load after cART interruption. Recent findings suggest DC also play a role in HIV-1 latency reversal. We have demonstrated that a DC-based therapeutic HIV vaccine increases residual viremia in cART-suppressed individuals after analytic treatment interruption. We therefore assessed the in vitro capacity of differentially polarized DC to induce HIV-1 latency reversal during cART. Methods: Monocyte derived immature DC (iDC) were generated with IL-4 and GM-CSF for 5 days. The iDC were then exposed for 2 days to a combination of IFN-γ, TNF-α, IL-1β, IFN-α and Poly(I:C) to generate high IL-12p70 producing, type-1 polarized DC (DC1), or TNF-α, IL-1β, IL-6 and PGE 2 to generate IL-12p70 deficient DC (DC2). Two models of HIV-1 latency reversal were tested: 1) Total resting CD4 + (rCD4) T cells from HIV-1 uninfected donors were treated with CCL19, infected with HIV-1 xxLAI , exposed to low-dose IL-2 with Efavirenz to establish latency, and subsequently stimulated with DC in the presence or absence of superantigen staphylococcal enterotoxin B (SEB). 2) DC derived from HIV-1 infected individuals on cART (viral RNA <20 copies/mL plasma) were co-cultured with autologous rCD4 T cells, in the absence or presence of Efavirenz and/or SEB. Cellular and culture supernatant viral RNA were quantified by qRT-PCR. Results: All antigen expressing DC resulted in the propagation of replication competent virus from rCD4 T cells from HIV-1 infected participants on cART. However, DC1 consistently induced significantly greater levels of replication of HIV-1 RNA in rCD4 T cells than either iDC or DC2 in both models of HIV-1 latency reversal. Importantly, the greatest amounts of latency reversal were mediated by antigen expressing DC1. Conclusions: While they are known to have a strong capacity to produce IL-12p70 and drive antigen specific CTL responses in vitro, this study demonstrates that DC1 are also superior to iDC and DC2 as a potential HIV-1 latency reversing tool. Although the mechanisms of this effect have yet to be elucidated, the enhanced ability of DC1 to drive HIV-1 latency reversal was antigen dependent. Information gathered from these studies will be used in the development of novel DC1-based vaccine strategies to facilitate both the “kick” and “kill” of the HIV-1 reservoir. 377 Naïve CD4+ T Cells Harbor a Large Inducible Reservoir of Latent HIV-1 Jennifer Zerbato ; Deborah McMahon; Michele D. Sobolewski; JohnW. Mellors; Nicolas Sluis-Cremer Univ of Pittsburgh, Pittsburgh, PA, USA Background: The latent viral reservoir in resting CD4+ T cells represents a major barrier to eradicating HIV-1 infection. Central memory (T CM ) and transitional memory CD4+ T cells are thought to constitute the major reservoirs of latent HIV-1 because the frequency of infected cells, as assessed by total HIV-1 DNA, is greater compared to the other resting CD4+ T cell subsets. By contrast, HIV-1 DNA is detected less frequently in naïve (T N ) CD4+ T cells, and consequently there has been little emphasis on studying the establishment and reversal of viral latency in these cells. Here, we compared ex vivo production of HIV-1 from T N and T CM cells after exposure to latency reversing agents (LRAs). Methods: Resting CD4+ T N and T CM cells were purified using antibody-coated beads from PBMCs of HIV-1-infected individuals obtained via leukapheresis following written consent who had been on suppressive ART for ≥ 5 years. Total HIV-1 DNA was measured by quantitative real-time PCR. Reversal of latency was assessed by measuring virion- associated HIV-1 RNA, using a single copy assay, following treatment with a panel of LRAs (anti-CD3/CD28 antibodies, PHA + IL-2, PMA + ionomycin, prostratin, panobinostat or romidepsin). Infectious virus production was quantified by the quantitative viral outgrowth assay (Q-VOA). Results: Levels of total HIV-1 DNA were higher in CD4+ T CM cells (mean: 2,215 copies/10 6 cells; range: 723-4,533) compared to T N cells (mean: 735 copies/10 6 cells; range: 181-1,023), although this difference was not significant (p=0.09). Following exposure to anti-CD3/CD28 antibodies, virion-associated HIV-1 RNA levels were similar between T CM cells (mean: 19,299 copies/mL; range: 2,260-71,350) and T N cells (mean: 22,866 copies/mL; range: 0-57,663). In cells from 3 of the 5 donors, the virion-associated HIV-1 RNA levels produced were higher for T N compared to T CM cells, independent of the LRA used. Replication-competent virus was recovered from both T N and T CM CD4+ T cells by Q-VOA. Conclusions: Although the frequency of HIV-1 infection is lower in CD4+ T N compared to T CM cells purified from individuals on long-term suppressive ART, as much, if not more, virus is produced from T N cells after exposure to LRAs. This finding shows that quantifying HIV-1 DNA alone may not be predictive of the size of the inducible latent reservoir in different CD4+ T cell subsets and that greater attention should be given to the reservoir of HIV-1 in T N cells.
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