NK Cell Activation In Response Research Articles

Abstract 4548: SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity

Abstract CD112R (PVRIG) is a recently identified inhibitory receptor of the PVR gene family, which includes TIGIT, CD96, and CD226, that is expressed on NK cells and T cells. CD112R suppresses immune cell activation through its association with the cell adhesion molecule CD112 (PVRL2), a ligand that it competes for with the activating receptor CD226. CD112 binding to CD112R induces downstream signaling via recruitment of SHP-1, SHP-2, and SHIP-1 phosphatases to the ITIM in the cytoplasmic tail, resulting in dampened effector cell activation. As CD112 is commonly upregulated on cancer cells and is also expressed on tumor infiltrating myeloid cells, it was hypothesized that disruption of the CD112:CD112R interaction would promote anti-tumor immune responses. Phenotypic screening of a panel of antibodies generated against CD112R led to the identification of SRF813, a high affinity, fully human CD112R antibody that blocks the interaction of CD112R with CD112. To explore the therapeutic potential of CD112R blockade, the ability of SRF813 to increase immune cell activation was evaluated in human peripheral blood mononuclear cells. In vitro assays showed that SRF813 treatment enhanced NK cell activation in response to multiple tumor cell lines. This activation was characterized by upregulation of several cell surface markers, including 4-1BB and ICAM-1, in addition to increased cytokine production and degranulation. The activity of SRF813 was highly dependent on the antibody isotype and Fc receptor engagement. Mechanistically, the enhanced immunologic responses observed in NK cells treated with SRF813 could be reversed in the presence of a CD226 blocking antibody, confirming the role of CD112R in regulating CD226 mediated signaling. Blockade of CD112R in mouse syngeneic tumor models using a murine surrogate of SRF813 demonstrated reduced tumor growth with an accompanying increase in TIL activation following treatment. In the CT-26 tumor model, anti-CD112R treatment resulted in complete tumor regression in a subset of treated mice. These mice rapidly rejected tumors upon re-challenge, signifying the development of an immunologic anti-tumor memory response in these animals. Additional depletion studies highlighted that the anti-tumor efficacy was dependent on both CD8 T cells and NK cells. Moreover, the combination of anti-CD112R with PD-1 blockade led to greater inhibition of tumor growth than either treatment alone. Collectively, these preclinical data demonstrated that CD112R is a negative regulator of immune responses and that CD112R blockade can potentiate anti-tumor responses in cancers that express CD112. Citation Format: Marisella Panduro, Roy M. Dornbrook, Kshama A. Doshi, Jing Hua, Jamie Strand, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill, James F. Mohan. SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4548.

Influenza Vaccination Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function.

Cytokine-induced memory-like (CIML) NK cells generated in response to proinflammatory cytokines in vitro and in vivo can also be generated by vaccination, exhibiting heightened responses to cytokine stimulation months after their initial induction. Our previous study demonstrated that in vitro human NK cell responses to inactivated influenza virus were also indirectly augmented by very low doses of IL-15, which increased induction of myeloid cell-derived cytokine secretion. These findings led us to hypothesize that IL-15 stimulation could reveal a similar effect for active influenza vaccination and influence CIML NK cell effector functions. In this study, 51 healthy adults were vaccinated with seasonal influenza vaccine, and PBMC were collected before and up to 30 d after vaccination. Myeloid and lymphoid cell cytokine secretion was measured after in vitro PBMC restimulation with low-dose IL-15, alone or in combination with inactivated H3N2 virus; the associated NK cell response was assessed by flow cytometry. PBMC collected 30 d postvaccination showed heightened cytokine production in response to IL-15 compared with PBMC collected at baseline; these responses were further enhanced when IL-15 was combined with H3N2. NK cell activation in response to IL-15 alone (CD25) and H3N2 plus IL-15 (CD25 and IFN-γ) was enhanced postvaccination. We also observed proliferation of less-differentiated NK cells with downregulation of cytokine receptors as early as 3 d after vaccination, suggesting cytokine stimulation in vivo. We conclude that vaccination-induced "training" of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response postvaccination.

FCGR3A and FCGR2A Genotypes Differentially Impact Allograft Rejection and Patients' Survival After Lung Transplant.

