Pretreatment Of NK Cells Research Articles

NK-клетки, предобработанные эстриолом и клетками комменсальной флоры, регулируют созревание Treg и Th17 из CD4+ лимфоцитов у больных рассеянным склерозом и здоровых доноров

Multiple sclerosis (MS) is an inflammatory chronic disease of the central nervous system, one of the pathogenetic mechanisms of which is an immune-mediated attack to the myelin sheath of nerve axons under the control of autoreactive T cells. NK cells, one of subtypes of ILC cells, have the ability to regulate autoimmune mechanisms through the production of cytokines or cytotoxic effects directly against effector cells, such as autoreactive T cells or antigen-presenting cells. At the same time, they can also lyse microglial, astrocyte, and oligodendrocyte cells, indicating a dual function of NK cells in MS. The influence of the pregnancy hormone estriol (E3) and commensal microflora on the ability of NK cells to modulate the level of T regulatory (Treg) and IL-17-producing (Th17) lymphocytes from patients with multiple sclerosis (MS) in comparison with healthy donors was studied. To activate NK cells, Ecsherichia coli K12 and Lactobacillus plantarum 8R-A3 strains were used. The phenotype of Treg and Th17 cells was assessed by flow cytometry using monoclonal antibody staining. It was found that NK cells incubated with E3 and E. coli stimulate the formation of Tregs, and pretreatment of NK cells with L. plantarum inhibits the formation of Th17 from CD4+ lymphocytes. In general, regulatory lymphocytes from MS patients turned out to be more sensitive to the influence of NK cells than cells from healthy donors.

Abstract 2903: Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma

Abstract Osteosarcoma is the most common primary malignant bone tumor. It metastasizes almost exclusively to the lung, and this constitutes the main cause of death in these patients. Immune therapy has shown limited benefit. We investigated whether epigenetic modulation of allogeneic NK cells by the HDAC inhibitor MS-275 will improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma (OS) lung metastases. Human NK cells were purified and expanded from donor buffy coats using recombinant human IL-2 and genetically engineered K562 feeder cells and treated with MS-275 before injection into mice. RNA seq was performed in untreated and MS-275 pre-treated NK cells to determine differentially expressed genes. We also used ChiPseq to determine the histone post-translational modifications associated with the differentially expressed genes induced by the treatment. Culture media from untreated and treated NK cells was collected for cytokine analysis using the Isolight/Isoplexis system. We used our OS-17 human OS experimental lung metastases mouse model and injected untreated or pre-treated NK cells (0.5uM MS-275) IV labeled with near-infrared DiR dye twice a week. Tumor burden was assessed, and NK cells were tracked by Bioluminescence imaging (BLI). Mice were either euthanized 24 hours after the final NK cell treatment for histologic analysis of the lungs or, in a second experiment, analyzed for survival across treatment groups. Some mice from each group were selected for 3D imaging using the Xerra cryofluorescence tomography (CFT) imaging system, allowing for greater resolution of NK cell distribution as compared to the IVIS imaging system. We identified 547 differentially expressed genes between the untreated and MS-275 treated NK cells. Four immune related genes (L1CAM, IFNG, C3 and CD28) were upregulated in the MS-275 treated NK cells and histone H3 and H4 acetylation was found at the identified immunomodulatory genes. Culture media from MS-275 treated NK cells showed significantly increased MIP-1b, IL-6 and IFN-g. Furthermore, there was a significant decrease in lung micro-metastases in the MS-275 pre-treated NK cell group as compared to the untreated control group (199 vs 97 p=0.05). Lastly, Xerra CFT imaging demonstrated greater DiR signal in the lungs of the mice that received MS-275 treated NK cells compared to those that received untreated NK cells, suggesting that MS-275 might lead to greater persistence of NK cells at tumor sites. We conclude that MS-275 has an immunomodulatory effect on NK cells (increase L1CAM, IFNG, C3 and CD28) and increases H3 and H4 acetylation which leads to transcriptional activation of the immune-related genes that contribute to NK cell increased cytolytic function. Pre-treatment of NK cells with MS-275 enhances NK cell therapeutic efficacy and localization and persistence in the lungs. Citation Format: Ariana Rupp Anjier, Nancy Gordon, Shinji Maegawa, Vidya Gopalakrishnan, Juan Bournat, Donghang Cheng, Yanwen Yang, Charles Kingsley, Adam Kulp. Epigenetic modulation of NK cells to improve tumor trafficking and enhance therapeutic efficacy against osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2903.

