Purified NK Cells Research Articles

IFN type I and type II independent enhancement of B cell TLR7 expression by natural killer cells

The PRR TLR7 plays a key role in the activation of autoantigen-reactive B cells. This response is increased markedly by IFN-α, produced by accessory cells, as a result of the up-regulation of TLR7. We report herein an alternative pathway by which TLR7 expression can be augmented. This finding was derived from continuation of ongoing studies to uncover interactions between NK and B cells. Here, we have compared gene expression profiles by microarray analysis of B cells before and after their interaction with purified NK cells. The most outstanding alteration of genes transcribed in B cells is a significant increase in the expression of many members of the ISG family, among which is TLR7. Further analysis revealed that the enhancement of TLR7 on B cells is not mediated via type I or type II IFN but by another cytokine, IL-28, a type III IFN, which acts in concert with contact-mediated interactions with NK cells. This increased expression allows B cells to respond more readily upon stimulation by its ligand and may increase in vivo responses to other TLR7 ligands, such as autoantigens, prior to or jointly with stimulation by other cytokines.

Altered natural killer cells' response to herpes virus infection in multiple sclerosis involves KIR2DL2 expression

The role of herpes viruses as potential triggers of multiple sclerosis (MS) is still debated. Peripheral blood mononuclear cells from MS patients and controls were treated with CpG sequences and infected in vitro with HSV-1. Samples were analyzed for viral yield, TLR9 pathways, cytokine secretion, NK cell activation and killer immunoglobulin-like receptor (KIR) expression. CpG treatment promoted an unexpected sensitivity to herpes virus infection in a subset of MS patients: TLR9 pathways did not show defects while NK cells presented decreased degranulation and cytotoxicity and up-regulated the inhibitory KIR2DL2 receptor. CpG treatment of purified NK cells affected directly KIR2DL2 modulation and cell activation. These data suggest potential implications for viral pathogenesis of MS.

Role of monocytes in mediating HIV-specific antibody-dependent cellular cytotoxicity

Antibodies (Abs) that mediate antibody-dependent cellular cytotoxicity (ADCC) activity against HIV-1 are of major interest. A widely used method to measure ADCC Abs is the rapid and fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay. Antibody-dependent killing of a labelled target cell line by PBMC is assessed by loss of intracellular CFSE but retention of membrane dye PKH26 (CFSE-PKH26+). Cells of this phenotype are assumed to be derived from CFSE+PKH26+ target cells killed by NK cells.We assessed the effector cells that mediate ADCC in this assay. Backgating analysis and phenotyping of CFSE-PKH26+ revealed that the RFADCC assay's readout mainly represents CD3-CD14+ monocytes taking up the PKH26 dye. This was confirmed for 53 HIV+plasma-purified IgG samples when co-cultured with PBMC from three separate healthy donors. Emergence of the CFSE-PKH26+ monocyte population was observed upon co-culture of targets with purified monocytes but not with purified NK cells. Image flow cytometry and microscopy showed a monocyte-specific interaction with target cells without typical morphological changes associated with phagocytosis, suggesting a monocyte-mediated ADCC process.We conclude that the RFADCC assay primarily reflects Ab-mediated monocyte function. Further studies on the immunological importance of HIV-specific monocyte-mediated ADCC are warranted.

Phenotypic and Functional Characterization of NK Cells in Human Immune Response against the Dimorphic Fungus Paracoccidioides brasiliensis

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56(+)granulysin(+) cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.

NK Cell Tolerance to TLR Agonists Mediated by Regulatory T Cells after Polymicrobial Sepsis

As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as TLRs. NK cells present in many tissues contribute to inflammatory processes, particularly through the production of IFN-γ. They may display a protective role during infection but also a detrimental role during sterile or infectious systemic inflammatory response syndrome. Nevertheless, the exact status of NK cells during bacterial sepsis and their capacity directly to respond to TLR agonists remain unclear. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors. The aim of this study was to gain insight into TLR2/TLR4/TLR9 expression on/in murine NK cells at the protein level and determine if their agonists were able to induce cytokine production. We show, by flow cytometry, a strong intracellular expression of TLR2 and a low of TLR4 in freshly isolated murine spleen NK cells, similar to that of TLR9. In vitro, purified NK cells respond to TLR2, TLR4, and TLR9 agonists, in synergy with activating cytokines (IL-2, IL-15, and/or IL-18), and produce proinflammatory cytokines (IFN-γ and GM-CSF). Finally, we explored the possible tolerance of NK cells to TLR agonists after a polymicrobial sepsis (experimental peritonitis). For the first time, to our knowledge, NK cells are shown to become tolerant in terms of proinflammatory cytokines production after sepsis. We show that this tolerance is associated with a reduction of the CD27(+)CD11b(-) subset in the spleen related to the presence of regulatory T cells and mainly mediated by TGF-β.

