Novel KIR3DL1 Alleles and Their Expression Levels on NK Cells: Convergent Evolution of KIR3DL1 Phenotype Variation?

Abstract Abstract 4683 Natural killer (NK) cells can kill malignant or virus-infected cells through the interaction of activating and inhibitory receptors without needing specific antigen recognition of target cells, and therefor have broad therapeutic applications for treatment of human malignancies. However, due to their limited life-span in vivo and poor expansion in vitro, production of sufficient numbers of NK cells for effective adoptive immunotherapy poses an obstacle. Genetically engineered artificial antigen presenting cells (aAPCs) consisting of K562 modified 4-1BBL and membrane bound IL-15 or IL-21 have been reported for their ability to support ex vivo NK cell proliferation. aAPCs with mbIL-21 were shown to promote increased proliferation of NK cells with shorter telomeres, but differences in in vivo survival or tumor or tissue migration have not been assessed. Tumor and/or tissue migration is primarily mediated by the expression of chemokine receptors. Using aAPCs bearing mbIL15 or mbIL21, we expanded NK cells for 3 weeks and assessed their expression of chemokine receptors, organ migration, and in vivo survival in a xenograft model. Propagated NK cells showed relatively similar levels of low to modest expression of CCR2, CCR7, CXCR4 and CXCR5, and high expression levels of CXCR3. Mean CCR5 expression levels were similar on cells that were positive, but CCR5 was expressed on a higher percentage of NK cells expanded with mbIL-15 than those expanded with mbIL-21. In contrast, about 20% of mbIL-21 expanded NK cells expressed CX3CR1 expression whereas mbIL-15 NK cells showed almost no expression of this receptor. Results from ongoing migration and survival experiments will also be presented. Disclosures: No relevant conflicts of interest to declare.

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Induction of the NKG2D Ligands Expression by Histone Deacetylase Inhibitor and Its Impact on the Susceptibility of Leukemic Cells to the Cytolytic Activity of NKG2D-Expressing Cells.

Blood ◽

10.1182/blood.v108.11.924.924 ◽

2006 ◽

Vol 108 (11) ◽

pp. 924-924

Author(s):

Naoko Kato ◽

Junji Tanaka ◽

Junichi Sugita ◽

Tomomi Toubai ◽

Yoko Miura ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Histone Deacetylase ◽

Immune Cells ◽

Histone Deacetylase Inhibitor ◽

Cytolytic Activity ◽

Leukemic Cells ◽

Expression Levels ◽

Nkg2d Ligands ◽

Deacetylase Inhibitor

Abstract Innate immune cells such as natural killer (NK) cells play a crucial rule in antitumor immune responses. NK cells have functionally opposite receptors for activating and inhibiting signals to exert cytotoxicity. NKG2D is a C-type lectin-like activating receptor and is expressed in various immune cells, including NK cells, NKT cells, CD8+α/β+T cells, and γ/δ+T cells. NKG2D recognizes its ligands, MHC class I-related chain A and B (MICA, MICB). NKG2D ligands are not present on normal cells but are induced by various stresses such as viral infection. Moreover NKG2D ligands are expressed in many malignant cells including hematological malignancies. It has been suggested that involvement of NKG2D in NK or CD8+Tcell-mediated cytotoxicity correlates with the expression levels of ligands on target cells. Therefore induction of NKG2D ligands may lead to enhance the sensitivity to NKG2D-mediated cytotoxicity. In this study, we could enhance expression levels of MICA and MICB by treatment with the histone deacetylase inhibitor trichostatin A (TsA) in lymphoid leukemic cell line BALL1 and some primary patients’ lymphoid leukemic cells. Treatment of BALL1 and patients’ leukemic cells with TsA for 12 hr increased MICA and MICB mRNA expression at least by more than 2 fold by Real-time PCR. Treatment of BALL1 with TsA for 12 hr increased MICA and MICB protein expression on the cell surface by more than 2 fold by flow cytometry analysis. These results suggested that expression of MICA and MICB is partly regulated by histone acetylation. Chromatin immunoprecipitation assay revealed that treatment with TsA resulted in increased acetylation of histone H3 and decreased association with the counteracting enzymes of histone acetyltransferases HDAC1 at the MICA and MICB promoter in BALL1 cell and patients’ leukemic cells. To examine the impact of the cytolytic activity of NKG2D-expressing cells on leukemic cells in which expression of NKG2D ligands was induced by TsA treatment, we performed standard 4 hr 51Cr release assays using BALL1 cells and patients’ leukemic cells. Up-regulation of NKG2D ligands by TsA treatment led to enhance the susceptibility of BALL1 and patients’ leukemic cells by 2 or 3 times to the cytolytic activity of NKG2D-expressing cells. Blocking experiment using specific antibodies for MICA and MICB inhibited the NKG2D-expressing cell-mediated cytolytic activity against BALL1 cells. Our results suggest that regulation of NKG2D ligands expression by treatment with chromatin-remodeling drugs may be an effective strategy to enhance the susceptibility of leukemic cells to the cytolytic activity of NKG2D-expressing cells for immunotherapy.