MRNA Transfection of NK Cells with Gain-of-Function CXCR4 As a Novel Method to Enhance the Homing of Adoptively Transferred NK Cells to the Bone Marrow for the Treatment of Hematological Malignancies

Introduction: Proof of principle that adoptively transferred NK cells can mediate regression of hematological malignancies has recently been established in the clinic. Despite recent advances in the field, the overall efficacy of NK-cell based immunotherapy remains limited. Directing cellular migration to tumor-bearing tissues could be used as a method to improve the efficacy of NK cell-based immunotherapy. As most hematological malignancies arise from bone marrow (BM) compartments, we investigated the potential of genetic modification of NK cells to express high levels of the BM homing chemokine receptor CXCR4 to improve NK cell migration to BM compartments in vivo.Methods: Human NK cells were expanded ex vivo in G-rex flasks for 14 days using irradiated EBV-LCL feeder cells and IL-2 containing media. Electroporation (EP) of NK cells with mRNA coding for the wild-type CXCR4 receptor (WT CXCR4) and the gain-of-function mutation CXCR4 receptor (CXCR4-R334X) was performed using the MaxCyte GT instrument. Cell viability and receptor expression was assessed by flow cytometry using a BD LSR II Fortessa. In vitro transwell migration assays towards the CXCR4 ligand SDF-1α were performed in serum-free media over 2 hours at +37¡C. Pretreatment of NK cells with 100 uM of plerixafor for 30 min at +4¡C prior to migration assays was used for CXCR4 blockade experiments. In vivo homing studies were performed with bioluminescence tracking of luciferase-transfected NK cells in NSG mice. Animals were imaged using an IVIS Bioluminescence imager 1 and 24 hours after adoptive NK cell transfer.Results: EP of ex vivo expanded NK cells with either WT CXCR4 or CXCR4-R334X mRNA both resulted in a substantial increase in CXCR4 surface expression for up to 36 hours compared to non-EP NK cell controls. In vitro assays showed CXCR4-R334X transfected NK cells had superior migration to SDF-1α compared to both WT CXCR4 transfected and control NK cells, with an average 40% increase in their migration capacity towards SDF-1α compared to non-transfected NK cells (n=10 donors). This augmented migration capacity was abrogated when the CXCR4 receptor was selectively blocked with plerixafor. To confirm that CXCR4-R334X modified NK cells had improved BM homing capacity in vivo, we compared the distribution of these cells using bioluminescent imaging (BLI) after transfection with luciferase mRNA and intravenous injection into NSG mice (n=3). Twenty-four hours after adoptive transfer, CXCR4-R334X mRNA EP NK cells had improved homing to BM compartments such as the vertebrae, sternum ribs, and femurs compared to their unmodified NK cells counterparts (figure).Conclusions: The data demonstrate that genetic modification of NK cells with CXCR4-R334X mRNA can be utilized to efficiently direct their homing of infused NK cells to BM compartments in vivo. We hypothesize that CXCR4-modified NK cells can be utilized to improve the efficacy of adoptive NK cell immunotherapy for patients with BM-residing malignancies such as leukemia and multiple myeloma.Emily R. Levy is a predoctoral candidate in the Molecular Medicine program of Institute for Biomedical Sciences at the George Washington University. This work is from a dissertation to be presented to the above program in partial fulfillment of the requirements for the Ph.D. degree. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Valproic acid upregulates NKG2D ligand expression and enhances susceptibility of human renal carcinoma cells to NK cell-mediated cytotoxicity