INF-γ production of NK cells is suppressed in cancer patients. (45.10)

Abstract Natural killer (NK) cells are the innate immune system that play an important role in killing viral infected and transformed cancer cells by direct cell to cell interaction. NK cells also secrete cytokines such as interferon (IFN)-γ and TNF-α which primes subsequent adaptive immune responses. However, tumor cells employ many different strategies to evade NK cells. Moreover, tumor cells secrete molecules to directly inhibit the function of immune cells of both innate and adaptive immunity. Here, we designed to investigate whether NK cells from cancer patients exhibit an altered ability to produce INF-γ. The results showed that peripheral blood mononuclear cells (PBMC) and purified NK cells of the cancer patients had suppressed ability to produce IFN-γ. Therefore, we propose that impaired IFN-γ production in peripheral blood mononuclear cells and in purified NK cells reflects immunosuppression and inadequate antitumor immune response in cancer patients. Thus, the measurement of INF-γ producing ability of NK cells in clinical blood samples may allow to assess the impact of immunosuppression on these effector cells, and this information may be helpful in understanding cancer and pathogenesis.

HLA-C Matching Status Does Not Affect Rituximab-Mediated Antibody-Dependent Cellular Cytotoxicity by Allogeneic Natural Killer Cells

Risk of leukemia relapse after T cell-depleted hematopoietic stem cell transplantation is lower in the “HLA-C mismatched” recipient-donor combinations. This might be attributable to increased natural killing by allogeneic NK cells carrying a KIR that does not bind to HLA-C on target cells (HLA-C-uncoupled KIR). Considering a new strategy of allogeneic NK cell transfer with rituximab to treat B-cell lymphomas, however, it is unknown whether the HLA-C matching status also affects rituximab-mediated antibody-dependent cellular cytotoxicity (ADCC). To address this issue, we investigated the levels of ADCC by purified NK cells carrying an HLA-C-uncoupled KIR, where the NK cell donors had either matched or mismatched HLA-C combination with target cells. Purified NK cells carrying an HLA-C-uncoupled KIR consistently showed enhanced ADCC against target cells when NK cell donors had an HLA-C-mismatch. When NK cell donors did not have an HLA-C mismatch, it was inconsistent whether HLA-C-uncoupled KIR caused ADCC enhancement. When the levels of ADCC by whole NK cells were compared, there were substantial differences among the donors regardless of the HLA-C matching status. Subjects with HLA-C mismatch may not have an advantage when cytoimmunotherapy using allogeneic NK cells is considered in combination with rituximab.

Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties

IntroductionResponse to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A.MethodsMGAH22 was compared to an identical anti-HER2 mAb except for a wild type Fc domain. Antibody-dependent cell cytotoxicity (ADCC) assays were performed with HER2-expressing cancer cells as targets and human PBMC or purified NK cells as effectors. Xenograft studies were conducted in mice with wild type murine FcγRs; in mice lacking murine CD16; or in mice lacking murine CD16 but transgenic for human CD16A-158F, the low-binding variant. The latter model reproduces the differential binding between wild type and the Fc-optimized mAb for human CD16A. The JIMT-1 human breast tumor line, derived from a patient that progressed on trastuzumab therapy, was used in these studies. Single and repeat dose toxicology studies with MGAH22 administered intravenously at high dose were conducted in cynomolgus monkeys.ResultsThe optimized Fc domain confers enhanced ADCC against all HER2-positive tumor cells tested, including cells resistant to trastuzumab's anti-proliferative activity or expressing low HER2 levels. The greatest improvement occurs with effector cells isolated from donors homozygous or heterozygous for CD16A-158F, the low-binding allele. MGAH22 demonstrates increased activity against HER2-expressing tumors in mice transgenic for human CD16A-158F. In single and repeat-dose toxicology studies in cynomolgus monkeys, a species with a HER2 expression pattern comparable to that in humans and Fcγ receptors that exhibit enhanced binding to the optimized Fc domain, MGAH22 was well tolerated at all doses tested (15-150 mg/kg) and exhibited pharmacokinetic parameters similar to that of other anti-HER2 antibodies. Induction of cytokine release by MGAH22 in vivo or in vitro was similar to that induced by the corresponding wild type mAb or trastuzumab.ConclusionsThe data support the clinical development of MGAH22, which may have utility in patients with low HER2 expressing tumors or carrying the CD16A low-binding allele.

