Human Natural Killer (NK) Cells: Differential Expression of Phosphatase and Tensin Homologue Deleted On Chromosome Ten (PTEN) During NK Cell Development Regulates Its Cytolytic Activity Against Leukemic Target Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 254-254
Author(s):  
Edward L. Briercheck ◽  
Rossana Trotta ◽  
Jordan P. Cole ◽  
Tyler D. Cole ◽  
Alex S. Hartlage ◽  
...  
Keyword(s):  
Nk Cells ◽  
Differential Expression ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Cell Development ◽  
Cytolytic Activity ◽  
Negative Regulator ◽  
Target Cells ◽  
Fluorescent Intensity ◽  
Stage 4

Abstract Abstract 254 Human NK cells are CD56(+)CD3(-) large granular lymphocytes characterized by the ability to directly kill virally infected or malignantly transformed cells. Five stages of human NK cell development can be identified in secondary lymphoid tissue. Stage 4 NK cells are immature CD56bright and have poor cytolytic activity against sensitive leukemic target cells at rest while stage 5 NK cells are mature CD56dim and have potent cytolytic activity against the same leukemic cells at rest. Both stage 4 CD56bright and stage 5 CD56dim NK cells can be found circulating in blood. We sought to determine the mechanism responsible for this different cytolytic activity by exploring changes in gene expression between CD56bright NK cells and CD56dim NK cells. We first observed that CD56bright NK cells expressed ∼5 fold greater amounts of PTEN protein over CD56dim NK cells by western blot (n=5, p < 0.04). Given that PTEN is a lipid phosphatase that opposes the PI3K/AKT pathway, we hypothesized that it may negatively regulate NK cell cytolytic activity. In order to test this, we used lentiviral infection to overexpress PTEN in the human NK cell line NK-92. Relative to cells infected with an empty lentiviral vector, NK-92 cells with overexpression of PTEN showed decreased cytotoxicity against sensitive leukemic target cells by at least 25% at all effecter:target ratios (n=4, p < 0.02). Next, we infected primary human NK cells with the same vector and showed an approximately 30% reduction in degranulation of cytolytic mediators as determined by CD107a mean fluorescent intensity (M.F.I.) when cultured with sensitive leukemic target cells (n=4, p<.08). Consistent with this, murine NK cells were isolated from FVB mice with a heterozygous germline deficiency in Pten (Pten+/−) and showed at least a 25% increase in cytotoxicity against sensitive lymphoma target cells (n=4, p<.0001). At the molecular level, when NK-92 cells overexpressing PTEN were bound to their sensitive leukemic target cells, they showed decreased activation along the AKT and ERK1/2 signaling pathways, which are known as positive regulators of NK cell cytotoxicity. Collectively, we identify a functional role for the tumor suppressor PTEN in normal human NK cell development. We demonstrate differential expression of PTEN in immature and mature human NK cells, show that it is a negative regulator of NK cell cytolytic activity, and suggest that this differential expression may in fact account for the difference in baseline cytolytic activity found in the CD56bright and CD56dim human NK cell subsets, possibly via the AKT and ERK1/2 pathways. Disclosures: No relevant conflicts of interest to declare.

Costimulatory Effects of Interleukin-18 (IL-18) on Human Natural Killer (NK) Cells Activated through Fc Receptors for Immunoglobulin (IgG)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2780-2780
Author(s):  
Shivani Srivastava ◽  
Hailin Feng ◽  
Menggang Yu ◽  
David Pelloso ◽  
Michael Robertson
Keyword(s):  
Monoclonal Antibodies ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Fc Receptors ◽  
Cytolytic Activity ◽  
Mitogen Activated Protein Kinase ◽  
Target Cells