IntroductionWe aimed to investigate the effect of valproic acid (VPA) on NKG2D ligand expression in human renal carcinoma cell lines and to investigate the mechanisms.Material and methodsDifferent concentrations of VPA from 0.5 mM to 8.0 mM were applied to 786-O and ACHN cell lines, respectively. Cell viability after treatment with VPA was determined by flow cytometry (FCM). Real-time PCR and FCM were used to detect the changes of mRNA and protein level of NKG2D ligands (MICA/B and ULBPs) in the two cell lines treated with 4 mM VPA. The cytotoxicity assay and CD107a mobilization assay were carried out to detect the cytotoxicity changes of NK cells against renal carcinoma cell lines after the same treatment.ResultsValproic acid can efficiently upregulate MICA/B, ULBP1 and ULBP2 expression in the renal carcinoma cell lines at the mRNA and protein level (p < 0.05). 786-O and ACHN cells treated with VPA were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the pretreatment of NK cells with anti-NKG2D monoclonal antibodies (p < 0.05).ConclusionsValproic acid can clearly induce the expression of NKG2D ligands of renal carcinoma cell lines, thereby enhancing the cytotoxicity of NK cells against renal carcinoma cell lines.

Effect of lenalidomide and pomalidomide combined with IgG1-isotype antibodies on antibody-dependent cellular cytotoxicity (ADCC) via cytokine signaling and effector cell granzyme B and FasL expression

3058 Background: Lenalidomide and pomalidomide (CC-4047) display anti-angiogenic and immunomodulatory activities in a variety of in vitro assay systems. Evidence from single agent patient studies indicate enhancement of T cell and NK cell function in patients either with hematological or solid tumors. There is also in vitro and preclinical evidence that both drugs can enhance NK cell mediated ADCC. Methods: We have utilized an in vitro ADCC system to investigate the mechanism(s) whereby lenalidomide and pomalidomide are able to enhance human NK cell and monocyte effector function in response to FCγ receptor signaling initiated by therapeutic antibodies. Results: Pre-treatment of NK cells or monocytes with pomalidomide or lenalidomide (but not thalidomide) greatly enhanced ADCC of Her2/neu overexpressing breast cancer cells pre-coated with trastuzumab and EGFR positive colorectal cancer cells pre-coated with cetuximab. NK cell and monocyte-mediated killing of antibody coated tumor cells was dose-dependent. There was minimal tumor cell killing by antibody alone or by either drug alone or in the presence of panitumumab, an anti-EGFR IgG2a antibody that does not efficiently engage NK cell FCγ receptors. NK cell-mediated killing is associated with distinct changes in intracellular signaling (decreased pSHIP-1, increased PLCγ2, pERK) and increased granzyme B and FasL expression. Monocyte-mediated ADCC takes longer to initiate (16 hrs vs 4 hrs) is also associated with increased granzyme B and FasL and is blocked by anti-IL-12mAb. ADCC is completely blocked by an inhibitor of granzyme B activity and partially blocked by an antibody that blocks FasL-Fas interaction. Conclusions: Pomalidomide and lenalidomide (but not thalidomide) strongly enhance the ability of therapeutic IgG1 antibodies to induce ADCC via NK cell/monocyte-mediated killing of tumor cells in vitro. Furthermore, granzyme B activity and concurrent cytokine signaling is crucial to enable the synergistic combination of either drug with antibodies to tumor-specific surface antigens the potential utility of which is being assesses in upcoming clinical studies. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Celgene Corp Celgene Corp

Rapid and potent induction of cell death and loss of NK cell cytotoxicity against oral tumors by F(ab')2 fragment of anti-CD16 antibody.