Dasatinib Has a Dual Mode of Action: Direct BCR-ABL1 Mediated Anti-Leukemic Effects Are Complemented by Promotion of Th1-Type and NK-Cell Mediated Cellular Immune Responses,

Abstract 3760 Background.Tyrosine kinase inhibitors (TKIs; imatinib, dasatinib, nilotinib) have dramatically improved outcome of CML. Besides inhibiting target kinases in leukemic cells, off-target kinases in immune effector cells are also affected. We have previously described that dasatinib therapy induces an oligoclonal expansion and mobilization of large granular lymphocytes (LGLs; CD8+ T-cells or NK-cells) in Ph+ leukemia patients. Importantly, LGL expansion is associated with improved therapy responses, but the actual mechanisms are unknown. In this study, we explored the function and anti-leukemic properties of LGLs. Methods.Peripheral blood samples from CML patients treated with dasatinib (n=10), imatinib (n=4), or nilotinib (n=7), or healthy controls (n=6) were used to analyze the activation and cytotoxicity of T- and NK-cells. Samples were collected before and after drug intake. The number of LGLs was determined from MGG stained blood smears and compared with granzyme B (GrB) positivity analyzed by flow cytometry.Th1-type cytokine (TNF-a, IFN-g) production was measured by flow cytometry after stimulation of mononuclear cells (MNCs) with a-CD3/CD28-antibodies. Unpurified and purified NK cells were cultured with K562 cells, and degranulation (CD107 analysis) and cytotoxicity were measured. Results.As GrB positivity correlated well (r=0.95, p<0.0001, n=17) with the number of LGLs counted from MGG stained blood smears, a GrB specific antibody was used to identify LGLs in further analyses.At diagnosis CML patients had more GrB+CD8+ T-cells than healthy controls (38 % vs. 11%, p=0.028). Also GrB+CD4+ T-cells were slightly increased, but did not differ significantly from healthy controls (3.6% vs. 0.8%, p=0.08). During dasatinib treatment the proportion of GrB+CD4+ (median at 6 months 28.1%, p=0.03) and GrB+CD8+ (70.9%, p=0.03) T cells increased significantly, whereas similar increase was not observed during imatinib (1.2% GrB+CD4+ and 30.0% GrB+CD8+ T-cells) or nilotinib (4.4% and 41.8%, respectively) therapies.In patients on dasatinib therapy, GrB+CD3+cells were more sensitive to CD3/CD28-antibody stimulation and a larger proportion of cells (13.7%) produced Th1-type cytokines (TNF-a+IFN-g) compared to imatinib (2.4%) or nilotinib patients (5.5%) or healthy controls (5%) under same conditions (p=0.015).As Th-1 cytokine-producing T cells are important in promoting cell-mediated immune responses, we next assessed whether dasatinib also enhances the cytolytic activity of NK cells. When MNC fraction was used as effector population (ratio 20:1), the median percentage of dead K562 cells was 18% in samples taken before dasatinib intake and 32% in samples taken 1h after dasatinib intake (p=0.004). Pre-dasatinib killing did not differ significantly from healthy volunteers (p=0.12). No increase in NK-cytotoxicity was observed after imatinib (11% vs. 8%) or nilotinib (10% vs. 10%) intake. Similar results were also obtained with purified NK-cells: the median percentage of dead K562 cells was 12% pre-dasatinib and 29% in post-dasatinib samples (p=0.06), whereas no differences were noticed with imatinib (30% vs. 28%) or nilotinib (14% vs. 15%) patients. The median percentage of dead K562 cells after incubation with pure NK-cells from healthy volunteers was 20%.Interestingly, the cytolytic ability of NK-cells differed significantly among dasatinib treated patients. When the patients were divided into two groups based on therapy response, patients who had achieved CMR within 12 months (n=4) had significantly higher cytotoxic capability compared to patients who had not (n=6): 46% vs. 28% of dead K562 cells in post-dasatinib samples (p=0.02). Conclusions.Dasatinib therapy resulted in increased numbers of GrB+ T-cells and generation of a Th1-type cellular immune response. In addition, 1h dasatinib exposure in vivo improved the cytotoxicity of NK-cells. These data support the dual mode of action of dasatinib: potent BCR-ABL1 inhibition in leukemic cells is accompanied by enhancement of cellular immunity, which likely have implications in be the long term control of Ph+ leukemia. Disclosures:Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria; Novartis: Honoraria.