Fc gamma receptors (FcγRs) play a major role in the regulation of humoral immune responses. Single-nucleotide polymorphisms (SNPs) of FCGR2A and FCGR3A can impact the expression level, IgG affinity and function of the CD32 and CD16 FcγRs in response to their engagement by the Fc fragment of IgG. The CD16 isoform encoded by FCGR3A [158V/V] controls the intensity of antibody-dependent cytotoxic alloimmune responses of natural killer cells (NK) and has been identified as a susceptibility marker predisposing patients to cardiac allograft vasculopathy after heart transplant. This study aimed to investigate whether FCGR2A and FCGR3A polymorphisms can also be associated with the clinical outcome of lung transplant recipients (LTRs). The SNPs of FCGR2A ([131R/H], rs1801274) and FCGR3A ([158V/F], rs396991) were identified in 158 LTRs and 184 Controls (CTL). The corresponding distribution of genotypic and allelic combinations was analyzed for potential links with the development of circulating donor-specific anti-HLA alloantibodies (DSA) detected at months 1 and 3 after lung transplant (LTx), the occurrence of acute rejection (AR) and chronic lung allograft dysfunction (CLAD), and the overall survival of LTRs. The FCGR3A [158V/V] genotype was identified as an independent susceptibility factor associated with higher rates of AR during the first trimester after LTx (HR 4.8, p < 0.0001, 95% CI 2.37–9.61), but it could not be associated with the level of CD16- mediated NK cell activation in response to the LTR's DSA, whatever the MFI intensity and C1q binding profiles of the DSA evaluated. The FCGR2A [131R/R] genotype was associated with lower CLAD-free survival of LTRs, independently of the presence of DSA at 3 months (HR 1.8, p = 0.024, 95% CI 1.08–3.03). Our data indicate that FCGR SNPs differentially affect the clinical outcome of LTRs and may be of use to stratify patients at higher risk of experiencing graft rejection. Furthermore, these data suggest that in the LTx setting, specific mechanisms of humoral alloreactivity, which cannot be solely explained by the complement and CD16-mediated pathogenic effects of DSA, may be involved in the development of acute and chronic lung allograft rejection.

NK cell recognition of hematopoietic cells by SLAM-SAP families.

The signaling lymphocyte activation molecule (SLAM) family of receptors (SFRs) are ubiquitously expressed on immune cells, and they regulate multiple immune events by recruiting SH2 (Src homology 2) domain-containing SAP family adapters, including SAP and its homologs, Ewing's sarcoma-associated transcript 2 (EAT-2) and EAT-2 related transducer (ERT). In human patients with X-linked lymphoproliferative (XLP) disease, which is caused by SAP mutations, SFRs alternatively bind other inhibitory SH2 domain-containing molecules to suppress immune cell activation and development. NK cells express multiple SFRs and all SAP family adapters. In recent decades, SFRs have been found to be critical for enhancing NK cell activation in response to abnormal hematopoietic cells in SAP-family-intact NK cells; however, SFRs might suppress NK cell activation in SAP-family-deficient mice or patients with XLP1. In this paper, we review how these two distinct SFR signaling pathways orchestrate NK cell activation and inhibition and highlight the importance of SFR regulation of NK cell biology and their physiological status and pathological relevance in patients with XLP1.

Increased HLA-E Expression Correlates with Early Relapse in Multiple Myeloma

Increased HLA-E Expression Correlates with Early Relapse in Multiple Myeloma

Co-stimulation of the fc receptor and interleukin-12 receptor on human natural killer cells leads to increased expression of cd25

