CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

1 McGill University, Montreal, QC, Canada, 2 Université de Montréal, Montreal, QC, Canada, 3 Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 4 Research Institute of McGill University Health Centre, Montreal, QC, Canada Background: KIR3DS1 (3DS1) is an activating NK cell receptor (NKR) implicated in several viral infections, cancer outcomes and autoimmune diseases. In the context of HIV, co-carriage of 3DS1 and HLA-Bw4*80I alleles is associated with slower time to AIDS while 3DS1 homozygotes (hmz) have a reduced risk of HIV infection. The ligand for 3DS1 is HLA-F, a non-classical MHC-1b antigen expressed on infected CD4+ T cells (iCD4). Methods: Purified NK cells from 5 3DS1hmz were stimulated with autologous iCD4 cells in duplicate. The frequency of 3DS1+/-CD3-CD56dim NK cells positive for IFN-, CD107a or CCL4 function and the sum of these functions was assessed using gating strategies that included and excluded NK cell subsets co-expressing other NKRs that may contribute to 3DS1+ NK cell function. We assessed the effect of blocking the interaction of 3DS1 and HLA-F on the function of 3DS1+ NK cells by using KIR3DS1-Fc chimeric protein and the anti-HLA-F specific mAb (3D11). Results: We confirmed that HLA-F is expressed on iCD4 cells. HIV infection partially downmodulates HLA-F expression, though sufficient HLA-F remains on iCD4 to bind 3DS1+NK cells and activate them. The frequency of cells exhibiting the sum of all functions tested, total CCL4 and IFN-γ secretion and total CD107a expression was higher among 3DS1+ than 3DS1- NK cells (p<0.002, for all, Wilcoxon matched-pairs test). After excluding NK cells co-expressing other NKRs such as KIR2DL1/L2/L3, KIR3DL2, KIR2DS1/S2/S3/S5, NKG2A and ILT-2, a higher frequency of 3DS1+ than 3DS1- NK cells were positive for total function, total CCL4, total IFN-γ and total CD107a (p<0.002, for all, Wilcoxon matched-pairs test). Blocking 3DS1-HLA-F interactions reduced the frequency of exclusively gated functional 3DS1+ NK cells to levels close to those observed for unstimulated NK cells. Conclusion: The interaction of 3DS1 on NK cells with HLA-F on iCD4 cells activates a higher frequency of 3DS1+NK cells. The exclusive gating strategy confirmed that the higher frequency of functional 3DS1+ NK cells was not due to co-expression of other NKRs. HLA-F levels on iCD4 are sufficient to activate 3DS1+ NK cells for anti-viral functions such as CCL4, which blocks HIV infection of new target cells, IFN-γ secretion, an important antiviral cytokine and CD107a expression, a marker of NK cell degranulation. These findings provide a mechanism explaining the association of the 3DS1hmz genotype with HIV protection. 325 IMPACT OF A NOVEL NATURAL KILLER CELL SUBSET ON IMMUNE RECONSTITUTION Jaewon Lee , Robert C. Güerri-Fernández, Richard Pollard, David M. Asmuth, Sungjin Kim University of California Davis, Davis, CA, USA Background: We have identified a novel subset of NK cells, called ‘g-NK cells’, which display adaptive immune features, including clonal-like expansion and long-term persistence. The presence of g-NK cells is associated with previous infection by cytomegalovirus and have enhanced response to broad range of viral-infected cells in the presence of virus-specific antibodies. We hypothesize that g-NK cells contribute to low CD4 counts in HIV patients and CD4 recovery during ART. Methods: In a cohort of 18 MSM chronically infected HIV patients naïve to treatment before and 12 months after starting a PI-based cART, the presence of g-NK cells, as well as their frequencies and phenotypic characteristics, were measured by flow cytometry after intracellular staining of FcR-gamma signaling protein following cell surface marker staining. 17 HIV-negative control underwent identical procedures. Plasma biomarkers of inflammation were measured by ELISA and cytokine production by g-NK cells and conventional NK cells after stimulation with HIV-infected cells in the presence or absence of HIV-seropositive plasma. Results: We observed that (1) HIV patients possessed higher frequencies of g-NK cells compared to HIV-negative control groups [39.9% and 10.33%, p=0.0320], (2) HIV patients with readily detectable g-NK cells show a trend toward lower CD4+ T cell count before [p<0.08] and 1 yr after cART [p<0.01]. g-NK cells did not change levels before and after the treatment, and (3) compared to conventional NK cells, g-NK cells produced greater amount of IFN- and TNF- in response to HIV-infected cells in the presence of HIV-seropositive plasma.