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TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals

European Journal of Immunology ◽

10.1002/eji.201545480 ◽

2015 ◽

Vol 45 (10) ◽

pp. 2886-2897 ◽

Cited By ~ 42

Author(s):

Feng Wang ◽

Hongyan Hou ◽

Shiji Wu ◽

Qing Tang ◽

Weiyong Liu ◽

...

Keyword(s):

Nk Cells ◽

Healthy Individuals ◽

Functional Heterogeneity ◽

Expression Levels

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Hsa_circ_0007456 regulates the natural killer cell-mediated cytotoxicity toward hepatocellular carcinoma via the miR-6852-3p/ICAM-1 axis

Cell Death and Disease ◽

10.1038/s41419-020-03334-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Min Shi ◽

Zhao-Yun Li ◽

Li-Ming Zhang ◽

Xiao-Yu Wu ◽

Shi-Hao Xiang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Nk Cells ◽

Cancer Progression ◽

Killer Cell ◽

Intercellular Adhesion Molecule ◽

Circular Rnas ◽

Intercellular Adhesion Molecule 1 ◽

Tumor Immune Evasion ◽

Expression Levels ◽

Tumor Tissues

AbstractCircular RNAs (circRNAs) is one type of important non-coding RNAs that participate in tumorigenesis and cancer progression. In our previous study, we performed a microarray analysis of circRNAs between the tumor tissues and the adjacent normal tissues of hepatocellular carcinoma (HCC) patients, and found that the circRNA hsa_circ_0007456 is significantly downregulated in the tumor tissues and correlated with the prognosis of HCC. We further investigated the relationship between the expression levels of hsa_circ_0007456 in HCC and the susceptibility of NK cells, and found that the expression levels of hsa_circ_0007456 in HCC cell lines significantly influenced their susceptibility to NK cells. Through a series of screening and validation, we found that hsa_circ_0007456 mainly functioned through sponging miR-6852-3p and regulating the expression of intercellular adhesion molecule-1 (ICAM-1) in HCC. The miR-6852-3p/ICAM-1 axis is essential for the NK cytotoxicity toward HCC mediated by hsa_circ_0007456. In conclusion, we identify here hsa_circ_0007456 as a promising biomarker of HCC, and highlight hsa_circ_0007456/miR-6852-3p/ICAM-1 axis as an important signaling pathway in the process of tumor immune evasion and the tumorigenesis of HCC.