ABSTRACT Natural killer (NK) cells serve a critical role in the immune response against microbes and developing tumors. We have demonstrated that NK cells produce stimulatory cytokines (e.g., IFN-γ) in response to potent stimulation via immobilized IgG (to engage Fc receptors) and interleukin (IL)-12. CD25 is a component of the high-affinity IL-2R, which promotes NK cell activation in response to low doses of IL-2 such as those released by activated T cells. We hypothesized that stimulation of NK cells via IgG and IL-12 would enhance CD25 expression and promote NK cell anti-tumor activity in response to low-dose IL-2. It was confirmed that this dual stimulation strategy significantly enhanced NK cell CD25 expression compared to unstimulated cells or cells treated with IgG or IL-12 alone. Dual stimulated NK cells also were more responsive to low-dose IL-2. Dual stimulated NK cells subsequently treated with low-dose IL-2 (10 pg/mL) displayed enhanced intracellular signaling as indicated by increased pSTAT5 levels. IFN-γ production and cytotoxicity against K562 cells by NK cells stimulated with low-dose IL-2 was comparable to that of cells treated with high-dose IL-2 (10 ng/mL). Importantly, cells isolated from head and neck cancer patients receiving the mAb cetuximab and IL-12 on a clinical trial displayed increased CD25 expression following combination therapy compared to baseline. Altogether, these findings suggest that FcR and IL-12R co-stimulation induces expression of the high-affinity IL-2R and promotes NK cell anti-tumor activity.

IL-18-dependent NKG2D ligand upregulation on accessory cells is mediated by the PI3K/GSK-3 pathway.

NK cells are critical for the control of viral infections. Studies have shown that efficient NK cell activation in response to infection with VV in vivo requires multiple pathways, including the NKG2D pathway. We have recently shown that IL-18 is necessary for the activation of NK cells through upregulation of the NKG2D ligand Rae-1 on DCs upon VV infection. However, how IL-18R signaling on the accessory cells contributes to Rae-1 up-regulation remains to be defined. In this study, we found IL-18-mediated Rae-1 up-regulation in accessory cells, including macrophages and DCs, to be dependent on the MyD88-PI3K pathway. We further found that IL-18 signaling through PI3K led to inhibition of GSK-3, which we found to be a negative regulator of Rae-1. Finally, we demonstrated that in vivo inhibition of GSK-3 could restore Rae-1 up-regulation on IL18R-/- DCs and partially rescue NK-cell activation against VV, leading to improved viral control in IL-18R-/- mice. Our results showed that IL18-dependent Rae-1 up-regulation on accessory cells is mediated by the MyD88-PI3K-GSK3 pathway. These observations may provide important insights into the design of effective NK cell-based immunotherapies.

Expansion of Dysfunctional CD56-Negative NK Cells Is a Hallmark of NK Cell Activation in Ph+ Leukemia Patients Treated with Dasatinib