Results of a Phase 1 Clinical Trial of Allogeneic Tumor-Primed Natural Killer Cells in AML

Abstract 946We have previously reported that resting human NK cells can be primed to lyse NK-resistant tumor cells, including AML, by co-incubation with membrane preparations from the leukemia cell line CTV-1. Tumor-primed NK cells can be cryopreserved and remain in a primed state when thawed. Following translation of this production method to GMP-compliance we designed a phase 1 trial for AML patients. PROTOCOL: AML patients meeting the inclusion criteria were enrolled and they and their children HLA-typed and screened for medical suitability for apheresis. KIR mismatched donors in the GvL direction were preferred when available. PATIENT INCLUSION CRITERIA: >60 years in PR (blasts >5<20% in BM) after 2nd course of induction chemotherapy; >60 years in CR2 after re-induction chemotherapy; >60 years in PR or CR after 2 courses of chemotherapy with poor risk disease by conventional cytogenetic criteria; <60 years beyond CR2 but unsuitable for transplantation. CONDITIONING: Fludarabine 25mg/m2/day for 3 days plus a single fraction (2Gy) TBI on day 4. MANUFACTURE OF Tumor-primed NK: T-pNK cells were generated from single 2hr apheresis from haploidentical related donors in 14 cases; 1 donor declined and the patient was not treated. NK cells were isolated with anti-CD56 microbeads (CliniMACS, Miltenyi Biotec) with (n=2) or without (n=12) prior depletion of CD3+ cells. Purified NK cells were co-incubated with a lysate of CTV-1 cells (DSMZ repository) overnight. Lysate was removed by density gradient separation on day 2 and cells were cryopreserved in dosed aliquots and released for infusion within 15 days of manufacture. TREATMENT: Three groups of 5 patients were to receive 1×10^6/kg, 5×10^6/kg or 1×10^7/kg haploidentical related donor T-pNK as a single i.v. infusion 24 hours after completion of TBI. RESULTS: 15 patients were enrolled between July 2008 and Dec 2009 – Table 1. Mean donor T-pNK yield was 6.73×10^9 and CD56 purity 99.3%. T cell contamination was <10^4/kg. All products achieved QC release criteria. Median time from enrolment to product release was 46 days. 8/15 patients received the T-pNK therapy; 7 untreated due to relapse during product manufacture or failure to remit (6) and donor refusal (1). No infusional toxicity was observed. 7/8 patients experienced profound bone marrow suppression with median time of neutrophil recovery 55 days (19–101). Two patients who previously had an allogeneic transplant were salvaged with a CD34+ top up stem cell transfusion from their original donors. All experienced neutropenic fevers requiring antibiotic support with 2 of them requiring ICU admission. Due to the toxicities observed the dose escalation was abandoned and all treated patients received 1×10^6 cell/kg. The median duration of CR post T-pNK was 222 days (55–845) compared to previous CR median of 47 days (0–90) – Table 1. 7/8 patients experienced a longer CR post T-pNK than their previous CR; median increase in duration of CR was 242 days (range 54–815). CONCLUSIONS: T-pNK can be easily generated and administered with acceptable toxicity which was easily managed. In this non-controlled trial there was evidence of clinical efficacy but delays in donor screening and processing led to 40% attrition of enrolled patients prior to drug administration.Table 1:UPNSexAgeCytogeneticsAlloHSCTStatus at treatmentDuration of CR pre-TpNK (days)Duration CR post-TpNK01F54Trisomy 21NoCR33084503M72N/KNoPR1N/A25004M51t(9;11)YesCR33028407M71ComplexNoCR1605509F73Trisomy 13NoCR14714108M67normalNoCR26211611M61Trisomy 9YesCR39035212F67normalNoCR23098 Disclosures:Lowdell:Coronado Biosciences: Consultancy.