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Influenza Virus Infection but Not H1N1 Influenza Virus Immunization Is Associated with Changes in Peripheral Blood NK Cell Subset Levels

Clinical and Vaccine Immunology ◽

10.1128/cvi.00194-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1291-1297 ◽

Cited By ~ 9

Author(s):

A. Juárez-Reyes ◽

D. E. Noyola ◽

A. Monsiváis-Urenda ◽

C. Alvarez-Quiroga ◽

R. González-Amaro

Keyword(s):

Influenza Virus ◽

Nk Cells ◽

Peripheral Blood ◽

Killer Cell ◽

Cell Subset ◽

Membrane Receptors ◽

Blood Samples ◽

Expression Levels ◽

Severe Influenza ◽

Nkt Lymphocytes

ABSTRACTThe innate immune system constitutes the first line of defense against viral agents, and NK cells seem to have an important protective role during the early phases of influenza virus infections. We decided to assess the levels of NK and NKT lymphocytes and the expression levels of different membrane receptors (NKp44, NKp46, NKG2A, killer cell immune-like receptor [KIR] 3DL1/DS1, KIR2DL1/DS1, and CD161) in peripheral blood samples of patients with influenza (n= 17) and healthy individuals immunized against this virus (seasonal and [H1N1]pdm2009 influenza vaccines;n= 15 and 12, respectively). Blood samples were obtained from all individuals, and NK and NKT cell subsets were analyzed by multiparametric flow cytometry. We found that the patients with severe influenza (n= 9) showed significant increases in the percentages of NKp46+NKp44+NK cells and the proportions of NK and NKT lymphocytes expressing KIR2DL1 and KIR3DL1 and reductions in the percentages of NKp46+NKp44−NK cells compared to those in the healthy controls (n= 27). In contrast, influenza immunization, against either the seasonal or the pandemic H1N1 virus, was not associated with important changes in the levels of NK and NKT lymphocytes or the expression levels of the different receptors by these cells. Our data suggest that severe influenza is associated with important and complex alterations on NK cells, which might contribute to the pathogenesis of this condition.

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STAT5A Overexpression Enhances Killer Immunoglobulin Receptor (KIR) Expression in Developing NK Cells and Is Associated with a Loss of Reverse Transcription from the Proximal KIR Promoter.

Blood ◽

10.1182/blood.v110.11.798.798 ◽

2007 ◽

Vol 110 (11) ◽

pp. 798-798

Author(s):

Frank M. Cichocki ◽

Todd Lenvik ◽

Stephen K. Anderson ◽

Jeffrey S. Miller

Keyword(s):

Dna Methylation ◽

Stem Cell ◽

Nk Cells ◽

Reverse Transcription ◽

Nk Cell ◽

Hla Class I ◽

Class I ◽

Proximal Promoter ◽

Rt Pcr ◽

Expression Levels

Abstract Down-regulation of HLA-class I molecules is commonly observed in virally infected and malignantly transformed cells and can contribute to the ability of these cells to escape recognition by the adaptive immune system. NK cells are able to detect the loss of even single HLA alleles on potential target cells through their clonally distributed HLA-class I-specific inhibitory receptors (KIR). While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides in areas surrounding the transcription initiation region, the mechanisms that regulate variegated KIR expression are largely unknown. The manipulation of KIR expression is of considerable interest due to the widely reported correlation between KIR ligand status and patient survival in hematopoietic stem cell transplant settings. Because the transcription factor STAT5 is activated downstream of the BCR/ABL oncogene, which we have previously shown to substantially augment KIR expression levels in primary NK cells, we hypothesized that STAT5 could directly influence KIR expression. To test this hypothesis, we purified CD34+ hematopoietic progenitor cells from umbilical cord blood and retrovirally transduced these cells with either a control murine stem cell virus (MSCV) vector encoding eGFP alone or an MSCV vector encoding both eGFP and a constitutively active form of STAT5A, termed STAT5A 1*6. CD34/eGFP-positive cells were then purified and cultured on the EL08-D1 stroma line, known to support NK cell development. After a period of 28 days in culture, 6.25±2.73% of the NK cells in eGFP control cultures were KIR-positive compared with 29.3±4.27% of STAT5A 1*6 transductants. To more closely examine individual KIR expression at the transcriptional level, we carried out a quantitative RT-PCR analysis with probe sets previously validated for amplification of individual KIR (Cooley et al., Blood). We observed a general increase in the mRNA expression levels of both inhibitory and activating KIR in STAT5A 1*6-transduced cells. In order to investigate the mechanism underlying the increased KIR expression observed in the STAT5A 1*6-transductants, we performed an RT-PCR specific for the reverse transcript originating from the proximal promoter of the KIR3DL1 gene. In a model proposed by Anderson et al., reverse transcription from the bidirectional proximal promoter of variegated KIR genes may be responsible for the silencing of the KIR locus through the production of dsRNA, which then induces DNA methylation in a siRNA-dependent manner. Our RT-PCR results from multiple donors clearly show an absence of reverse proximal transcript in cells transduced with the STAT5A 1*6 construct in contrast to high levels of transcript in eGFP controls. These results provide evidence for a role of STAT5A in the induction of KIR expression and support a model of KIR regulation involving intergenic transcription and possibly siRNA-mediated gene silencing. A better understanding of how to manipulate these KIR expression patterns may be of benefit to exploit the potential of NK cell therapy to improve transplant outcomes.

