Antigen-specific memory NK cell responses against HIV and influenza use the NKG2/HLA-E axis.

Multiple studies have broadened the roles of natural killer (NK) cells functioning as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including HIV-1 and influenza. However, the mechanisms underlying antigen specificity remain unknown. Here, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism largely dependent on the activating CD94/NKG2C receptor and its ligand HLA-E. We validated the permanent acquisition of antigen specificity by individual memory NK cells by single-cell cloning. We identified elevated expression of KLRG1, α4ß7, and NKG2C as biomarkers of antigen-specific NK cell memory through complex immunophenotyping. Last, we uncovered individual HLA-E-restricted peptides that may constitute the dominant NK cell response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms contributing to antigen-specific memory NK cell responses and suggest that they could be potentially targeted therapeutically for vaccines or other therapeutic interventions.

Nanodepots Encapsulating a Latency Reversing Agent and Broadly Neutralizing Antibody Enhance Natural Killer Cell Cytotoxicity Against an in vitro Model of Latent HIV.

Purpose: Current antiretroviral therapies (ART) for human immunodeficiency virus (HIV) are not curative, as the virus persists in latent reservoirs, requiring lifelong adherence to ART and increasing the risk of co-morbidities. "Shock and kill" approaches to reactivate HIV from latent reservoirs followed by administration of anti-HIV drugs represent a promising strategy for eradicating latent HIV. To achieve effective shock and kill, we describe a strategy to eradicate the HIV reservoir that combines latency reversing agents (LRAs), broadly neutralizing antibodies (bnAbs), and natural killer (NK) cells. This strategy utilizes a polymer nanodepot (ND) that co-encapsulates the LRA and bnAb to reactivate latent infection and elicit enhanced cytotoxicity from co-administered NK cells. Methods: Poly(lactic-co-glycolic acid) (PLGA) NDs were synthesized using the nanoprecipitation method to co-encapsulate an LRA (TNF-α) and a bnAb (3BNC117) (TNF-α-3BNC117-NDs). ACH-2 cells were used as a cellular model of latent HIV infection. An NK92 subline, genetically modified to constitutively express the Fc receptor CD16, was administered to ACH-2 cells in combination with TNF-α-3BNC117-NDs. ACH-2 cell death and extracellular p24 were measured via flow cytometry and ELISA, respectively. Results: Stable PLGA NDs co-encapsulated TNF-α and 3BNC117 with high efficiencies and released these agents in physiological conditions. NK92 phenotype remained similar in the presence of TNF-α-3BNC117-NDs. TNF-α released from NDs efficiently reactivated HIV in ACH-2 cells, as measured by a 3.0-fold increase in the frequency of intracellular p24 positive cells. Released 3BNC117 neutralized and bound reactivated virus, targeting 57.5% of total ACH-2 cells. Critically, TNF-α-3BNC117-NDs significantly enhanced NK92 cell-mediated killing of ACH-2 cells (1.9-fold) and reduced extracellular levels of p24 to baseline. Conclusion: These findings suggest the therapeutic potential of our novel ND-based tripartite strategy to reactivate HIV from latently infected cells, generate an HIV-specific site for bnAb binding, and enhance the killing of reactivated HIV-infected target cells by NK92 cells.

NK Cells Contribute to the Immune Risk Profile in Kidney Transplant Candidates.

Background: A previously proposed immune risk profile (IRP), based on T cell phenotype and CMV serotype, is associated with mortality in the elderly and increased infections post-kidney transplant. To evaluate if NK cells contribute to the IRP and if the IRP can be predicted by a clinical T cell functional assays, we conducted a cross sectional study in renal transplant candidates to determine the incidence of IRP and its association with specific NK cell characteristics and ImmuKnow® value. Material and Methods: Sixty five subjects were enrolled in 5 cohorts designated by age and dialysis status. We determined T and NK cell phenotypes by flow cytometry and analyzed multiple factors contributing to IRP. Results: We identified 14 IRP+ [CMV seropositivity and CD4/CD8 ratio < 1 or being in the highest quintile of CD8+ senescent (28CD-/CD57+) T cells] individuals equally divided amongst the cohorts. Multivariable linear regression revealed a distinct IRP+ group. Age and dialysis status did not predict immune senescence in kidney transplant candidates. NK cell features alone could discriminate IRP- and IRP+ patients, suggesting that NK cells significantly contribute to the overall immune status in kidney transplant candidates and that a combined T and NK cell phenotyping can provide a more detailed IRP definition. ImmuKnow® value was negatively correlated to age and significantly lower in IRP+ patients and predicts IRP when used alone or in combination with NK cell features. Conclusion: NK cells contribute to overall immune senescence in kidney transplant candidates.

