Natural Killer Cell Receptors and Ligands Are Associated With Markers of HIV-1 Persistence in Chronically Infected ART Suppressed Patients.

The latent HIV-1 reservoir represents a major barrier to achieving a long-term antiretroviral therapy (ART)-free remission or cure for HIV-1. Natural Killer (NK) cells are innate immune cells that play a critical role in controlling viral infections and have been shown to be involved in preventing HIV-1 infection and, in those who are infected, delaying time to progression to AIDS. However, their role in limiting HIV-1 persistence on long term ART is still uncharacterized. To identify associations between markers of HIV-1 persistence and the NK cell receptor-ligand repertoire, we used twin mass cytometry panels to characterize the peripheral blood NK receptor-ligand repertoire in individuals with long-term antiretroviral suppression enrolled in the AIDS Clinical Trial Group A5321 study. At the time of testing, participants had been on ART for a median of 7 years, with virological suppression <50 copies/mL since at most 48 weeks on ART. We found that the NK cell receptor and ligand repertoires did not change across three longitudinal samples over one year-a median of 25 weeks and 50 weeks after the initial sampling. To determine the features of the receptor-ligand repertoire that associate with markers of HIV-1 persistence, we performed a LASSO normalized regression. This analysis revealed that the NK cell ligands CD58, HLA-B, and CRACC, as well as the killer cell immunoglobulin-like receptors (KIRs) KIR2DL1, KIR2DL3, and KIR2DS4 were robustly predictive of markers of HIV-1 persistence, as measured by total HIV-1 cell-associated DNA, HIV-1 cell-associated RNA, and single copy HIV-RNA assays. To characterize the roles of cell populations defined by multiple markers, we augmented the LASSO analysis with FlowSOM clustering. This analysis found that a less mature NK cell phenotype (CD16+CD56dimCD57-LILRB1-NKG2C-) was associated with lower HIV-1 cell associated DNA. Finally, we found that surface expression of HLA-Bw6 measured by CyTOF was associated with lower HIV-1 persistence. Genetic analysis revealed that this was driven by lower HIV-1 persistence in HLA-Bw4/6 heterozygotes. These findings suggest that there may be a role for NK cells in controlling HIV-1 persistence in individuals on long-term ART, which must be corroborated by future studies.

Generation and preclinical characterization of an NKp80-Fc fusion protein for redirected cytolysis of natural killer (NK) cells against leukemia.

The capacity of natural killer (NK) cells to mediate Fc receptor-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), largely contributes to their clinical application. Given that activation-induced C-type lectin (AICL), an identified ligand for the NK-activating receptor NKp80, is frequently highly expressed on leukemia cells, the lack of therapeutic AICL-specific antibodies limits clinical application. Here we explore a strategy to reinforce NK anti-leukemia reactivity by combining targeting AICL-expressing leukemia cells with the induction of NK cell ADCC using NKp80-Fc fusion proteins. The NKp80-Fc fusion protein we generated bound specifically to leukemia cells in an AICL-specific manner. Cell binding assays between NK and leukemia cells showed that NKp80-Fc significantly increased NK target cell conjugation. In functional analyses, treatment with NKp80-Fc clearly induced the ADCC effect of NK cells. NKp80-Fc not only promoted NK-mediated leukemia cell apoptosis in the early stage of cell conjugation but also enhanced NK cell degranulation and cytotoxicity activity in the late stage. The bifunctional NKp80-Fc could redirect NK cells toward leukemia cells and triggered NK cell killing in vitro. Moreover, NKp80-Fc enhanced the lysis of NK cells against tumors in leukemia xenograft non-obese diabetic/severe combined immunodeficiency mice. Taken together, our results demonstrate that NKp80-Fc potently amplifies NK cell anti-leukemia effects in vitro and in vivo through induction of the NK cell ADCC effect. This method could potentially be useful for molecular targeted therapy, and the fusion proteins may be a promising drug for immunotherapy of leukemia.

Direct and indirect antitumor effects by human peripheral blood lymphocytes expressing both chimeric immune receptor and interleukin-2 in ovarian cancer xenograft model.

Human peripheral blood lymphocytes (PBLs) electroporated with RNA encoding anti-Her-2/neu-specific chimeric immune receptor (CIR) have been reported to elicit potent immune responses against SKOV3 tumors in a nude mouse model. However, CIR-electroporated PBL (CIR-PBL) did not proliferate, and the cell number rapidly decreased in the absence of exogenous interleukin-2 (IL-2). In this study, PBLs electroporated with both CIR and IL-2 RNA (CIR/IL-2-PBL) were studied to determine whether antitumor effects could be improved by adoptive immunotherapy. CIR and IL-2 were expressed in CIR/IL-2-PBL at levels similar to PBLs electroporated, with IL-2 RNA (IL-2-PBL) or CIR-PBL. Transfer of IL-2 RNA induced proliferation and prolonged survival of PBLs in vitro. In a xenograft model, both IL-2-PBL and CIR/IL-2-PBL showed significantly higher antitumor effects than CIR-PBL. The number of tumor-infiltrating natural killer (NK) cells was significantly increased in IL-2-PBL and CIR/IL-2-PBL. After NK cell depletion, IL-2-PBL showed significantly lower antitumor effects than CIR/IL-2-PBL. These results suggest that transfer of IL-2 RNA to CIR-PBL can promote NK cell infiltration of tumors and prolong survival of infused PBLs in vivo. RNA electroporated PBLs may represent efficient tools for delivery of functional molecules to tumors by multiple gene transfer.