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The Role of Proteinase Inhibitor 9 (PI-9) and Granzyme B in Peripheral Leukocytes for GVL and GVHD After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽

10.1182/blood.v118.21.1984.1984 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1984-1984

Author(s):

Osamu Horie ◽

Kaori Minami ◽

Sawako Toji ◽

Kenji Yonezawa ◽

Hiroshi Gomyo ◽

...

Keyword(s):

Nk Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Polymorphonuclear Cells ◽

Hematopoietic Stem ◽

Allogeneic Hsct ◽

Expression Levels ◽

Proteinase Inhibitor 9 ◽

And Gender

Abstract Abstract 1984 The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)- and perforin (PFR)-induced cytotoxicity and apoptosis by specifically inhibiting GrB. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects of the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least in part through GrB and PFR produced by donor-derived cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, GrB and PFR expressions with respect to a GVL effect, and the role of PI-9 in both GVHD and GVL, are unknown. In this study we analyzed the expression of GrB, PFR and PI-9 in acute leukemia patients to whom allogeneic HSCT was performed during remission. This study was performed under the approval of the ethical committee of Kobe University Graduate Schools of Medicine and Health Sciences, and all human samples were obtained from whom a written informed consent was obtained. We first investigated the age- and gender-related differences of expressions of PI-9 and GrB mRNA by quantitative RT-PCR in mononuclear and polymorphonuclear cells from healthy volunteers. GrB and PFR mRNAs were expressed almost exclusively in mononuclear cells that included CTLs and NK cells, while PI-9 was expressed both in mononuclear and polymorphonuclear cells. GrB and PFR expression levels were comparable among all age and gender groups. Intriguingly, however, the expression of PI-9 mRNA in lymphocytes gradually increased with age, and the PI-9 expression in the volunteer group aged 60 years old or older was significantly higher than the one in the group aged 20 to 39 years old (p=0.002). Meanwhile, the PI-9 expression in polymorphonuclear cells was comparable among all age groups, and there was no gender difference in PI-9 expression levels. Next, we analyzed the expressions of PI-9, GrB and PFR mRNAs in mononuclear and polymorphonuclear cells of 3 patients (male 2, female 1) with acute GVHD and 11 patients without acute GVHD (male 6, female 5). The GrB, PFR and PI-9 expression levels were comparable between patient groups with and without acute GVHD, both before transplantation and a week after engraftment. However, the GrB and PFR expressions prominently augmented when acute GVHD became overt in all 3 patients, while PI-9 expression level increased mildly. As a result, the ratio of GrB (or PFR) and PI-9 expressions was stable in patients with and without acute GVHD. Among them, 4 cohort patients eventually relapsed and 10 cohorts remained in remission. The GrB mRNA expression in lymphocytes one week after engraftment was 3-fold higher in 10 patients without relapse of primary disease than in 4 patients who eventually relapsed (P=0.044). This might suggest that GrB plays a role in eliciting a GVL effect as well as acute GVHD. On the other hand, PFR and PI-9 mRNA expressions were then relatively stable among patients both with and without later relapse (P=0.667; P=0.103), and the ratio of GrB (or PRF) and PI-9 expressions did not change. The expressions of GrB and PFR mRNAs in polymorphonuclear cells of all patients were under the detectable level throughout the course. Finally, we analyzed the expressions of GrB, PFR and PI-9 in leukocytes 6 months after HSCT, to see if these expressions might change in patients with chronic GVHD. Among 14 patients with chronic GVHD, the GrB expression increased (up to 6.2-fold) in 10 patients, but the PFR expression did not change. In contrast, the expression of PI-9 decreased profoundly in all of the cases both with and without chronic GVHD, relative to healthy volunteers (P=0.028). Importantly, the ratio of GrB and PI-9 expressions was significantly higher in patients with chronic GVHD than in those without GVHD (P = 0.035). In conclusion, (1) high PI-9 expression in elderly people might explain a mechanism of escape from tumor immunity in them, where PI-9 might inhibit functions of cytotoxic T cells and NK cells; (2) a high expression of GrB shortly after engraftment may be a novel biomarker for a GVL effect; and (3) a low level of PI-9 expression or an increased ratio of GrB/PI-9 later after allogeneic HSCT might be an important biomarker for cGVHD. Monitoring of both GrB and PI-9 mRNAs in peripheral blood after allogeneic HSCT could be useful for predicting the GVL effect, as well as for monitoring both acute and chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