Brief Report: Decreased JC Virus-Specific Antibody-Dependent Cellular Cytotoxicity in HIV-Seropositive PML Survivors.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is an often fatal disease caused by JC virus (JCV) in severely immunocompromised patients, including HIV patients. Development of therapeutics to prevent or treat PML is an urgent medical need. While JCV-specific T cells are crucial to control JCV and recover from PML, the role played by antibodies remains unclear. Anti-JCV antibodies, including potent neutralizing antibodies, can be detected in most infected adults, yet in PML patients, JCV seems to escape from neutralization. Whether antibodies can contribute to JCV control by eliciting Fc-mediated effector functions activity has not been evaluated. METHODS: We measured the capacity of plasma anti-JCV VP1 antibodies to recruit Fc receptor (FcR)-bearing effector cell functions in 28 HIV patients, comparing subjects without PML with PML survivors (PML S) who were alive 1 year after disease onset or PML progressors (PML P) who succumbed within the first year. Antibody titers against JCV VP1 and HIV gp140 trimer were determined by end-point titer dilution ELISA. FcR-mediated natural killer cell degranulation and IFN-γ production were measured as surrogate for in vitro antibody-dependent cellular cytotoxicity (ADCC). RESULTS: PML S had higher JCV antibody titers than PML P and patients without PML. However, anti-JCV antibodies had a higher ability to functionally engage FcR in PML P than PML S. Antibody titers and ADCC activity did not vary over time in PML S. Anti-HIV antibody titers and ADCC activity were similar among groups. CONCLUSIONS: The ability of anti-JCV antibodies to stimulate FcR-bearing effector cell activity might contribute to the outcome of PML. Further studies are warranted to define Fc-mediated functions of anti-JCV antibodies and evaluate whether ADCC can contain JCV replication.

Semaphorin 7A modulates cytokine-induced memory-like responses by human natural killer cells.

Cytokine-induced memory-like (CIML) NK cells are endowed with the capacity to mediate enhanced effector functions upon cytokine or activating receptor restimulation for several weeks following short-term preactivation with IL-12, IL-15, and IL-18. Promising results from a first-in-human clinical trial highlighted the clinical potential of CIML NK cells as adoptive immunotherapy for patients with hematologic malignancies. However, the mechanisms underlying CIML NK cell differentiation and increased functionality remain incompletely understood. Semaphorin 7A (SEMA7A) is a potent immunomodulator expressed in activated lymphocytes and myeloid cells. In this study, we show that SEMA7A is substantially upregulated on NK cells stimulated with cytokines, and specifically marks activated NK cells with a strong potential to release IFN-γ. In particular, preactivation of NK cells with IL-12+IL-15+IL-18 resulted in greater than tenfold upregulation of SEMA7A and enhanced expression of the ligand for SEMA7A, integrin-ß1, on CIML NK cells. Strikingly, preactivation in the presence of antibodies targeting SEMA7A lead to significantly decreased IFN-γ production following restimulation. These results imply a novel mechanism by which cytokine-enhanced SEMA7A/integrin-ß1 interaction promotes CIML NK cell differentiation and maintenance of increased functionality. Our data suggest that targeting SEMA7A/integrin-ß1 signaling might provide a novel immunotherapeutic approach to potentiate antitumor activity of CIML NK cells.

Human vascular progenitor cells derived from renal arteries are endothelial-like and assist in the repair of injured renal capillary networks.

Vascular progenitor cells show promise for the treatment of microvasculature endothelial injury. We investigated the function of renal artery progenitor cells derived from radical nephrectomy patients, in animal models of acute ischemic and hyperperfusion injuries. Present in human adventitia, CD34positive/CD105negative cells were clonal and expressed transcription factors Sox2/Oct4 as well as surface markers CXCR4 (CD184)/KDR(CD309) consistent with endothelial progenitor cells. Termed renal artery-derived vascular progenitor cells (RAPC), injected cells were associated with decreased serum creatinine after ischemia/reperfusion, reduced albuminuria after hyperperfusion, and improved blood flow in both models. A small population of RAPC integrated with the renal microvasculature following either experimental injury. At a cellular level, RAPC promoted local endothelial migration in co-culture. Profiling of RAPC microRNA identified high levels of miRNA 218; also found at high levels in exosomes isolated from RAPC conditioned media after cell contact for 24 hours. After hydrogen peroxide-induced endothelial injury, RAPC exosomes harbored Robo-1 transcript; a gene known to be regulated by mir218. Such exosomes enhanced endothelial cell migration in culture in the absence of RAPC. Thus, our work shows the feasibility of pre-emptive pro-angiogenic progenitor cell procurement from a targeted patient population and potential therapeutic use in the form of autologous cell transplantation.