Activating NK cell receptor expression/function (NKp30, NKp46, DNAM‐1) during chronic viraemic HCV infection is associated with the outcome of combined treatment

Specific NK cell killer inhibitory receptor (KIR):HLA haplotype combinations have been associated with successful clearance of acute and chronic HCV infection. Whether an imbalance of activating NK cell receptors also contributes to the outcome of treatment of chronic HCV infection, however, is not known. We studied peripheral NK cell phenotype and function in 28 chronically viraemic HCV genotype I treatment-naïve patients who underwent treatment with pegylated IFN-α and ribavirin. At baseline, chronically infected patients with sustained virological response (SVR) had reduced CD56(bright) CD16(+/-) cell populations, increased CD56(dull) CD16(+) NK cell proportions, and lower expression of NKp30, DNAM-1, and CD85j. Similarly, reduced NK cell IFN-γ production but increased degranulation was observed among nonresponding (NR) patients. After treatment, CD56(bright) CD16(+/-) NK cell numbers increased in both SVR and NR patients, with a parallel significant increase in activating NKp30 molecule densities in SVR patients only. In vitro experiments using purified NK cells in the presence of rIL-2 and IFN-α confirmed upregulation of NKp30 and also of NKp46 and DNAM-1 in patients with subsequent SVR. Thus, differences in patient NK cell receptor expression and modulation during chronic HCV-1 infection are associated with subsequent outcome of standard treatment. Individual activating receptor expression/function integrates with KIR:HLA genotype carriage to determine the clearance of HCV infection upon treatment.

Abstract 787: The role of monocytes in promoting natural killer cell activation in response to antibody-coated tumor cells and IL-12

Abstract Introduction: Previous studies from our group has demonstrated that co-stimulation of NK cells via the interleukin-12 receptor (IL-12R) and the FcgRIIIa activates the extracellular signal-regulated kinase (ERK) signaling pathway in vitro, in murine tumor models and in phase I clinical trials. The ERK pathway in turn promotes the secretion of interferon-gamma (IFN-g) thereby mediating potent anti-tumor effects. Hypothesis: NK cell cytokine secretion would be significantly enhanced following simultaneous stimulation of the NK cells via the activating receptor, NKG2D and their ligands (such as MICA/B) expressed on monocytes. Methods: Purified NK cells, Monocytes and peripheral blood mononuclear cells (PBMC) were co-cultured with antibody (Ab)-coated tumors cells in the presence of IL-12. Cell free supernatants were analyzed for the presence of inflammatory cytokines. Results: Co-stimulation of purified NK cells with trastuzumab-coated HER2+ SKBR3 breast cancer cells and IL-12 (10 ng/mL) resulted in synergistic production of IFN-g (&amp;gt;20,000 pg/mL) as compared to the single conditions (&amp;lt;3000 pg/mL). Cytokine production in response to antibody-coated tumor cells and IL-12 was significantly enhanced in the presence of autologous monocytes (2-3-fold, P&amp;lt;0.05) but not T cells or B cells. Maximum cytokine secretion was observed at 48 hours, and preceded by a 6-fold increase in IFN-g transcript. This enhancement of cytokine response was dependent on cell-cell contact as determined by a Transwell assay. NK cells co-cultured with monocytes (1:1=200,000 cells per well) also secreted significantly higher amounts of TNF-a (&amp;gt;2 fold) and MIP1 alpha (&amp;gt;4 fold) as compared to un-supplemented NK cells exposed to Ab and IL-12. A dose-response effect was observed with increasing numbers of added monocytes. Pre-treatment of monocytes with LPS and/or IFN-alpha led to increased expression of NKG2D ligands (MICA and MICB) and increased the ability of monocytes to act as co-stimulators of NK cell cytokine secretion. The stimulatory effects of MICA/B+ monocytes were duplicated by the use of a MICA over-expressing cell line (C1R-MICA) but not the parental MICA-negative cell line (2-3.2 fold increase). Incubation of C1R MICA cells with a MICA neutralizing antibody prior to co-culture with NK cells led to a significant reduction in IFN-g secretion. The stimulatory effects of monocytes were also observed in whole PBMC in that depletion of monocytes from PBMC markedly inhibited the production of IFN-g by the NK cell compartment whereas supplementation of PBMC with additional monocytes led to dose-dependent increases in cytokine production. Conclusions: These data suggest that stimulation of NKG2D by monocyte ligands can enhance the NK cell cytokine response to Ab-coated targets. Enhancement of NK monocyte interactions could increase the efficacy of Ab-based anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 787. doi:10.1158/1538-7445.AM2011-787

CpG ODN induced NK cell IFNγ production is positively regulated by IL-12 producing neutrophils and suppressed by IL-10 producing B cells (108.8)