Abstract Abstract 2780 NK cells play an important role in innate and adaptive immune responses. Most human NK cells express CD16, an Fc receptor for IgG that mediates lysis of antibody-coated target cells and costimulates interferon (IFN)-g production in response to cytokines. IL-18 is an immunostimulatory cytokine with antitumor activity in preclinical animal models. The effects of IL-18 on human NK cell function were examined. Here we show that NK cells stimulated with immobilized IgG in vitro secreted IFN-g; such IFN-g production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-g production by NK cells stimulated with immobilized IgG or CD16 antibodies (Figure 1). NK cell IFN-g production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk, extracellular signal-related kinases (ERK), p38 mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3-K). Stimulation with IL-18 or immobilized IgG could augment IL-12-induced IFN-g production by STAT4-deficient lymphocytes obtained from lymphoma patients after autologous stem cell transplantation (Figure 2). IL-18 also augmented the in vitro lysis of rituximab-coated Raji cells by human NK cells (Figure 3). These observations that IL-18 can co stimulate IFN-g production and cytolytic activity of NK cells activated through Fc receptors makes it an attractive cytokine to combine with monoclonal antibodies for treatment of cancer. Disclosure: No relevant conflicts of interest to declare.

scholarly journals GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1802
Author(s):  
Nayoung Kim ◽  
Mi Yeon Kim ◽  
Woo Seon Choi ◽  
Eunbi Yi ◽  
Hyo Jung Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Negative Regulator ◽  
Target Cells ◽  
Dependent Manner ◽  
Cell Functions ◽  
Cell Based Therapy ◽  
Activating Receptors ◽  
Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

Pseudoexpression of Glucocorticoid-Induced TNF-Related (GITR) Ligand Upon Coating of Cancer Cells by Platelets Impairs NK Cell Anti-Tumor Reactivity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3193-3193 ◽  
Author(s):  
Theresa Placke ◽  
Lothar Kanz ◽  
Helmut R. Salih ◽  
Hans-Georg Kopp
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Substantial Reduction ◽  
Cell Activity ◽  
Target Cells ◽  
Tumor Immune Escape ◽  
Tumor Spread

Abstract Abstract 3193 NK cells as part of the innate immune system substantially contribute to cancer immune surveillance. They prevent tumor progression and metastasis due to their ability to mediate cellular cytotoxicity and to produce cytokines like IFN-γ, which, among others, stimulates subsequent adaptive immune responses. NK reactivity results from an integrative response emerging upon recognition of multiple ligands for activating and inhibitory NK cell receptors including various members of the TNFR family. Apart from the direct interaction with their target cells, NK cell activity is further influenced by the reciprocal interplay with various other hematopoietic cells like e.g. dendritic cells. Metastatic tumor spread in experimental animals is dramatically reduced in thrombopenic mice. Additional depletion of NK cells reverses this effect, indicating that platelets may impair NK anti-tumor reactivity. However, the underlying mechanisms have not been fully elucidated, especially in humans. Recently, we demonstrated that NK anti-tumor immunity is impaired by platelet-derived TGF-β, which is released upon interaction of platelets with tumor cells (Kopp et al., Cancer Res. 2009). Here we report that the ligand for the TNFR family member GITR (GITRL) is upregulated on megakaryocytes during maturation resulting in substantial GITRL expression by platelets. Since we recently identified GITR as inhibitory NK receptor involved in tumor immune escape (e.g., Baltz et al., Blood 2008, Baessler et al., Cancer Res. 2009) we investigated how platelet-derived GITRL influences platelet function and NK immune surveillance. Signaling via GITRL into platelets upon interaction with NK-expressed GITR or recombinant GITR-Ig fusion protein did not alter platelet activation as revealed by analysis of the activation marker CD62P and release of TGF-β. Interestingly, we found that GITRL-negative tumor cells rapidly get coated by platelets, which confers a seemingly GITRL-positive phenotype. “GITRL pseudoexpression” on tumor cells caused a substantial reduction of NK cell cytotoxicity and cytokine production. This reduced NK reactivity was not due to induction of apoptosis via GITR and could be restored by addition of a blocking GITR antibody. Thus, coating of tumor cells by platelets inhibits NK reactivity, which is in part mediated by platelet-derived GITRL. Our data provide a functional basis for the previously observed finding that platelets increase metastasis i.e. by enabling evasion of tumor cells from NK-mediated immune surveillance. Disclosures: No relevant conflicts of interest to declare.