[Background] Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are key drugs for the treatment of Philadelphia chromosome-positive (Ph+) leukemia. Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) including NK cells in peripheral blood, which associates with a better prognosis. We previously showed that dasatinib expands CMV-associated highly differentiated NK cells in Ph+ leukemia patients through reactivation of CMV (Ishiyama, et al., Leukemia, 2016). NK cells consist of conventional CD56bright NK cells and CD56dim NK cells, and also rare CD56-negative (CD56neg) NK cells, which have been reported to increase in chronic viral infection such as HIV and HCV. Here, we show that CD56neg NK cells increase in CMV seropositive (CMV+) patients treated with dasatinib (DA), but not in those treated with other TKIs (OT). However, characteristics and clinical implications of CD56neg NK cells in CMV+DA patients remain unknown. Therefore, we sought to examine the phenotypic and functional properties of CD56neg NK cells in these patients.[Methods] We assessed NK cell subsets in 36 DA and 26 OT patients. NK cells were defined as lin-CD16+ or lin-CD56+ cells in peripheral blood. NK-cell marker expression and cytotoxic activity were compared between CD56neg NK cells and CD56dim NK cells in CMV+ DA patients with 5% or more of CD56neg NK cells. In vivo phosphorylation of intracellular signaling molecules in NK cells were evaluated by flow cytometry combined with phospho-specific antibodies (BD PhosflowTM).[Results] CD56neg NK cells exclusively increased in CMV+ DA group, compared to CMV- DA and CMV+ OT groups (median count: 20.9/μl, 1.1/μl, 1.5μl; p < 0.001. Median percentage: 4.5%, 0.8%, 0.4% of total NK cells; p < 0.001). CD56neg NK cell counts strongly correlated with total NK cell counts in CMV+ DA (r = 0.720, p < 0.001). CD56neg NK cells gradually increased over a period of one year after starting dasatinib. When we compared CD56neg NK cells and CD56dim NK cells in CMV+ DA, CD56neg NK cells showed decreased expression of activating NK cell markers including NKG2C, CD57, NKG2D, NKp30 and NKp46. In contrast, expression of PD-1 increased. CD56neg NK cells also showed lower rate of degranulation and IFN-γ production in functional assays. CMV+ DA patients with 4% or more of CD56neg NK cells significantly achieved deep molecular responses at 1 year after starting dasatinib than those with lower CD56neg NK cells (10/14, 3/14; p < 0.05). Phosflow analysis showed enhanced phosphorylation of ZAP70 in NK cells from CMV+ DA compared to CMV+ healthy controls (MFI/Isotype = 24.6 vs. 7.4, p < 0.01).[Discussion] CD56neg NK cells exclusively increased in CMV+ DA, and their increase persisted during dasatinib therapy. This suggests the similarity with the previous findings that CMV-associated differentiation in NK cells is enhanced during dasatinib therapy in CMV+ DA, which reflects the NK cell activation in response to CMV reactivation. In addition, CD56neg NK cells exhibited downregulation of activating receptors, upregulation of PD-1, and lower cytotoxic activity, indicating that these cells are the anergic and exhausted population as seen in chronic viral infection. These findings suggest that CD56neg NK cells accumulate owing to chronic stimulation by reactivated CMV during dasatinib therapy. ZAP70 is an immediate downstream tyrosine kinase of activating NK cell receptors, and is regulated by Src-family kinases, which is potentially inhibited by the kinase inhibitor property of dasatinib. Intriguingly, phosphorylation of ZAP70 in CD56dim NK cells was enhanced in CMV+ DA, although they had taken dasatinib a few hours before collecting blood samples. This finding suggests that strong activation of NK cells by CMV reactivation in CMV+ DA may overcome the direct suppressive effect of dasatinib on NK cell activation.[Conclusion] The accumulation of CD56neg NK cells is a consequence of NK cell activation specifically observed in dasatinib-treated patients. The NK cell activation is likely to be a response to the CMV reactivation induced by dasatinib treatment. Assessing CD56neg NK cells in peripheral blood could be a practical clinical indicator for the immunomodulatory effect of dasatinib, which significantly affects the prognosis. DisclosuresTakaori-Kondo:Chugai Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Merck Sharp and Dohme: Speakers Bureau; Pfizer: Research Funding; Toyama Chemical: Research Funding; Takeda Pharmaceutical: Research Funding; Kyowa Kirin: Research Funding; Eisai: Research Funding; Cognano: Research Funding; Janssen Pharmaceuticals: Speakers Bureau; Shionogi: Research Funding; Mochida Pharmaceutical: Research Funding; Alexion Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Speakers Bureau.

Functional anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies in patients with systemic lupus erythematosus.

Recently we serendipitously identified a patient with systemic lupus erythematosus (SLE) who was positive for autoantibodies to CD94/natural killer receptor group 2A (NKG2A). The present study was undertaken to investigate the occurrence and function of autoantibodies targeting lectin-like NK cell receptors in SLE. Sera from 203 SLE patients and 90 healthy individuals were analyzed, by flow cytometry, for Ig binding to Ba/F3 cells transfected with CD94/NKG2A, CD94/NKG2C, or NKG2D. Autoantibodies identified were characterized with regard to interference with HLA-E binding, effect on NK cell activation in response to HLA-E-transfected K562 cells, and capacity to facilitate antibody-dependent cell-mediated cytotoxicity (ADCC). Levels of autoantibodies were determined in longitudinally sampled sera, and correlations with disease activity (SLE Disease Activity Index 2000) and severity (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index) were investigated. Anti-CD94/NKG2A autoantibodies were identified in 7 SLE patients. The autoantibodies from 6 patients inhibited binding of HLA-E to CD94/NKG2A, whereas those from the seventh patient augmented this binding. Autoantibodies from 2 patients also reacted with the activating receptor CD94/NKG2C, with inhibition of the binding of HLA-E to CD94/NKG2C observed in 1 case and enhancement of this binding in the other. None of the sera contained anti-NKG2D autoantibodies. The levels of anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies correlated with disease activity and with a more severe SLE phenotype. Mechanistically, anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies both interfered with HLA-E-mediated regulation of NK cell activation and facilitated the elimination of target cells expressing CD94/NKG2A or CD94/NKG2C through ADCC. Anti-CD94/NKG2A and anti-CD94/NKG2C autoantibodies occur in a subset of patients with clinically active SLE. Given their capacity to deplete certain NK cell subsets and interfere with particular NK cell function, such autoantibodies may promote the pathogenesis of SLE.