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Heterogeneous Expression of the 158V and 158F Alleles in FcγRIIIA Heterozygous Donors.

Blood ◽

10.1182/blood.v104.11.1362.1362 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1362-1362

Author(s):

Michael Dechant ◽

Thomas Poellot ◽

Ulrich Kunzendorf ◽

Thomas Valerius

Keyword(s):

Nk Cells ◽

Clinical Studies ◽

Nk Cell ◽

Mrna Level ◽

Receptor Expression ◽

Allelic Polymorphism ◽

Effector Cells ◽

Expression Levels ◽

Protein Levels ◽

Immunofluorescence Studies

Abstract Recent clinical studies demonstrated that a bi-allelic polymorphism in the human FCGRIIIA gene critically determines the clinical outcome of rituximab therapy in certain hematologic malignancies and in autoimmune diseases. Thus, patients homozygous for the 158V allele of the FcγRIIIa receptor demonstrated significantly better response rates to rituximab therapy compared to homozygous 158F donors. However, confliciting data were reported for heterozygous patients - which account for approximately 45 % of the investigated population. Furthermore, most of the in vitro reports about functional effects of this polymorphism did not address the function of 158V/F heterozygotes. In all these studies, genomic DNA was used for allotyping of the FCGRIIIA 158V/F polymorphism. Thus, these studies were not set up to investigate potential allele- specific differences at mRNA or protein levels, which were demonstrated for several other NK cell- related genes. Therefore, we were interested to analyze receptor expression and function in 158V/F heterozygous donors in more detail. First, we established FCGRIIIA allotyping at the mRNA level. Thus, isolated mRNA was reversely transcribed, PCR amplified and sequenced using BigDye terminator mix. Results from homozygous donors were consistent and homogeneous, whereas sequencing profiles from heterozygotes appeared heterogeneous. Some 158V/F heterozygotes demonstrated similar expression of both alleles, whereas sequencing profiles in others were almost similar to either homozygous 158V/V or homozygous 158F/F donors. Next, we analyzed the quantitative expression of the corresponding proteins by immunofluorescence experiments with antibodies 3G8 and MEM-154. While 3G8 binds similarly to both allelic forms of FcγRIIIa, MEM-154 preferentially recognizes the 158V allele, as confirmed by immunofluorescence studies with FcγRIIIa 158F or 158V transfected cell lines. These experiments again demonstrated that FcγRIIIA 158V/F heterozygous donors are a heterogeneous group - with 158V protein levels ranging from similar to 158F/F homozygotes to similar to 158V/V homozygous donors. Furthermore, we investigated whether 158V expression levels have functional implications in classical 51Cr- release assays against ARH-77 B cells. We used isolated NK cells as effector cells, and rituximab as sensitizing antibody. Effector cells were isolated from matched sets of FCγRIIIA allotyped donors to directly compare 158V/V or 158F/F homozygotes with 158V/F heterozygotes. As described by others, we observed statistically significant differences between 158V/V and 158F/F homozygous donors at suboptimal antibody concentrations. Interestingly, NK cells from 158F/V heterozygous donors, which appeared similar by sequencing profiles and by immunofluorescence studies to effector cells from 158V/V homozygotes, also demonstrated killing levels similar to those from 158V/V homozygous donors. In conclusion, our data demonstrate that donors heterozygous for FcγRIIIA at position 158 are a heterogeneous population - potentially explaining controversial data published about 158F/V heterozygotes in clinical studies. In order to further assess the biological impact of our results, clinical trials may address whether FcγRIIIA 158V expression levels will correlate with clinical responses to rituximab therapy. These studies could help to predict which patients may optimally benefit from rituximab therapy.