Over-Expression of CD200 In Acute Myeloid Leukemia Mediates the Expansion of Regulatory T-Lymphocytes and Directly Inhibits Natural Killer Cell Tumor Immunity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 491-491 ◽  
Author(s):  
Steven J Coles ◽  
Stephen Man ◽  
Robert Hills ◽  
Eddie CY Wang ◽  
Alan Burnett ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Nk Cells ◽  
Tumor Immunity ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Over Expression ◽  
Regulatory T Lymphocytes ◽  
Acute Myeloid

Abstract Abstract 491 CD200 is a type-1 transmembrane glycoprotein which suppresses inflammatory and autoimmune responses by signalling through its cognate transmembrane receptor homologue (CD200R). Normally, CD200 expression is restricted to immune privileged sites where it enhances immune tolerance through mechanisms that include modulating the expansion of FOXP3+ regulatory T-lymphocytes (T-regs) and suppressing macrophage cytolytic activity. Furthermore, leukocyte associated CD200 has been reported to suppress Natural Killer (NK) cell activity in vivo. Pathologically, we have previously shown that CD200 over-expression on leukemic blasts in around 50% of acute myeloid leukemia (AML) patients is significantly associated with a poor overall survival (Tonks et al, Leukemia, 2007). Given the existing evidence that T-reg frequency and NK cell function influence blast clearance and long-term survival in AML, we investigated the possibility that CD200 expression in AML may be directly suppressing anti-tumor immunity in this disease. Here we present evidence that CD200+ AML can suppress host anti-tumor responses by augmenting the frequency of AML patient T-regs and by direct inhibition of NK cell anti-tumor activity. We also show that targeting the interaction between CD200 and its receptor might provide a new strategy for the treatment of AML. Bone marrow aspirates from 91 diagnostic AML patients were analysed by multiparameter flow cytometry for blast CD200 protein expression. We found that the level of blast CD200 expression directly correlated with an increased frequency of T-regs (CD4+CD25++FoxP3+; R=0.78, p=0.0008). Measuring 3H-thymidine incorporation, we show that T-regs isolated from AML patients by MACS® separation inhibited T-cell proliferation (induced by CD3 and CD28 stimulation) at ratios <0.1%, thus confirming that patients T-regs were functional. In contrast to T-regs, NK cell frequency (CD45+CD19−CD3−CD56+) did not correlate with the level of AML blast CD200 expression (R=0.15, p=0.851), however, NK cell subpopulation bivariate analysis using CD56 and CD16 demonstrated that the CD56dimCD16+ (the principle active NK population) was significantly reduced by over 50% in CD200+ AML patients (36±5% compared to 15±5%, p=0.009). Furthermore, CD200 expression on target cells appeared to have a direct effect on the cytotoxic activity of NK cells; co-culture of NK cells with CD200+ targets resulted in decreased CD107a expression (a marker for cytolytic granules) in NK cells (23±4% vs 12±5%, p=0.038) and decreased apoptosis of the target cells (19±1% vs 10±1%, p=0.041). Since CD200R was detected on NK cells in AML patients, it was likely that CD200 was having a direct effect on suppression of NK cytotoxicity. This was supported by the significant recovery of NK cytolytic activity against CD200+ blasts in the presence of a CD200 blocking antibody (5±1% vs 11±2% CD107a+ NK cells, p=0.046) whereas there was no change seen with CD200− blasts (19±4% vs 19±3%). In conclusion, these findings suggest that CD200 expression on leukemic blasts plays an influential role in suppressing anti-tumor immunity in AML patients through modulating the expansion of functionally suppressive T-regs and directly suppressing NK cell cytolytic activity. In this study blocking CD200 interaction with its receptor was able to recover a significant proportion of patient NK activity, making CD200 a potential therapeutic target for CD200+ AML. Disclosures: No relevant conflicts of interest to declare.

Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1537-1537 ◽  
Author(s):  
Julia Hilpert ◽  
Katrin Baltz-Ghahremanpour ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Gundram Jung ◽  
...  
Keyword(s):  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 1537 The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures: No relevant conflicts of interest to declare.

Expression and Immunomodulatory Function of RANKL in Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 245-245
Author(s):  
Benjamin J Schmiedel ◽  
Tina Baessler ◽  
Miyuki Azuma ◽  
Lothar Kanz ◽  
Helmut R. Salih
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Immune Surveillance ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cytotoxic Lymphocytes ◽  
Hematopoietic Malignancies ◽  
Immunomodulatory Function