NK cell-extrinsic IL-18 signaling is required for efficient NK-cell activation by vaccinia virus.

NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR-dependent and -independent pathways, as well as the NKG2D activating receptor that recognizes host stress-induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study using C57BL/6 mice, we first showed that IL-18 is critical for NK-cell activation and viral clearance. We then demonstrated that IL-18 signaling on both NK cells and DCs is required for efficient NK-cell activation upon VV infection in vitro. We further showed in vivo that efficient NK-cell activation in response to VV is dependent on DCs and IL-18 signaling in non-NK cells, suggesting an essential role for NK cell-extrinsic IL-18 signaling in NK-cell activation. Mechanistically, IL-18 signaling in DCs promotes expression of Rae-1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell-extrinsic IL-18 signaling in NK-cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK-cell-based therapies for viral infections and cancer.

CTLA-4 Is Expressed by Activated Mouse NK Cells and Inhibits NK Cell IFN-γ Production in Response to Mature Dendritic Cells

NK cells express an array of activating and inhibitory receptors that determine NK cell responses upon triggering by cognate ligands. Although activating NK cell receptors recognize mainly ligands expressed by stressed, virus-infected, or transformed cells, most inhibitory receptors engage MHC class I, preventing NK cell activation in response to healthy cells. In this study, we provide insight into the regulation and function of additional receptors involved in mouse NK cell responses: CTLA-4 and CD28. CTLA-4 and CD28 engage the same ligands, B7-1 and B7-2, which are primarily expressed by APCs, such as dendritic cells. Our data demonstrate that activation of mouse NK cells with IL-2 induces the expression of CTLA-4 and upregulates CD28. CTLA-4 expression in IL-2-expanded NK cells was further up- or downregulated by IL-12 or TGF-β, respectively. Using gene-deficient NK cells, we show that CD28 induces, and CTLA-4 inhibits, IFN-γ release by NK cells upon engagement by the recombinant ligand, B7-1, or upon coculture with mature dendritic cells. Notably, we show that mouse NK cells infiltrating solid tumors express CD28 and CTLA-4 and respond to stimulation with recombinant B7-1, suggesting that the NK cell responses mediated by the CD28/CTLA-4:B7-1/B7-2 system could be of importance during malignant disease. Accordingly, our study might have implications for immunotherapy of cancer based on blocking anti-CTLA-4 mAbs.

Activation of NK cells by HIV-specific ADCC antibodies

HIV-specific ADCC antibodies could play a role in providing protective immunity. We have developed a whole blood ADCC assay that measures NK cell activation in response to HIV peptide epitopes. These HIV peptide-specific ADCC responses are associated with escape from immune recognition and slower progression of HIV infection and represent interesting HIV vaccine antigens. However, the mechanism by which these epitopes are expressed and whether or not they induce NK-mediated killing of cells expressing such peptide-antigens is not understood. Herein, we show that fluorescent-tagged ADCC peptide epitopes associate with blood granulocytes. The peptide-associated granulocytes become a specific target for antibody-mediated killing, as shown by enhanced expression of apoptosis marker Annexin and reduction in cell numbers. When HIV Envelope gp140 protein is utilized in the ADCC assay, we detected binding to its ligand, CD4. During the incubation, cells co-expressing gp140 and CD4 reduce in number. We also detected increasing Annexin expression in these cells. These data indicate that blood cells expressing HIV-specific ADCC epitopes are targeted for killing by NK cells in the presence of ADCC antibodies in HIV+ plasma and provide a clearer framework to evaluate these antigens as vaccine candidates.

