scholarly journals Anti-Leukemia Effects of NK Cell-Derived Exosomes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3223-3223
Author(s):  
Michael Boyiadzis ◽  
Chang Sook Hong ◽  
Theresa L Whiteside
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Granzyme B ◽  
Cell Activity ◽  
Leukemia Cells ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Transmission Electron ◽  
Healthy Donors

Introduction: Exosomes are 30-150 nm-sized extracellular vesicles originating from the endocytic compartment of parent cells. The exosome molecular cargo reflects the content of its cells of origin and is delivered to recipient cells in a protective glycol-lipid bilayer without degradation. Because of their small size, exosomes freely circulate within the body, can reach the bone marrow, and can cross biological barriers. Natural killer (NK) cells play a critical role in the innate immune response through their capacity to lyse malignant cells without prior antigen-specific priming. Importantly, NK cell activity is reduced in patients with acute myeloid leukemia (AML) relative to that in healthy donors. To overcome the decreased NK cell activity in AML, several therapeutic strategies have been evaluated for safety and efficacy, both in transplant and non-transplant settings, using autologous and allogeneic activated NK cells. Since NK cell-derived exosomes acquire tumor-killing abilities from the parent NK cells, we hypothesize that NK cell-derived exosomes by transferring exosome content to leukemia blasts can induce the death of these target cells. In the current study, we evaluated the in vitro anti-leukemia effects of NK cell-derived exosomes. Methods: Exosomes were isolated from the supernatants of NK cells obtained from healthy donors (n=12) using mini-size exclusion chromatography (mini-SEC). Protein levels, number and size (qNano), and exosome morphology using transmission electron microscopy were determined. The exosome cargo was studied by Western blots and on-bead flow cytometry for NK cell activating and inhibitory receptors, immune inhibitory molecules, and for perforin and granzyme B. Cytotoxicity of the NK cell-derived exosomes for AML cell lines (Kasumi, MLL-1) and primary leukemia blasts was measured using flow cytometry-based assays. Results: Activated human NK cells produced large quantities of exosomes. Transmission electron microscopy showed the presence of vesicles that were uniform in size (30-150nm in diameter) by NanoSight measurement. Confocal imaging of labeled NK cell-derived exosomes interacting with leukemia cells showed that they are rapidly internalized by leukemic targets. NK cell-derived exosomes carried activating NK cell receptor NKG2D, natural cytotoxicity receptors, perforin, granzyme B, transforming growth factor beta (TGF-β), killer-cell immunoglobulin-like receptors, and PD-1. NK cell-derived exosomes were co-incubated with target cells, AML cell lines and primary leukemia cells, at different exosome:target (E:T) ratios using escalating doses of exosomes (10-70 µg). NK cell-derived exosomes mediated strong anti-leukemic activity against AML cell lines and primary leukemic blasts. Importantly, with higher doses of exosomes, higher levels of cytotoxicity were observed, suggesting that exosome-mediated lysis is concentration dependent. NK cell-derived exosomes mediated leukemia killing via different cell death pathways including apoptosis and necroptosis. Conclusion: NK cell-derived exosomes mediating cytotoxicity against leukemic targets represents a novel therapeutic modality for patients with AML. Disclosures No relevant conflicts of interest to declare.

IPH2101, a Novel Anti-Inhibitory KIR Monoclonal Antibody, and Lenalidomide Combine to Enhance the Natural Killer (NK) Cell Versus Multiple Myeloma (MM) Effect.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3870-3870 ◽  
Author(s):  
Don Benson ◽  
Courtney E Bakan ◽  
Shuhong Zhang ◽  
Lana Alghothani ◽  
Jing Liang ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Nk Cells ◽  
Tumor Cells ◽  
Nk Cell ◽  
Phase 1 ◽  
Granzyme B ◽  
Cell Activity ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Immune Complex Formation