Abstract CpG-containing oligodeoxynucleotides (CpG ODN) stimulates spleen cells in vitro and induces IFNγ secretion mostly from NK cells, whereas purified NK cells are not stimulated by CpG ODN. IL-12 neutralization inhibits NK cell IFNγ production, and IL-12 knockout mouse spleen cells stimulated with CpG ODN produce very little IFNγ, indicating that IL-12 is required. Depletion of macrophages and DC has no effect, whereas neutrophil depletion dramatically decreases both IL-12 and IFNγ production by spleen cells, indicating that neutrophils are a key source of IL-12. NK cells in B cell-deficient mouse spleen secrete higher levels of IFNγ than wild type spleen cells, and B cell depletion enhances NK cell IFNγ production, indicating that B cells are suppressive. CD5+ B cells secrete IL-10 in response to CpG ODN. The neutralization of IL-10 increases IFNγ and IL-12 production by CpG ODN-stimulated spleen cells, and B cell-deficient splenocytes produce more IL-12 than WT splenocytes. Furthermore, exogenously added IL-10 effectively inhibits CpG ODN-induced IL-12 production by purified neutrophils, and IL-12/IL-18 induced stimulation of purified NK cells. Therefore, CD5+ B cells produce IL-10 and suppress CpG ODN-mediated NK cell IFNγ production by inhibiting neutrophil IL-12 production as well as by directly acting on activated NK cells themselves.

Impaired IFN-γ production and proliferation of NK cells in Multiple Sclerosis

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56(dim)CD16(+)) or immunoregulatory (CD56(bright)CD16(-)) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56(bright)CD16(-) and CD56(dim)CD16(+) NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-γ production of CD56(bright)CD16(-) NK cells but not of their CD56(dim)CD16(+) counterparts were substantially diminished in MS. Impaired expansion of CD56(bright)CD16(-) NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4(+) T cell line CEM and allogeneic primary CD4(+) T-cell blasts was unchanged. Thus, characteristic functions of CD56(bright)CD16(-) NK cells, namely cytokine-induced NK cell expansion and IFN-γ production, are compromised in the NK cell compartment of MS patients.

An ADCC-Independent Mechanism of Rituximab-Mediated B-NHL Cells Depletion Via Sensitization to Cell Death through the Expression of TRAIL on NK Cells

AbstractAbstract 4919Rituximab, a chimeric anti-CD20 mAb, has being used, alone or in combination with chemotherapy, in the treatment of patients with B-NHL and rheumatoid arthritis. It is also being tested clinically in the treatment of other B cell malignancies. The mechanisms by which the antibody depletes the B cells have been shown to be mediated via ADCC, CDC, and apoptosis. In addition, the antibody also signals the cells and modifies various survival pathways and sensitizes the resistant tumor cells to various apoptotic stimuli (Jazirehi and Bonavida, Oncogene 24:2121, 2005). The role of the host innate cytotoxic cells, such as NK cells, in cooperation with rituximab in the depletion of B-NHL cells has been poorly explored. Studies by us and others have reported that rituximab sensitizes resistant B-NHL tumor cells to both Fas ligand and TRAIL-induced apoptosis (Bonavida, Oncogene 26:3629, 2007; Daniel, D. et al., Blood 110:4037, 2007). Since NK cells express on the surface TRAIL, we hypothesized that rituximab may also sensitize the TRAIL-resistant tumor cells to NK-mediated cytotoxicity. Accordingly, we have examined various TRAIL-resistant B-NHL cell lines and used peripheral blood-derived purified human NK cells. Treatment of various B-NHL cell lines with rituximab sensitized the cells to TRAIL-induced apoptosis. The mechanism of TRAIL-induced cytotoxicity was found to be the result of TRAIL-induced inhibition of NF-κB and downstream inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) as well as inhibition of anti-apoptotic gene products such as Bclxl. Treatment of various B-NHL cell lines with rituximab, unlike treatment with control IgG1, resulted in significant cytotoxicity in the presence of purified NK cells. The extent of the cytotoxic activity was a function of the E:T ratios used. We then examined the contribution of TRAIL expressed on the NK cell surface for its role in NK-mediated cytotoxicity of rituximab-pretreated B-NHL cells. We used a neutralizing TRAIL antibody that was added in the reaction mixture and demonstrated that the NK cytotoxic activity was significantly reduced compared to controls. These studies with rituximab were also confirmed with other CD20 mAbs. We are currently examining the sensitization of freshly-derived B-NHL and CLL cells that are treated with rituximab and other anti-CD20 mAbs to NK-mediated cytotoxicity for validation of the findings with cell lines. The present findings suggest that, in vivo, patients who are treated with rituximab may recruit NK and other effector cells to mediate, independently of ADCC, cytotoxicity via the TNF-family ligands (e.g. TNF-α, Fas-L, TRAIL). The studies also suggest that this B cell-depletion mechanism by NK cells may contribute to the mechanism of rituximab- mediated depletion of B-NHL cells in vivo. Noteworthy, the proposed host cytotoxic mechanism may not be functional if the therapeutic treatment consists of the combination of rituximab and immunosuppressive chemotherapeutic drugs that may lead to depletion or inactivation of host cytotoxic cells. Disclosures:No relevant conflicts of interest to declare.