Impact of Commonly Used Transplant Immunosuppressive Drugs on Human NK Cell Function Is Dependent upon Stimulation Condition

Lung transplantation is a recognised treatment for patients with end stage pulmonary disease. Transplant recipients receive life-long administration of immunosuppressive drugs that target T cell mediated graft rejection. However little is known of the impact on NK cells, which have the potential to be alloreactive in response to HLA-mismatched ligands on the lung allograft and in doing so, may impact negatively on allograft survival. NK cells from 20 healthy controls were assessed in response to Cyclosporine A, Mycophenolic acid (MPA; active form of Mycophenolate mofetil) and Prednisolone at a range of concentrations. The impact of these clinically used immunosuppressive drugs on cytotoxicity (measured by CD107a expression), IFN-γ production and CFSE proliferation was assessed in response to various stimuli including MHC class-I negative cell lines, IL-2/IL-12 cytokines and PMA/Ionomycin. Treatment with MPA and Prednisolone revealed significantly reduced CD107a expression in response to cell line stimulation. In comparison, addition of MPA and Cyclosporine A displayed reduced CD107a expression and IFN-γ production following PMA/Ionomycin stimulation. Diminished proliferation was observed in response to treatment with each drug. Additional functional inhibitors (LY294002, PD98059, Rottlerin, Rapamycin) were used to elucidate intracellular pathways of NK cell activation in response to stimulation with K562 or PMA-I. CD107a expression was significantly decreased with the addition of PD98059 following K562 stimulation. Similarly, CD107a expression significantly decreased following PMA-I stimulation with the addition of LY294002, PD98059 and Rottlerin. Ten lung transplant patients, not receiving immunosuppressive drugs pre-transplant, were assessed for longitudinal changes post-transplant in relation to the administration of immunosuppressive drugs. Individual patient dynamics revealed different longitudinal patterns of NK cell function post-transplantation. These results provide mechanistic insights into pathways of NK cell activation and show commonly administered transplant immunosuppression agents and clinical rejection/infection events have differential effects on NK cell function that may impact the immune response following lung transplantation.

Subcapsular sinus macrophages promote NK cell accumulation and activation in response to lymph-borne viral particles

AbstractNatural killer (NK) cells become activated during viral infection in response to cytokines or to engagement of NK cell activating receptors. However, the identity of cells sensing viral particles and mediating NK cell activation has not been defined. Here, we show that local administration of a modified vaccinia virus Ankara vaccine in mice results in the accumulation of NK cells in the subcapsular area of the draining lymph node and their activation, a process that is strictly dependent on type I IFN signaling. NK cells located in the subcapsular area exhibited reduced motility and were found associated with CD169+-positive subcapsular sinus (SCS) macrophages and collagen fibers. Moreover, depletion of SCS macrophages using clodronate liposomes abolished NK cell accumulation and activation. Our results identify SCS macrophages as primary mediators of NK cell activation in response to lymph-borne viral particles suggesting that they act as early sensors of local infection or delivery of viral-based vaccines.

Frequent and Strong Antibody-Mediated Natural Killer Cell Activation in Response to HIV-1 Env in Individuals with Chronic HIV-1 Infection

Natural killer (NK) cells play a critical role in the control of HIV-1 infection, and NK cells that respond to HIV-1 peptides have been recently described. However, the mechanisms by which NK cells recognize HIV-1 antigens are not fully understood. We investigated NK cell activation in response to HIV-1 peptides during early and chronic HIV-1 clade B infection using a whole-blood assay and multiparameter flow cytometry. Antibody-mediated NK cell activation in response to HIV-1 peptides was not detected in HIV-1-uninfected individuals. In contrast, 79% of individuals with chronic infection and 22% of individuals with early infection had detectable gamma interferon (IFN-γ) NK cell responses to HIV-1 antigens (P < 0.00001). IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing NK cells most frequently targeted Env gp120 (median of 4% and range of 0 to 31% of all NK cells). NK cells rarely targeted other HIV-1 proteins such as Gag, Pol, and Nef. Antibody-mediated NK cell responses to peptides mapped predominantly to Env protein, required the presence of plasma or plasma IgG, and resulted in lower CD16 expression on NK cells, suggesting an antibody-mediated activation of NK cells. Further studies are needed to assess the consequences of these antibody-mediated NK cell responses for HIV-1 disease progression and vaccine-induced protection from infection.