Abstract Abstract 3870 Poster Board III-806 Background NK cell activity against tumor cells is regulated by a balance of inhibitory and activating signals mediated by receptors on NK cells that recognize inhibitory and activating ligands expressed by cancer cells. IPH2101 (1-7F9) is a novel monoclonal anti-inhibitor KIR blocking antibody that has been shown to augment NK cell function against MM targets. Moreover, lenalidomide has been shown to expand and activate NK cells in vivo and in vitro. We have previously reported that the combination of IPH2101 and lenalidomide enhances NK cell mediated cytotoxicity against MM cells compared to each agent alone (Zhang et al., AACR 2009). We expand our studies to investigate potential mechanisms for the enhancement of NK cell activity by the combination of IPH2101 and lenalidomide. Methods The effects of IPH2101 and lenalidomide alone and in combination were studied using primary human NK cells from healthy donors as well as from MM patients. The MM cell lines U266 and RPMI 8226 as well as primary tumor cells from marrow aspirates of MM patients served as target cells. The effect of lenalidomide on MM activating and inhibitory ligand expression was studied by flow cytometry. NK cell trafficking was investigated with standard transwell plate migration assay. Immune complex formation between NK cell effectors and MM tumor targets was characterized by flow cytometry in control conditions and with NK cells pre-treated with IPH2101 and lenalidomide. The effects of IPH2101 and lenalidomide were studied regarding interferon-gamma and granzyme B production by ELISPOT and target-specific cytotoxicity studies were conducted to complement effector-based assays. Results IPH2101 (30 ug/ml) significantly enhanced cytotoxicity against U266 cells and primary MM tumor cells by both purified NK cells at effector:target (E:T) ratios of 10:1 or less, and also of freshly isolated peripheral blood mononuclear cells (PBMC) at E:T ratios of 60:1 or less, from more than 10 random donors. In addition, treatment of PBMC with 5-10 μmol/L lenalidomide for 72h without interleukin (IL)-2 increased NK cell lysis of U266. Treatment of PBMC from normal donors did not enhance the expression of the NK receptors KIR, NKG2D, NCR, TRAIL, and DNAM-1. Incubation of U266 cells with lenalidomide (5 uM) for 3-5 days resulted in significant enhancement of cytotoxicity by normal donor NK cells. This was associated with upregulation of the activating ligands, MICA, ULBP-2, DR4, and CD112. Using blocking antibodies to NKG2D, TRAIL, and DNAM-1, lenalidomide enhancement of MM cell killing was abrogated indicating the importance of the modulation of the ligands to the latter receptors by lenalidomide. Although IPH2101 and lenalidomide did not significantly increase NK cell migration into normal media, migration was enhanced 2.98-fold (+/− 0.36, p < 0.05) towards U266 cell targets (n= 3, p < 0.05) and MM patient serum 3.2-fold (+/− 0.4, n=3, p < 0.05). IPH2101 and lenalidomide also led to a 2.3-fold (+/− 0.43, p < 0.05) increase in immune complex formation between NK cells and MM tumor cells. IPH2101 and lenalidomide also augmented NK cell interferon gamma production against MM (control mean 303 spots/well +/− 13 versus 525 +/− 83, n=3, p < 0.05) and granzyme B production (control mean 115 +/− 98 versus 449 +/−72, n=3, p < 0.05). Importantly, in all experiments described herein, the effects of IPH2101 and lenalidomide together were greater than either agent alone. Conclusions Taken together, our data suggest that IPH2101 and lenalidomide may exert complementary mechanisms on both effector and target cells to enhance NK cell mediated killing of MM cells. Moreover, these agents have no predicted clinical cross-toxicities. A single-agent phase 1 clinical trial of IPH2101 has shown the mAb to be safe and well tolerated in MM patients. These findings support a phase 1/2 clinical trial of IPH2101 with lenalidomide as a first dual-innate immunotherapy for patients with MM. Disclosures: Andre: Innate Pharma: Employment. Squiban:Innate pharma: Employment. Romagne:Innate Pharma: Employment.

Thermal stresses reduce natural killer cell cytotoxicity

1995 ◽  
Vol 79 (3) ◽  
pp. 732-737 ◽  
Author(s):  
S. J. Won ◽  
M. T. Lin
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Cell Activity ◽  
Cortisol Concentration ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Colonic Temperature ◽  
Effector Target

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7–10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.

scholarly journals Patients with a deficiency of natural killer cell activity lack the VEP13-positive lymphocyte subpopulation

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 65-70 ◽  
Author(s):  
HW Ziegler-Heitbrock ◽  
H Rumpold ◽  
D Kraft ◽  
C Wagenpfeil ◽  
R Munker ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Natural Killer Cell Activity ◽  
Killer Cell ◽  
Cell Activity ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Target Cells ◽  
Nk Cell Activity