Freshly isolated untreated NK cells undergo rapid apoptosis and lose their cytotoxic function upon the addition of F(ab')2 fragment of anti-CD16 antibodies. Loss of NK cell cytotoxic function after treatment with F(ab')2 fragment of anti-CD16 antibody can be seen against K562 and UCLA-2 oral tumor cells when either added immediately in the co-cultures of NK cells with the tumor cells or after pre-treatment of NK cells with the antibody before their addition to the tumor cells. Addition of Interleukin-2 (IL-2) in combination with anti-CD16 antibody to NK cells delayed the induction of DNA fragmentation in NK cells, and even though decreased cytotoxicity could still be observed against K562 and UCLA-2 oral tumors when compared to IL-2 alone treated NK cells, the cytotoxicity levels remained relatively higher and approached those obtained by untreated NK cells in the absence of antibody treatment. No increases in IFN-gamma, Granzymes A and B, Perforin and TRAIL genes could be seen in NK cells treated with anti-CD16 antibody. Neither secretion of IFN-gamma nor increased expression of CD69 activation antigen could be observed after the treatment of NK cells with anti-CD16 antibody. Furthermore, IL-2 mediated increase in CD69 surface antigens was down-modulated by anti-CD16 antibody. Finally, the addition of anti-CD16 antibody to co-cultures of NK cells with tumor target cells was not inhibitory for the secretion of VEGF by oral tumor cells, unlike those co-cultured with untreated or IL-2 treated NK cells. Thus, binding and triggering of CD16 receptor on NK cells may enhance oral tumor survival and growth by decreased ability of NK cells to suppress VEGF secretion or induce tumor cell death during the interaction of NK cells with oral tumor cells.

Low Levels of Circulating CS1, a Newly Identified Multiple Myeloma (MM) Antigen for a Novel Humanized HuLuc63 Monoclonal Antibody, Is Detected in MM Patient Sera and Correlates with Active Disease.

A new humanized anti-CS1 mAb HuLuc63 significantly induces antibody-dependent cellular cytotoxicity (ADCC) against autologous CS1-expressing patient MM cells. Here we examined whether soluble CS1 is detectable in MM patient sera and affects HuLuc63-induced cytotoxicity against MM cells. Using a sandwich ELISA, CS1 was detected in 44% (23/52) of sera of 52 MM patients (1.2 to 35.3 ng/ml). Immunoprecipitation of sera from CS1 ELISA-positive and -negative MM patients using anti-CS1 mAbs (HuLuc63 and ChLuc90) followed by immunoblotting with HuLuc63 and 1G9 recognizing different epitopes of CS1, further confirmed two forms of CS1 only in CS1-positive MM patient sera. Detection of CS1 is associated with MM (p < 0.0001), since it is detected in MM patient sera, but not in MGUS (n=15) or in healthy donors (n=100). In additional sera of 199 MM patients with newly diagnosed MM, 90% (181/199) of MM patients have detectable CS1 (1 to >80 ng/ml). Median CS1 levels for International Stage System (ISS) I (n=100), II (n=53), and III (n=46) patients are 5.87, 9.37, and 8.37 ng/ml, respectively. The correlation between ISS and CS1 is moderate (spearman correlation coefficient = 0.197, p=0.005). Patients with ISS II/III had significantly higher CS1 levels compared with those with ISS I (median 9.0 vs. 5.9 ng/ml, p=0.006), suggesting a correlation of serum CS1 with active MM. Since patients with ISS II and III require therapy, while those with ISS I do not, these results suggest that circulating CS1 may indicate need for therapy and further support clinical investigation of anti-CS1 therapy using HuLuc63 in MM. Importantly, HuLuc63 has demonstrated significant dose-dependant activity, leading to reduced or eliminated human myeloma tumors (MM1S, OPM2, and L363) in xenograft mice models. Anti-MM activity of HuLuc63 in mice could be observed at sustained serum levels of >2 mg/ml, which is well above the levels of circulating CS1 protein observed in MM patients. Thus, it is proposed that serum CS1 will be an unlikely antibody sink in patients treated with optimal doses of HuLuc63. Since NK cells also express CS1, albeit at lower levels than MM cells, we next determined the effects of HuLuc63 on NK cell function. Pretreatment of NK cells with HuLuc63 (0.1 mg/ml) for 3 days did not alter NK-mediated ADCC against CS1-expressing MM cells (MM1S, H929, INA-6) via HuLuc63. Moreover, recombinant CS1-Fc at physiological serum levels (<200 ng/ml) did not significantly inhibit HuLuc63-induced ADCC against MM cells. Inhibition of HuLuc63-induced MM cell lysis by CS1-Fc was observed at higher concentrations (>200 ng/ml), confirming specific HuLuc63-CS1 binding. Together, these studies strongly support continued clinical investigation of HuLuc63 in MM.