Adenovirus type 35, but not type 5, stimulates NK cell activation via plasmacytoid dendritic cells and TLR9 signaling

In hematopoietic stem cell transplant (HSCT) recipients, disseminated adenoviral infections during the first two months after HSCT can lead to severe complications and fatal outcome. Since NK cells are usually the first lymphocytes to reconstitute after HSCT and have been implicated in the clearance of adenovirus-infected cells, it was investigated whether NK cells are activated by adenovirus in vitro.Exposure of PBMC to human adenovirus type 5 (HAdV5) or HAdV35 resulted in the up-regulation of the activation marker CD69 on NK cells and enhanced the cytolytic activity of NK cells. HAdV5-induced NK cell activation relied on the contribution of T cells as the depletion of T cells from PBMC abolished NK cell activation. In contrast, NK cell activation in response to HAdV35 occurred in the absence of T cells. Plasmacytoid dendritic cells (pDC) were necessary and sufficient to mediate NK cell activation. HAdV35 induced significantly more interferon-α (IFN-α) production by pDC than HAdV5. The increased IFN-α production and NK cell activation correlated with a higher infection efficiency of viruses with the type 35 fiber. The IFN-α response of pDC was enhanced by the presence of NK cells, suggesting a reciprocal interaction between pDC and NK cells. Incubation with a TLR9 antagonist impaired the IFN-α production by pDC as well as NK cell activation, implying that TLR9 signaling is critically involved in the IFN-α response of pDC and NK cell activation after HAdV35 exposure.In conclusion, two human adenovirus serotypes from two different species differ considerably in their capacity to stimulate pDC and NK cells.

NK cells require type I IFN receptor for antiviral responses during genital HSV-2 infection

Type I interferon (IFN) signalling, NK cells and NK cell-derived IFN-γ are critical in the early control of genital HSV-2 infection. We have recently reported that NK cells are the source of early IFN-γ in the genital tract in response to HSV-2. However, the response of NK cells to genital HSV-2 infection is not well defined in the context of type I IFN signalling. Here we show that HSV-2 replication was significantly higher in mice deficient in the type I IFN receptor or NK cells compared to wild type controls. There was no detectable IFN-γ production in the genital washes from IFN-α/βR −/− mice or NK cell depleted mice in response to HSV-2 infection compared to control mice. Absence of the type I IFN receptor does not alter homing of NK cells to the genital mucosa. Moreover, the absence of IL-12 had no significant effect on NK cell-derived IFN-γ. Surprisingly, IFN-α/βR −/− mice had more IL-15 positive cells in the genital mucosa in response to HSV-2 infection compared to control mice. We then examined the expression of IL-15 receptors on NK cells. There was no significant differences in the levels of IL-15 receptor expression on NK cells from IFN-α/βR −/− or control mice. Our data clearly suggest that type I IFN receptor signalling is essential for NK cell activation in response to genital HSV-2 infection, and propose that NK cell activation by IL-15 may involve type I IFNs.

SHP-2 Expression Negatively Regulates NK Cell Function

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human NK cells upon recruitment to killer cell Ig-like receptors (KIR). To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN-gamma production, whereas cells overexpressing wild-type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts microtubule organizing center-based cytoskeletal rearrangement and granule release.

Direct Action of Type I IFN on NK Cells Is Required for Their Activation in Response to Vaccinia Viral Infection In Vivo

Type I IFN plays an important role in the activation of NK cells. However, the mechanism underlying type I IFN-dependent NK cell activation remains largely unknown. A recent report suggested that type I IFN acted on accessory dendritic cells, leading to IL-15 production, and that subsequent trans-presentation of IL-15 was required for NK cell activation upon stimulation with synthetic TLR ligands. It is not clear how type I IFN regulates NK cell activation in response to live pathogens. Using a murine model of infection with vaccinia virus (VV), we previously demonstrated a critical role for type I IFN in the innate immune control of VV infection. In this study, we first showed that type I IFN did not directly protect L929 cells from VV infection in vitro and that type I IFN-dependent innate immune control of VV infection in vivo was mediated by activated NK cells. We further demonstrated that direct action of type I IFN on NK cells, but not on dendritic cells, is required for the activation of NK cells in response to VV infection both in vitro and in vivo, leading to efficient VV clearance. Our findings may help design effective strategies for the control of poxviral infections in vivo.

Interleukin-21 Enhances NK Cell Activation in Response to Antibody-Coated Targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.