Abstract Abstract 245 The TNF family member RANKL and its receptors RANK and osteoprotegerin (OPG) are key regulators of bone remodelling, but have also been shown to influence progression of malignancies like breast cancer (Tan et al., Nature 2011), myeloma (Sordillo et al., Cancer 2003) and CLL (Secchiero et al. J Cell Physiol. 2006). NK cells are cytotoxic lymphocytes that play an important role in tumor immune surveillance especially of hematopoietic malignancies. Their reactivity is influenced by a variety of activating and inhibitory molecules expressed by their target cells including several members of the TNF family. Recently, we reported that RANK, upon interaction with RANKL which can be expressed by malignant hematopoietic cells, mediates signals that impair NK reactivity (Schmiedel et al., Blood 2010 116,21:893–893). Here we extended these analyses and comprehensively studied the expression and immunomodulatory function of RANKL in leukemia. Analysis of primary leukemia cells revealed substantial RANKL surface expression in a high proportion of the investigated cases (AML, 47 of 65 (72%); ALL, 16 of 21 (76%); CML, 6 of 10 (60%); CLL, all 54 (100%)). Signaling via surface-expressed RANKL into the malignant cells mediated the release of cytokines like TNF, IL-6, IL-8 and IL-10 which have been shown to act as autocrine and paracrine growth and survival factors in leukemia. Moreover, the factors released upon RANKL signaling upregulated RANK expression on NK cells. In line, NK cells from leukemia patients (n=75) displayed significantly (p<0.001, Mann-Whitney U-test) higher RANK expression compared to healthy controls (n=30) confirming our notion that RANK-RANKL interaction may contribute to leukemia pathophysiology. We further found that RANK-RANKL interaction, beyond directly inhibiting NK cell function via RANK, may contribute to evasion of leukemia cells from NK immunosurveillance by creating an NK inhibitory cytokine milieu. This was revealed by impaired cytotoxicity and degranulation in response to leukemia targets following exposure of the NK cells to the factors released upon RANKL signaling by leukemia cells. Notably, the RANKL-mediated cytokine release of leukemia cells could be disrupted by the clinically approved RANKL antibody Denosumab/AMG162. Thus, RANKL signaling may trigger a “vicious cycle” comprising of release of immunosuppressive cytokines and also upregulation of RANK on NK cells. The latter both directly inhibits NK reactivity and may result in augmented RANKL signaling into leukemia cells. Our data suggest that therapeutic modulation of the RANK/RANKL system e.g. with Denosumab/AMG162, which is approved for treatment of osteolysis, may be a promising strategy to reinforce NK reactivity against hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Engineering Anti-AML Antibodies for Improved NK Cell ADCC

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3629-3629
Author(s):  
Vladimir Senyukov ◽  
William Kelton ◽  
Nishant Mehta ◽  
George Georgiou ◽  
Dean Lee
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Adoptive Immunotherapy ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Cell Culture Supernatant ◽  
Current Therapy ◽  
Target Cells ◽  
Wild Type ◽  
Related Donor