Lenalidomide and pomalidomide strongly enhance tumor cell killing in vitro during antibody-dependent cellular cytotoxicity (ADCC) mediated by trastuzumab, cetuximab and rituximab

3023 Background: Potential mechanisms of action of lenalidomide and pomalidomide (CC-4047) include anti-angiogenic, anti- proliferative and immunomodulatory activities, e.g., enhancement of T cell and NK cell function. Both drugs appear to enhance T cell activation and Th1-type cytokines in cancer patients. Also, both drugs have been shown to enhance rituximab-mediated protection in a mouse lymphoma model. Methods: We have utilized an in vitro ADCC system to assess the ability of these drugs to enhance human NK cell function in response to the approved therapeutic antibodies trastuzumab, cetuximab and rituximab. Results: Pre-treatment of NK cells with either pomalidomide or lenalidomide greatly enhanced IFN-γ production by NK cells in response to IgG in the presence of either IL-2 or IL- 12. In a series of functional ADCC assays, Her2/neu overexpressing breast cancer cells (SKBR3 &amp; MCF-7) pre-coated with trastuzumab, EGFR positive colorectal cancer cells (HCT-116) pre-coated with cetuximab and NHL cell lines (Namalwa, Farage &amp; Raji) pre-coated with rituximab, were exposed to NK cells pre-treated with pomalidomide, lenalidomide or thalidomide. Both pomalidomide and lenalidomide synergistically increased (up to 6-fold) NK cell-mediated killing of antibody coated tumor cells in a dose-dependent manner. Thalidomide had no effect in this system. There was minimal tumor cell killing by antibody alone or in the absence of antibody. The presence of IL-2 or IL-12 was required to see enhanced killing by either drug. Enhanced ADCC was associated with increased signaling in NK cells, specifically with inhibition of the negative regulator SHIP-1. Other downstream effects on signaling pathways are also being investigated. Monocyte-mediated tumor cell ADCC was also enhanced by both drugs. Conclusions: We have shown that pomalidomide and lenalidomide (but not thalidomide) strongly enhance the ability of therapeutic antibodies to induce ADCC via NK cell/monocyte-mediated killing of tumor cells in vitro. These results provide a strong rationale for combination of either lenalidomide or pomalidomide with antibodies to tumor-specific surface antigens in cancer patients. No significant financial relationships to disclose.

Involvement of interaction between Fractalkine and CX3CR1 in cytotoxicity of natural killer cells against tumor cells

Many chemokine receptors are typically found on natural killer cells, including CX3CR1, the receptor for the chemokine fractalkine (FKN). This study explored whether interaction between CX3CR1 and FKN is relevant for NK cell functions in cytotoxicity against tumors. FKN expression was examined by polymerase chain reaction and CX3CR1 expression in NK cells was analyzed by flow cytometry. NK cell cytotoxicity was examined by 4-h 51Cr-release assay. FKN was expressed in a variety of tumor cell lines such as K562 cells, an NK-sensitive cell line. Approximately 90% of peripheral blood NK cells and almost all of the NK cell line, NK-92 cells, expressed CX3CR1. Anti-CX3CR1 antibody strongly neutralized the cytotoxicity of NK cells against K562 cells, and pretreatment of NK cells with recombinant soluble FKN improved the cytolytic function on tumor cells. This study demonstrates that an interaction between CX3CR1 on NK cells and FKN on tumor cells is involved in the natural cytotoxicity of NK cells against tumors.

Human NK cells express Fc receptors for IgA which mediate signal transduction and target cell killing.