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Role of NOTCH1 in Aggressive NK-Cell Leukemia.

Blood ◽

10.1182/blood.v110.11.4231.4231 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4231-4231

Author(s):

Hideki Makishima ◽

Masao Ota ◽

Takefumi Suzuki ◽

Kozo Nakayama ◽

Fumihiro Ishida

Keyword(s):

Cell Proliferation ◽

T Cell ◽

Nk Cells ◽

Cell Lines ◽

Western Blotting ◽

Nk Cell ◽

Cell Leukemia ◽

Expression Levels ◽

Healthy Donors ◽

Enhanced Expression

Abstract Back ground: Aggressive NK-cell leukemia (ANKL) is a malignant NK-cell proliferative disorder resistant to most chemotherapies and frequently shows rapidly progressive clinical course. ANKL is characterized with hepatosplenomegaly, hemophagocytic syndrome, and strong association with EBV, and is mostly observed among Asian populations. Oncogenic mechanism of ANKL is not clear. NOTCH1 is a gene encoding a transmembrane receptor that regulates T-cell development and is a major oncogene in T-cell lymphoblastic leukemia (T-ALL). Recently, the potential involvement of NOTCH signaling in the development of NK-cells has been reported. We discuss here that the NOTCH1 protein might play a significant role in the pathogenesis of ANKL. Patients, materials, and methods: 6 ANKL cases, 7 Chronic NK-cell lymphocytosis (CNKL) cases, and 3 NK tumor cell lines (NKL, NK-92, and KHYG) derived from ANKL patients were enrolled. Chromosomal translocation (7;9) was not found in any subjects. The expression levels of NOTCH1 intracellular subunit (NOTCH-IC) were examined with flowcytometry and western blotting. NK-cell lines were cultured with g-secretase inhibitor (GSI) (L-685,458) for evaluation of the effect. Cell proliferation and apoptosis were measured with XTT assay and annexinV positivity, respectively. The mutation analysis of NOTCH1 genome was performed with nested PCR and direct sequencing. mRNA expression of NOTCH1 gene was measured with real time PCR using the corresponding TaqMan probes. Results: NOTCH-IC was detected in CD2+CD3− fractions from healthy donors, CNKL patients, ANKL patients, NK-cell lines, and Cos7 cells transfected with NOTCH1 gene coding NOTCH-IC, but not in CD2-CD3− fractions. The expression levels were significantly high in ANKL patients, compared to healthy donors (P < 0.05). With western blotting, cleaved form (110kDa) of NOTCH1 protein was recognized in all NK-cell lines. NOTCH-IC expression was reduced with the treatment of GSI in NKL and NK-92, but not in KHYG. Cell proliferation was inhibited with GSI in NKL and NK-92, whereas the inhibitory effect was not seen in KHYG. There was no significant difference in the increase ratio of apoptotic cells after the treatment of GSI among 3 NK-cell lines. Mutations in HD, TAD, or PEST domain, where frequent mutations are detected in T-ALL, were not found in NK-cell lines, and no enhanced expression of NOTCH1 mRNA in ANKL cells, compared with healthy donor NK-cells. Conclusion: These results indicate that NOTCH-IC was increased in ANKL cells and might be an important role in the proliferation. Genomic or transcriptional dysregulation would not be the cause for enhanced expression of NOTCH1 in ANKL contrast with T-ALL. Further investigation is required for the relationship with NOTCH1 ligands. In the future, NOTCH1 might be a novel therapeutic target in ANKL.