Abstract Abstract 3629 Acute myeloid leukemia (AML) is an aggressive malignancy for which current therapy fails to provide durable remission in approximately half of cases. Natural killer (NK) cells, as a key component of innate immunity, have recently shown clinical potential for adoptive immunotherapy against AML, particular when the donor and recipient are KIR mismatched. In addition to patients who do not have a suitable related donor, approximately 30% of patients bear all three families of KIR ligands and therefor cannot benefit from KIR mismatch. Thus, finding a related donor with predicted KIR mismatch is a major obstacle for adoptive NK cell immunotherapy. The majority of peripheral blood NK cells express CD16a (FcγRIIIa), which is the most potent receptor among the activating receptors that NK cells posses. NK cells express CD16a in association with disulflde-linked homo- or hetero-dimers of FcRγ or CD3ζ. Clustering of CD16a initiated by binding to the Fc-portion of IgG1 or IgG3 that opsonize target cells induces signals strong enough to overcome KIR inhibition. Thus, combining NK cell adoptive immunotherapy with Abs against tumor antigens could help overcome the limitations of KIR mismatching. Indeed, many promising anticancer Abs have failed in clinical trials because of insufficient efficacy, which, at least in part, may result from low affinity CD16a binding. Indeed, it was shown that the affinity between Fc and FcγRs correlates with cytotoxicity in cell-based assays and that the Abs with optimized FcγR affinity induced strong cytotoxicity against targeted tumor cells. CD33 is expressed on the blast cells of most cases of AML and represents a suitable antigen for antibody-based therapies. Lintuzumab, an unconjugated, humanized anti-CD33 mAb (HuM195), failed to improve patient outcomes in two randomized trials when combined with conventional chemotherapy. Gemtuzumab ozogamicin, an anti-CD33 mAb conjugated to the calicheamicin, in combination with chemotherapy, improved survival in a subset of AML patients, but has been withdrawn from US market by safety concerns. We optimized the FcγR affinity of HuM195 mAb (mNuM195) by cloning into pMaz-IgH Herceptin recipient vector containing S239D, A330L, I332E mutations that, as previously shown, leads to significant improvement of IgG1 binding to CD16a. To generate control wild type variant (wHuM195) we cloned the variable domains of HuM195 into pMaz-IgH Herceptin. Plasmids were transfected into HEK293F, and Abs were purified from cell culture supernatant with protein A resin, eluted with glycine HCL, and then the samples were buffer exchanged into PBS pH 7.4 for long-term storage. This S239D-A330L-I332E triple mutation in Fc portion of IgG1 did not affect antigen-biding affinity for CD33 target protein but showed more than 14-fold higher binding to CD16a than the wild type variant. The mHuM195 Abs increased cytotoxic activity of expanded human NK cells in Calcein AM-release assay when used in concentration as low as 0.01 μg/ml to pretreat murine thymoma EL-4 cells gene-modified to express human CD33 (ADCC, Mean±SD: 38.7±2.25% vs 11.7±3.49% for optimized vs wild type HuM195, and 5±3.15% without Abs, E:T ratio 2:1). We obtained the similar results when using K562 as targets, which naturally express CD33. K562 cells pretreated with mHuM195 Abs induced degranulation in 34±5.25% of NK cells where wHuM195 did so only in 17±4.6% of NK cells. Thus, optimization of HuM195 Ab to improve CD16a affinity results in dramatic increases NK cell cytotoxic activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evidence for the involvement of CD56 molecules in alloantigen-specific recognition by human natural killer cells.

1991 ◽  
Vol 173 (6) ◽  
pp. 1451-1461 ◽  
Author(s):  
N Suzuki ◽  
T Suzuki ◽  
E G Engleman
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Target Molecules

In recent reports we have described the generation of natural killer (NK) lines devoid of CD3/TCR structures but with apparent specificity for allogeneic target cells. Using one such NK line as an immunogen, we now report the generation of two monoclonal antibodies (mAbs), designated 2-13 and 5-38, which bind selectively to the majority of CD3-, CD16+, CD56+ lymphocytes and inhibit the lysis of specific allogeneic target cells by a panel of alloreactive NK lines. By contrast, these mAbs had no effect on classical NK cell mediated lysis of K562 cells or major histocompatibility-restricted T cell-mediated cytolysis. Immunoprecipitation of radiolabeled NK lines followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the target molecules of both mAbs have a molecular mass of approximately 180 kD. Leu 19, a well-described anti-CD56 mAb, precipitated a 180 kD protein from NK cells, and the binding of Leu 19 to NK cells was blocked by pretreatment with both 2-13 and 5-38. However, in contrast to these mAbs, Leu 19 had no effect on the cytolytic activity of allospecific NK cells. Sequential immunoprecipitation analysis revealed that all three mAbs recognized distinct molecular species of CD56. We interpret these findings as indicating that multiple isoforms of CD56 are differentially expressed on NK lines and play critical roles in the recognition/interaction of these cells with their specific allogeneic targets.

scholarly journals Developmental regulation of a mucinlike glycoprotein selectively expressed on natural killer cells.

1993 ◽  
Vol 178 (6) ◽  
pp. 2023-2033 ◽  
Author(s):  
E Vivier ◽  
J M Sorrell ◽  
M Ackerly ◽  
M J Robertson ◽  
R A Rasmussen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Developmental Regulation ◽  
Interleukin 2 ◽  
Keratan Sulfate ◽  
Apparent Molecular Weight ◽  
Surface Expression ◽  
Cytolytic Activity ◽  
Target Cells