Receptors for the Fc region of IgG (FcgammaRIIIa, FcgammaRIIc) and IgM (FcmicroR) were previously described on NK cells. In this work the expression of Fc receptors for IgA (FcalphaR) on human NK cells and the signaling events were investigated. The FcalphaR was demonstrated by flow cytometry using secretory IgA (sIgA) and anti-human IgA antibody. The percentage of NK cells (CD3(-)CD56(+)CD16(+)) expressing FcalphaR ranged between 55.7% and 95.7%, with a mean +/- SD of 75.2+/-11.8. The association constant and the number of (125)I-labeled sIgA ((125)I-sIgA) molecules bound per cell, calculated by Scatchard analysis, were 2 x 10(7) M(-1) and 1.7 x 10(4), respectively. The binding specificity was proved by inhibition experiments. Cold sIgA but not IgA Fab fragments were able to inhibit (125)I-sIgA binding in a concentration-dependent manner. Binding of sIgA to NK cells was neither inhibited by anti-mannose receptor antibody, nor by L-fucose, D-galactose, D-glucose, D-mannose or N-acetyl-D-glucosamine. Pretreatment of NK cells with polymeric IgA inhibited their capacity to kill (51)Cr-labeled K562 target cells by 34.8%, whereas with monomeric IgA only by 13.1%. Ligand-induced clustering of the FcalphaR resulted in activation of tyrosine kinases Lck, Syk and phosphatidylinositol 3-kinase. The present studies support the concept that human NK cells bind preferentially sIgA and polymeric IgA with moderate affinity via FcalphaR, which is different from the FcalphaRI/CD89 and other carbohydrate-recognizing receptors like mannose receptor/CD206. This novel structure mediates signal transduction and cell killing.

Recruitment of Pleckstrin and Phosphoinositide 3-Kinase γ into the Cell Membranes, and Their Association with Gβγ After Activation of NK Cells with Chemokines

Abstract The role of phosphoinositide 3 kinases (PI 3-K) in chemokine-induced NK cell chemotaxis was investigated. Pretreatment of NK cells with wortmannin inhibits the in vitro chemotaxis of NK cells induced by lymphotactin, monocyte-chemoattractant protein-1, RANTES, IFN-inducible protein-10, or stromal-derived factor-1α. Introduction of inhibitory Abs to PI 3-Kγ but not to PI 3-Kα into streptolysin O-permeabilized NK cells also inhibits chemokine-induced NK cell chemotaxis. Biochemical analysis showed that within 2–3 min of activating NK cells, pleckstrin is recruited into NK cell membranes, whereas PI 3-Kγ associates with these membranes 5 min after stimulation with RANTES. Recruited PI 3-Kγ generates phosphatidylinositol 3,4,5 trisphosphate, an activity that is inhibited upon pretreatment of NK cells with wortmannin. Further analysis showed that a ternary complex containing the βγ dimer of G protein, pleckstrin, and PI 3-Kγ is formed in NK cell membranes after activation with RANTES. The recruitment of pleckstrin and PI 3-Kγ into NK cell membranes is only partially inhibited by pertussis toxin, suggesting that the majority of these molecules form a complex with pertussis toxin-insensitive G proteins. Our results may have application for the migration of NK cells toward the sites of inflammation.

Preferential involvement of Go and Gz proteins in mediating rat natural killer cell lysis of allogeneic and tumor target cells.

IL-2-activated NK cells from PVG rats potently lyse target cells expressing allo-MHC class I determinants. Here, we investigated the role that G proteins play in mediating this activity. Pretreatment of NK cells with pertussis toxin (PT) or cholera toxin (CT) inhibited NK cell killing of tumor (YAC-1 or P815), and allogeneic target cells. ADP ribosylation assay revealed that PT ADP ribosylates a 39-kDa G protein, whereas CT ADP ribosylates a 45 to 47-kDa G protein in PVG NK cell membranes. Membranes prepared from intoxicated NK cells with either PT or CT lost their ability to incorporate [32P]NAD. These membranes possess Gi, Go, Gs, and Gz as demonstrated by immunoblot analysis. However, Gq was not clearly detected by this method. IL-2-activated NK cells were permeabilized with streptolysin O. Permeabilized cells incorporated Abs to Gi, Go, Gz, Gs, and Gq as determined by flow cytometric analysis. When Abs to Go or Gz, but not to Gi, Gs, or Gq, were incorporated inside permeabilized NK cells, a significant reduction in the lysis of tumor or allo-MHC target cells was observed, suggesting that Go and Gz play important roles in transducing the signals necessary to lyse target cells. Our results show for the first time a role for G proteins in mediating NK cell killing of allo-MHC-encoded target cells, and provide evidence for Gz protein involvement in NK cell recognition of target cells. The effect of Gz is novel and has not been previously described in any other system or cell type.