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Expression of the ULBP Ligands for NKG2D by B-NHL Cells Plays An Important Role in Determining Their Susceptibility to Rituximab-Induced ADCC

Blood ◽

10.1182/blood.v112.11.218.218 ◽

2008 ◽

Vol 112 (11) ◽

pp. 218-218

Author(s):

Atsushi Inagaki ◽

Takashi Ishida ◽

Hiroki Yano ◽

Fumiko Mori ◽

Asahi Ito ◽

...

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Mhc Class I ◽

Cell Lines ◽

Associated Factors ◽

Rank Correlation ◽

Class I ◽

51Cr Release ◽

Expression Levels ◽

Nkg2d Ligands

Abstract Purpose: ADCC is a major antitumor mechanism for the action of therapeutic monoclonal antibodies (mAbs) such as rituximab, trastuzumab and cetuximab. Therefore, a better understanding of ADCC will allow the development of novel, more effective treatment strategies, and may help overcome the resistance which can develop against the effects of the therapeutic mAbs. However, the tumor-associated factors which determine susceptibility to rituximab-induced ADCC have not been identified. In the present study, we focused on this issue, especially focused on the molecules expressed by the tumor cells that interact with NK cells, such as NKG2D ligands, because of the importance of NK cells for rituximab-induced ADCC. The aim of this study was to identify tumor associated factors which determine susceptibility to rituximab-induced ADCC. Experimental Design: 30 different CD20+ non-Hodgkin lymphoma (B-NHL) cell lines were phenotyped for characteristics of cell surface protein: expression levels of CD20, MHC class I, NKG2D ligands (ULBP1-3, MICA, and MICB), CD48 (2B4 ligands), HLA-G, cathepsin B, and complement inhibitors (CD46, CD55, and CD59), and the influence thereof on susceptibility to rituximab-induced ADCC was established. Result: The degree of rituximab-induced ADCC were correlate with the expression levels of CD20 and ULBP1-3, and inversely correlate with the expression levels of MHC class I among 30 different CD20+ B-NHL cell lines. The importance of the ULBPs was confirmed using antibody blockade. In the presence of blocking mAb to ULBP1, 2 or 3, a decrease of rituximab-induced ADCCs against B-NHL cell lines were observed. In addition, the present study clearly identified the key mechanism of rituximab-induced ADCC as antibody-dependent target-specific cytotoxicity mediated by highly activated NK cells. Strong NK cells activation was due to the combination of Fc„dR stimulation via the Fc portion of rituximab, together with stimulation of activating NK cell receptors via their ligands expressed on the tumor cells, particularly ULBPs, which occurred in a robustly synergistic manner. Conclusions: Tumor cell susceptibility to rituximab-induced ADCC was determined by three major tumor-associated factors: the amount of the target molecule, CD20; the amount of the ligands for inhibitory killer Ig-like receptors, MHC class I; and the amounts of some of the NKG2D ligands, especially ULBP1-3. This is the first report to show the importance of ULBPs on tumor cells for rituximab-induced ADCC. The ULBPs could be valuable diagnostic biological makers and significant targets for immunotherapy to improve efficacy not only of rituximab but also of other therapeutic mAbs. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel. Figure. Correlations between the expression of the CD20, MHC class I, and NKG2D ligands ULBP1-3, and rituximab-induced ADCC in B-NHL cell lines. ADCC in the presence of 10μg/mL rituximab against 30 different CD20+ B-NHL cell lines determined by 51Cr release assays. Y-axis: % lysis. X-axis: MFI ratio of CD20 (upper left panel), MHC class I (upper right panel), ULBP-1 (lower left panel), ULBP-2 (lower middle panel), and ULBP-3 (lower right panel). Each dot plot in each panel represents one cell line. The coefficients and P values assessed by Spearman rank correlation coefficient testing are indicated in each panel.

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