Natural killer (NK) cells are CD3:TCR-, CD16+, CD56+ large granular lymphocytes capable of recognizing and eliminating a variety of virus-infected, malignant, and antibody-coated target cells. Two functionally distinct populations of peripheral blood NK cells can be differentiated by their surface expression of an isoform of the neural cell adhesion molecule (CD56). CD56bright NK cells have the attributes of an undifferentiated cell, in that they proliferate in response to exogenous cytokines, but exert poor cytolytic activity. CD56dim NK cells have the attributes of a more differentiated cell, in that they proliferate poorly in response to exogenous cytokines, but are potent cytolytic effector cells. Here we describe the molecular characterization of a NK cell restricted epitope (PEN5) that is selectively expressed on the functionally differentiated CD56dim NK cells. PEN5+ NK cells proliferate poorly in response to interleukin 2 (IL-2), but are potent cytolytic effectors, whereas PEN5- NK cells proliferate in response to IL-2, but are poor cytolytic effectors. Biochemical and immunochemical analyses reveal the PEN5 epitope to be an unusual sulfated poly-N-lactosamine carbohydrate related to keratan sulfate glycosaminoglycans. Immunoprecipitates prepared using a monoclonal antibody reactive with PEN5 include two polydisperse membrane-bound glycoproteins, PEN5 alpha (120-170 kD) and PEN5 beta (210-245 kD). Enzymatic deglycosylation reduces the apparent molecular weight of both PEN5 isoforms by 80-90%, and classifies PEN5 beta as a mucinlike glycoprotein. The surface expression of the PEN5 epitope is downmodulated by stimuli that induce NK cell proliferation, and it is absent from leukemic NK cells of patients with granular lymphocyte proliferative disorder. Taken together, these results indicate that PEN5 is a developmentally regulated poly-N-lactosamine epitope associated with a mucin-type glycoprotein, whose expression is restricted to the population of nonproliferative NK cells fully committed to cytolytic effector function.

GA101-Coated Target Cells Are More Effective Than Rituximab-Coated Target Cells at Activating NK Cells When Complement Is Present.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2707-2707 ◽  
Author(s):  
Britnie Spaunhorst ◽  
George J Weiner
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Autologous Serum ◽  
Target Cells ◽  
Type I ◽  
Raji Cells ◽  
Anti Cd20

Abstract Abstract 2707 Poster Board II-683 Rituximab has had a major impact on the treatment of B cell malignancies. The mechanisms responsible for mediating the anti-tumor effects of rituximab are complex. For example, complement can have both positive and negative effects on the ability of rituximab to induce target cell lysis. In particular, we recently reported that rituximab-mediated complement activation results in C3b deposition on the rituximab Fc. C3b then impedes interaction between rituximab and NK cell CD16, thereby limiting NK cell activation and ADCC. GA101 is a type II anti-CD20 monoclonal antibody that mediates enhanced direct cell death induction. It has significantly reduced CDC activity compared to type I anti-CD20 antibodies such as rituximab. In addition, GA101 was engineered to mediate increased ADCC (Umana et al., ASH 2007). The current studies were designed to assess whether the decreased ability of GA101 to activate complement results in an enhanced ability of GA101 to activate NK cells when complement is present. Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors and added to Raji cells (Burkitt lymphoma cell line) at a 1:1 ratio. Various concentrations of rituximab or GA101 were added along with media, 20% autologous serum or 20% heat-inactivated autologous serum (heated to 57°C for 30 minutes). Samples were cultured for 20 hours. NK cell (CD3−, CD56+) activation, as determined by phenotypic changes, was evaluated by flow cytometry based on prior studies demonstrating that downmodulation of CD16, and upregulation of CD54 and CD69 are reproducible surrogates for mAb-induced NK activation and ADCC. Raji cells coated with either rituximab or GA101 were able to activate NK cells when cultures were performed in media alone or with heat-inactivated serum (left panel). In contrast, serum blocked the ability of rituximab to activate NK cells, but not the ability of GA101 to activate NK cells (right panel). Similar results were found when upregulation of CD69 or downmodulation of CD16 were evaluated as markers of NK activation and using PBMCs from two other donors. We conclude that the presence of complement does not limit the ability of GA101-coated target B cells to activate NK cells. This is in contrast to rituximab-coated target B cells which are unable to activate NK cells in the presence of serum. These results suggest that the decreased ability of GA101 to fix complement could, paradoxically, enhance the efficacy of GA101 by resulting in enhanced activation of NK cells and increased ADCC. Disclosures: No relevant conflicts of interest to declare.

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