323 HIV RESERVOIR SIZE IS CORRELATED TO NK-MEDIATED KILLING OF EFFECTOR MEMORY CD4 CELLS Antonio Astorga Gamaza 1 , Judith Grau-Expósito 1 , Laura Luque-Ballesteros 1 , Ariadna Torrella 2 , Bibiana Planas 2 , Rosa Badía 2 , Esteban Ribera 2 , Joaquin Burgos 2 , Jordi Navarro 2 , Adrià Curran 2 , Vicenç Falcó 2 , María J. Buzón 1 1 Vall d’Hebron Research Institute, Barcelona, Spain, 2 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain Background: The identification of immune control mechanisms of the viral reservoir might help to develop new strategies to cure HIV. Antibody-dependent cell-mediated cytotoxicity (ADCC), largely mediated by natural killer (NK) cells, has been reported highly relevant to control HIV. However, the role of ADCC-NK at controlling the cells that compose the viral reservoir is currently unknown. Methods: The intrinsic susceptibility of Naïve (NA), Stem Cell Memory (SCM), Central Memory (CM), Effector Memory (EM) and CD20dim CD4 T cells, to ADCC NK-mediated killing was measured by a novel flow cytometry-based assay. n=10 ART-suppressed and 5 elite controllers (EC) patients were included. Isolated CD4 T cells were stained with the markers eF670 and PKH67 and coated with the gp120Bal protein. Cells were incubated with plasma from an HIV positive patient and autologous NK cells at ratio 1:1 for 3 hours. Then, cells were stained with CD3, CCR7, CD45RO, CD95, CD20 and HLA DR antibodies. NK- mediated killing was calculated as the disappearance of cells measured with the addition of flow cytometry particles. Total HIV-DNA and HIV-RNA was quantified by qPCR. Results: Results from all analyzed patients showed that each subset had a different susceptibility to killing, being CD20dim>CM>NA>EM>SCMmore prone to be killed (ANOVA Friedman test p<0.0001). Moreover, whereas no differences in the killing of the whole CD3 population were observed between cohorts (Mann-Whitney test, p=0.5122), EC showed higher potency to kill CD20dim and EM (median 61.6, 52.0, 48.0, 33.6 and 33.4% for CD20dim, EM, CM, NA, SCM, respectively) (ANOVA Friedman test p=0.0081), while ART-suppressed patients were more efficient at killing CM and CD20dim cells (median 48.5, 38.7 ,37.6, 35.7 and 28.9% for CM, CD20dim, NA, EM, SCM, respectively) (ANOVA Friedman test p=0.0005). A more efficient killing of CD20dim cells was detected in EC compared to ART-suppressed patients (Mann-Whitney test, p=0.0383). Importantly, an inverse correlation between the capacity of NK cells to kill the EM subset and the viral reservoir was observed (rho=-0.6000 p=0.0261 for viral DNA and rho=-0.8857 p=0.0333 for viral RNA). Conclusion: The susceptibility of different CD4 T subsets to ADCC-NK killing differs. The ADCC activity against EM cells, one of the most HIV-transcriptionally active cell subsets, was highly correlated to the size of the persistent HIV- reservoir. Inducing the specific killing of EM cells might significantly help to diminish the persistent reservoir. 324 WITHDRAWN LIGATION OF KIR3DS1 BY HLA-F ON ICD4 ACTIVATES PRIMARY NK CELLS FOR ANTI-HIV ACTIVITY Zahra Kiani 1 , Julie Bruneau 2 , Daniel E. Geraghty 3 , Nicole Bernard 4

Poster Abstracts

CROI 2019 118