CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

282 FUNCTIONAL MECHANISMS OF MEMORY-LIKE NK CELLS IN SIV-INFECTED MACAQUES Spandan Shah 1 , Cordelia Manickam 1 , Daniel Ram 1 , R. Keith Reeves 2 1 Beth Israel Deaconess Medical Center, Boston, MA, USA, 2 Harvard University, Cambridge, MA, USA Background: Burgeoning evidence indicates a broader functional repertoire for NK cells beyond innate immunity including memory and other memory-like functions. One recent example is memory-like NK cells identified by lack of the FcR intracellular γ-signaling chain (FcRΔg-NK cells) which still require antibody to grant antigen-specificity, but are pre-sensitized and capable of rapid mobilization and more robust responses against viral antigens. Interestingly, FcRΔg-NK cells are initially expanded by CMV infection as part of innate-priming but execute memory-like killing against other pathogens through incompletely understood mechanisms. Methods: Fifty-three rhesus macaques were used: twenty-one specific pathogen-free, rhCMV–; ten rhCMV+ but otherwise experimentally naïve; and twenty-two chronically SIVmac-infected macaques. NK cell analyses were performed using polychromatic, functional, and phospho-flow cytometry assays. Results: FcRΔg-NK cells were systemically distributed in mucosal and secondary lymphoid organs, but increased two- and four-fold in CMV+ and HIV/SIV-infected individuals. FcRΔg-NK cells displayed little difference in binding affinity to virus-antibody immunocomplexes compared to traditional NK cells, but exhibited two-fold more robust IFN-γ secretion and cytotoxicity, suggesting disparate signaling or activation could account for improved function. To that end, FcRΔg-NK cells showed significantly reduced expression of Helios and Eomes and clustered independently from traditional NK cells in multidimensional t-SNE. The γ-chain adaptor, Syk, was reduced or inactively dephosphorylated in FcRΔg-NK cells, but the expression of active ζ-chain, phosphorylated by increased adaptor Zap70, was significantly upregulated, suggesting these cells may exploit the ζ-chain/Zap70 pathway in the absence of γ-chain/Syk to achieve greater functional potency. Conclusion: Collectively, our work presents the first description of a combinatorial mechanism of innate-priming and alternative signaling cascade to explain the functional potency of memory-like NK cells. This mechanism could explain, at least in part, the improved functional potency of NK cell- mediated ADCC in rhCMV+ animals and impaired functions observed in chronic HIV/SIV infections. Future studies targeted at harnessing these pathways could open up newmodalities for vaccine and curative therapy. 283 IFN-ALPHA ENHANCES NK CELL AND CD8+ T CELL MEDIATED SUPPRESSION OF HIV REPLICATION Abena Kwaa , Joel Blankson Johns Hopkins University, Baltimore, MD, USA Background: Current shock and kill strategies have shown evidence of blips of viremia but no decrease in the size of the latent reservoir. One possible explanation for this is that the immune system is incapable of killing infected cells following the reversal of latency. Furthermore, some studies have suggested that certain latency reversing agents may inhibit CD8+ T, and NK, cell responses. In this study, we asked whether pulses of IFN-alpha could improve the function of NK and CD8+ T cells, in elite suppressors (ES) and chronic progressors (CPs). Methods: NK or CD8+ T cells were isolated from 10 ES PBMCs, pulsed for 6hrs with varying concentrations of IFN-alpha, washed, then co-cultured with autologous CD4+ T cells infected with GFP expressing pseudotyped virus. Viral suppression (determined by reduction of GFP expression) was quantified by flow cytometry at 72 hours. To bypass our inability to perform this suppression assay in CPs due to residual antiretroviral drugs in their CD4+ T cells, we employed a surrogate assay. Briefly, NK and CD8+ T cells were isolated from 9 CP PBMCs, treated with IFN-alpha as above, and then stimulated with K562 cells or anti-CD3/28 antibodies respectively. The effector function of the cells was then assessed by CD107a, MIP-1-beta, IFN-gamma, TNF-alpha and IL-2 production. Results: IFN-alpha treatment enhanced the HIV suppressive capacity of ES NK cells (0.012≤ p ≤0.043) and CD8+ T cells (0.007≤ p ≤0.035). In the surrogate assays, IFN-alpha treatments resulted in significant increases in CD107a (0.0001≤ p ≤0.0006), MIP-1-beta (0.0003≤ p ≤0.0032) and IFN-gamma (0.0003≤ p ≤0.0073) expression by CP NK cells. Furthermore, CP CD8+ T cells produced significantly more CD107a (0.0005≤ p ≤0.0022), MIP-1-beta (0.0001≤