Stimulation of protein tyrosine phosphorylation by interaction of NK cells with fibronectin via alpha 4 beta 1 and alpha 5 beta 1.

Recent evidence indicates that integrins do transduce signals that are involved in the regulation of a number of cellular processes. We have shown previously that human NK cells express alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate their adhesion to fibronectin (FN). Here we investigate whether cross-linking of beta 1 FN receptors on human NK cells stimulates tyrosine kinase activation. Our results indicate that cross-linking of beta 1 integrins on NK cells, either freshly isolated from PBL or generated from 10-day coculture of nonadherent PBMC with an irradiated EVB+ lymphoblastoid B cell line (long-term activated NK cells), altered the tyrosine phosphorylation pattern. In particular, we found stimulation of tyrosine phosphorylation of two proteins migrating with an apparent mass of 105 and 115 kDa, which was not observed after CD16 engagement. Phosphorylation of pp105-115 was already observed at 1 min, raised maximal values at 3 to 5 min, and persisted until 20 min after stimulation. Tyrosine phosphorylation of pp105-115 was also observed upon engagement of alpha 4 beta 1 and alpha 5 beta 1 FN receptors either with mAb directed against the alpha subunits or after NK cell adhesion to FN or its 120- and 40-kDa fragments. Pretreatment of NK cells with the tyrosine kinase inhibitor herbimycin A resulted in marked decrease of phosphorylation stimulated through alpha 4 beta 1 and alpha 5 beta 1 integrins, indicating that ligation of FN receptors on NK cells activates tyrosine kinase(s). Overall our results suggest that beta 1 integrins on NK cells play a major role as signaling molecules in the regulation of NK cell functions.

GTP-binding proteins transduce signals generated via human FC gamma receptor IIIA (CD16).

This study demonstrates that GTP-binding proteins regulate Fc gamma RIII-mediated signal transduction and inositol phosphate (IPn) generation in human NK cells. In addition the cross-linking of CD16 by mAb, guanosine 5'-o-3-thiophosphate induced 1,4,5 inositol trisphosphate (IP3) release in permeabilized NK cells and their membranes. By contrast, guanosine 5'-o-2-thiophosphate, almost completely inhibited IP3 generation induced by cross-linking with anti-CD16 mAb. Pretreatment of NK cells with 10 to 100 ng/ml Vibrio cholerae toxin (Ctx) almost completely inhibited the generation of IP3 and of other Ipn as well as Fc gamma RIII-operated cell functions such as antibody-dependent cell-mediated cytotoxicity against antibody-coated P815 mastocytoma cells. Isolated B subunit of Ctx was inactive. Bordetella pertussis toxin (0.1 to 1 microgram/ml) only marginally affected IP3 release and antibody-dependent cell-mediated cytotoxicity. Ctx increased cAMP levels in NK cells. However, inhibition of IP3 release preceded the rise of cAMP. Moreover, cAMP analogues (8-chlor-cAMP, 8-bromo-cAMP, dibutiryl-cAMP), as well as intracellular cAMP-enhancing agents (PGE1, PGE2, and forskolin) did not mimicked the effects of Ctx on IP3 generation, suggesting that the adenylate cyclase pathway is not responsible for the early effects of Ctx on Fc gamma RIII-mediated signalling. Overall these results demonstrate that signal transduction via Fc gamma RIII is mediated by Ctx-sensitive cellular membrane GTP-binding protein.