NK cell dysfunction with down-regulated CD16 and up-regulated CD56 molecules in patients with esophageal squamous cell carcinoma

NK cells can be divided into two subsets, CD56(dim) and CD56(bright) NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56(dim) or CD56(bright) NK cells gated on CD56(+)CD3(-) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)-expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56(dim) and CD56(bright) NK cells between ESCC patients and healthy donors, down-regulated CD16 and up-regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab-mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down-regulated CD16 and up-regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF-beta1 partially contributed to down-regulation of CD16 on NK cells. Down-regulated CD16 and up-regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.

Clusterin synergizes with IL-2 for the expansion and IFN-γ production of natural killer cells

CLU is a secreted, multifunctional protein implicated in several immunologic and pathologic conditions. As the level of serum CLU was shown to be elevated during inflammatory responses, we questioned if CLU might interact with circulating lymphocytes leading to functional consequences. To assess this possibility directly, mouse splenocytes and purified NK cells were cultured with varying dose of CLU, and its effect on cell proliferation was examined. Our data showed that CLU up-regulated DNA synthesis and expansion of NK cells significantly in response to a suboptimal, but not maximal, dose of IL-2, and CLU alone did not exhibit such effects. This CLU-mediated synergy required the co-presence of CLU at the onset of IL-2 stimulation and needed a continuous presence during the rest of the culture. Importantly, NK cells stimulated with CLU showed increased formation of cell clusters and a CD69 activation receptor, representing a higher cellular activation status compared with those from the control group. Furthermore, these NK cells displayed elevated IFN-γ production upon RMA/S tumor target exposures, implying that CLU regulates not only NK cell expansion but also effector function of NK cells. Collectively, our data present a previously unrecognized function of CLU as a novel regulator of NK cells via providing costimulation required for cell proliferation and IFN-γ secretion. Therefore, the role of CLU on NK cells should be taken into consideration for the previously observed, diverse functions of CLU in chronic inflammatory and autoimmune conditions.

Influence of NKG2D/DNAM-1 interaction with tumor cell ligands on the ability of lenalidomide to enhance ADCC of antibody-coated solid tumor cells.

e21008 Background: Lenalidomide has been shown to enhance the antibody- dependent cellular cytotoxicity (ADCC) of rituximab-treated B cell chronic lymphocytic leukemia (B-CLL) and non-Hodgkin lymphoma (NHL) tumor cells. The combination appears to be especially active in patients so we sought to build on these observations by determining lenalidomide's ability to enhance ADCC of solid tumor cells. Methods: Purified NK cells were used as effector cells and tumor cell lines were used as targets after pre-treatment with cetuximab or trastuzumab. 23 tumor cell lines were used; 11 colorectal (CRC), 3 breast, 3 ovary, 2 lung, 2 head and neck and 2 bone sarcoma. Cytotoxicity was assessed using a standard lactate dehydrogenase release assay and specific lysis calculated. NK cell expression of activating receptors from 30 donors and tumor cell expression of NK cell ligands were assessed by flow cytometry. Results: Lenalidomide increased NK cell-specific lysis of cetuximab-coated CRC cells; HCT- 116, from 19% to 39% (p < 0.01), HT-29, 32% to 50% (p < 0.05) and also of trastuzumab-coated breast cancer cells; SKBR-3, 32% to 44% (p < 0.01), MCF-7, 30% to 70% (p < 0.01). Variable sensitivity of CRC cell lines was dependent on EGFR expression (range 5%-96%) but independent of either KRAS or BRAF mutational status or FCγRIIIa genotype. CRC cell line expression of NK cell ligands, PVR, MIC-A, ULBP2 and ULBP3 correlated with sensitivity to ADCC. NK cell expression of DNAM-1, NKG2D, but not NKp46, correlated with the extent of ADCC and its enhancement by lenalidomide. Antibody blocking of NKG2D, and to a lesser extent DNAM-1, inhibited CRC cell killing from 70% to 31% and 48% respectively (both p < 0.05), whereas blocking of individual cognate ligands had minimal effect. Lenalidomide enhanced NK cell production of gmcsf, IL-8, MIP-1α/βm RANTES, MCP-1 and IP-10 but reversed phosphorylation of Src family kinases induced by antibody alone. Conclusions: Lenalidomide enhances the ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D/NKG2D ligand interactions, providing a rationale for exploratory clinical studies and assessment of potential biomarkers predictive lenalidomide-mediated ADCC. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Celgene Celgene