(p=0.033), MM (p<0.001), and MMP (p=0.024). The MM of the tetramer+ cells was also found to negatively correlate with their capacity to proliferate after stimulation (r=-0.681, p=0.013), even after adjustment for %PD-1+ or %CD127+ tetramer+ CD8 T cells (p≤0.025) or for clinical group (p=0.046). Conclusion: These findings suggest that PD-1+ HIV-specific cells have a state of metabolic stress, which is associated with poor proliferative capacity, that may be attenuated by CD127 expression. While it remains unclear if metabolic stress is a cause or consequence of PD-1 or CD127 expression, these findings highlight a potential role of metabolic stress in HIV-specific CD8 T cell exhaustion.

Poster Abstracts

281 S100A14, NOVEL NK CROSS TALK PROTEIN FOR MONOCYTE-NK ACTIVATION IN HESN-IDU SUBJECTS

Krystal Colon-Rivera 1 , Costin Tomescu 1 , David Metzger 2 , David W. Speicher 1 , Luis Montaner 1 1 Wistar Institute, Philadelphia, PA, USA, 2 University of Pennsylvania, Philadelphia, PA, USA Background: High-risk HIV-exposed uninfected individuals that share needles (HESN-IDU) have increased NK cell activation/function when compared to no-risk no-drug user controls and to low-risk no-sharing IV-drug users (NS-IDU). Using proteomic analysis of NK cells from HESN-IDU and controls, it was demonstrated that HESN-IDU have an increased expression of S100A14 and other members of the S100 family, along with other Interferon-induced proteins. These findings lead to hypothesize that S100A14 may be part of an immune mechanism of resistance in HESN-IDU. Methods: Plasma levels of S100A14 in HESN-IDU were measured using ELISA. Recombinant S100A14 was added to isolated PBMCs obtained from normal donors. Lysates were collected after 18hrs stimulation with S100A14. We measured CD69 to assess NK activation. To measure monocytes activation, PBMCS were stimulated for 5hrs then TNF-alpha was measured using flow cytometry. NK cells and monocytes were isolated by negative selection using magnetic beads. For ensuring the highest purity, the NKs enriched preparation was further sorted for CD56+ cells. Expression of MX1 in PBMCs was measured using western blot. TAK-242, a TLR4 inhibitor, was used to determine the signaling pathway of S100A14 in monocytes. Results: Increased levels of S100A14 were found in the plasma of HESN-IDU (n=15) in comparison with controls (n=10; p<0.01) and NS-IDU subjects (n=15; p<0.05). When added in vitro, S100A14 caused increased expression of CD69 in NKs (n=9; p<0.01) and increased expression of TNF-alpha in monocytes (n= 5; p<0.01). S100A14 induced the activation of isolated monocytes but not isolated NKs, but when in co-culture with monocytes, NKs activation was induced. S100A14 did not increase the expression of MX1, an IFN-induced gene, in PBMCs. S100A14 effects were inhibited when TAK-242 was added to PBMCs (n=7; p<0.001). Conclusion: Identified as over-expressed in NK in HESN-IDUs, S100A14 is now identified as increased in HESN-IDU sera as well as able to induce TLR-4- dependent monocyte responses that modulate NK cell activation. An increase of NK-derived S100A14 is a novel positive feedback protein identified in HESN-IDUs with direct monocyte cross talk potential to further re-engage NK immune activation in HESN-IDU subjects.

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CROI 2018