Chronic Cigarette Smoke Exposure Primes NK Cell Activation in a Mouse Model of Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a debilitating, progressive lung disease punctuated by exacerbations of symptoms. COPD exacerbations are most often associated with viral infections, and exposure to cigarette smoke (CS) followed by viral infection has been shown experimentally to enhance lung inflammation, tissue destruction, and airway fibrosis. Despite this, however, the cellular mechanisms responsible for this effect are unknown. In this study, we examined NK cell function in a mouse model of COPD given the vital role of NK cells following viral infection. Ex vivo stimulation of lung leukocytes with poly(I:C), ssRNA40, or ODN1826 enhanced production of NK cell-derived IFN-gamma in CS-exposed mice. NK cells from CS-exposed mice exhibited a novel form of priming; highly purified NK cells from CS-exposed mice, relative to NK cells from filtered air-exposed mice, produced more IFN-gamma following stimulation with IL-12, IL-18, or both. Further, NK cell priming was lost following smoking cessation. NKG2D stimulation through overexpression of Raet1 on the lung epithelium primed NK cell responsiveness to poly(I:C), ssRNA40, or ODN1826 stimulation, but not cytokine stimulation. In addition, NK cells from CS-exposed mice expressed more cell surface CD107a upon stimulation, demonstrating that the NK cell degranulation response was also primed. Together, these results reveal a novel mechanism of activation of the innate immune system and highlight NK cells as important cellular targets in controlling COPD exacerbations.

Abstract 2448: h224G11, a humanized whole antibody targeting the c-Met receptor, induces c-Met down-regulation and triggers ADCC functions

Abstract c-Met is the prototypic member of a sub-family of receptor tyrosine kinases (RTK) which also includes RON and SEA. The c-Met RTK family is structurally different from other RTK families and is the only known high-affinity receptor for hepatocyte growth factor (HGF), also called scatter factor (SF). While the controlled regulation of c-Met and HGF are essential for in mammalian development, tissue maintenance and repair, their deregulation is implicated in the progression of cancers. Aberrant signalling of c-Met can arise by ligand-dependent and independent mechanisms such as over-expression of c-Met, paracrine or autocrine activations, or through gain of function mutations. However, an oligomerization of the c-Met receptor, either in the presence or absence of the ligand, is required to regulate the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. An inappropriate c-Met activation plays a crucial role in tumorigenesis and metastasis. We have generated, a humanized anti-c-Met IgG1 monoclonal antibody (h224G11) that displays fully antagonist activities when used as a whole antibody. The h224G11 Mab exhibits a high affinity for the receptor, blocks HGF binding, inhibits c-Met phosphorylation and c-Met dimerization. These effects are presumably responsible for the inhibition of the major functions of c-Met including cell proliferation, migration, invasion but also cell scattering and angiogenesis, observed in vitro with different cell lines. In addition to these in vitro properties, h224G11 dramatically inhibits in vivo growth of autocrine (U87-MG), partially autophosphorylated (NCI-H441) and c-Met amplified cell lines (MKN45, EBC1 and Hs746T). Recent in vitro experiments demonstrate that h224G11 down-regulates c-Met on many cells including A549 and NCI-H441. Using an ELISA immunoassay, it has been shown that this down-regulation does not result from a shedding process but that h224G11 induces c-Met internalization and degradation. Similar results were obtained in ex-vivo experiments using Hs764T tumors from xenografted mice. In addition to its direct effect on c-Met modulation, the humanized antibody also triggers effector functions. A549, NCI-H441 and Hs746T were used as target cells in an ADCC chromium-released assay with human purified NK cells. In these assays, the h224G11 demonstrates a significant ADCC activity. Taken together the in vitro and in vivo data suggest that the h224G11 antibody targeting c-Met is a promising candidate for the treatment of ligand-dependent and ligand-independent tumors as a single or combined therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2448.