Many patients with B-type chronic lymphocytic leukemia (CLL) exhibit a profound defect in their natural killer (NK) cell activity, the basis of which is still obscure. Hence, we analyzed the NK cells from peripheral blood samples from 11 patients with CLL for phenotype and function, after removal of the leukemic cells with a monoclonal antibody (BA-1) plus complement. Phenotypic analysis of these nonleukemic cells with monoclonal antibodies (MoAbs) against NK cells revealed that the CLL patients had higher percentages of HNK-1-positive cells (23.5% compared to controls with 14.7%). In contrast, VEP13- positive cells were absent or low in seven patients (0.8% compared to controls with 11.2%) and normal in four patients (10.5%). When testing NK cell activities against K562 or MOLT 4 target cells, patients with no or minimal numbers of VEP13-positive cells were found to be deficient, while patients with normal percentages of VEP13-positive cells had NK cell activity comparable to controls. Isolation by fluorescence-activated cell sorter of HNK-1-positive cells from patients lacking VEP13-positive cells and NK cell activity indicated that the majority of the HNK-1-positive cells in these patients had the large granular lymphocyte morphology that is characteristic of NK cells. Thus, the deficiency of NK cell activity in CLL patients appears to result from the absence of cells carrying the VEP13 marker.

Increase In NK Cell Lysis of Leukemic Blasts Due to Loss of Mismatched HLA Haplotype After Haplo-Identical Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2540-2540
Author(s):  
Yoshiyuki Takahashi ◽  
Itzel Bustos Villalobos ◽  
Sayoko Doisaki ◽  
Hideki Muramatsu ◽  
Akira Shimada ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Cell Activity ◽  
Leukemic Cells ◽  
Target Cells ◽  
Chromosome 6 ◽  
Nk Cell Activity ◽  
Before And After ◽  
Hla Loss

Abstract Abstract 2540 Introduction: Hematopoietic stem cell transplantation (HSCT) from HLA haplo-identical family donors is promising as a therapy for patients with leukemia who are at high risk for relapse. The lower relapse rates and improved survival, especially for patients with acute myelogenous leukemia (AML) who have received HSCT from killer cell immunoglobulin-like receptor (KIR) ligand-mismatched donors, suggest that donor NK cells that are alloreactive against the recipient's cells cause graft-versus-leukemia effects. An Italian group and we identified genomic loss of the patient-specific HLA haplotype in leukemic cells after haplo-identical HSCT. Analysis using SNP arrays revealed that the HLA loss in 29 to 66% of relapsed patients after haplo-identical HSCT was caused by segmental uniparental disomy (UPD) of the HLA region on chromosome 6. This suggested that leukemic cells often escape immunosurveillance through the loss of the mismatched HLA haplotype via the UDP mechanism after haplo-identical HSCT (Vago et al. N Engl J Med. 2009, Villalobos, IB et al. Blood 2010). Since NK cell effector function is tightly regulated by inhibitory KIRs on NK cells that bind to MHC class I on target cells, the escape of leukemic cells from immune surveillance by losing a mismatched HLA antigen might enhance the cytotoxicity of NK cells towards target cells. We examined alterations in donor-derived alloreactive NK cell activity against leukemic blasts of AML patients who relapsed after HLA haplo-identical HSCT. Patients and methods: We enrolled three patients with AML, aged 2, 3 and 12 years, who relapsed after HLA haplo-identical HSCT with T cell depletion of rabbit ATG in vivo. Two patients had AML M7 and the other had M0. Only one donor was KIR ligand-mismatched. Engraftment was achieved in all three patients within 28 days. Relapse occurred 35, 372 and 445 days after HSCT. Mononuclear cells were obtained from both donors and patients before and after HSCT. The NK cells were purified using NK cell selection kits (DYNAL) and measured by conventional 51Cr release assays of leukemic blasts from the patients and the control cell line K562. The mismatched HLA expression between patients and donors on hematopoietic cells was monitored by flow cytometry using anti-HLA antibody (One Lambda). Results: The mean of NK cell killing of the patients' leukemic cells at the same effector target ratio of 10 was significantly higher in donors (50.3%) than in relapsed patients (9.7%). Monitoring of alloreactive NK cell activity after HSCT showed that donor-derived NK cell killing against the patients' leukemic cells gradually decreased in the KIR ligand-mismatched setting. Relapse was minimal but obvious according to HLA monitoring of hematopoietic cells on day 98, which was 7 days after alloreactive NK cell activity was diminished in the patients. Monitoring HLA expression after HSCT also revealed that one patient had leukemic blasts at relapse with loss of the patient's specific HLA haplotype caused by UDP of the HLA region on chromosome 6. Notably, primary leukemic blasts in this patient were not killed by the donor NK cells (2.3%), but leukemic blasts at relapse were efficiently killed (69.0%) after HLA loss of leukemic cells. On the other hand, killing of leukemic blasts at relapse by the patient's NK cells after transplantation was much less effective (19.0%) than that by donor NK cells, although they originated from the same donor (Figure). Because haplotype loss of HLA caused by UDP does not change the status of the KIR ligand in patients with homozygous HLA-Cw, we further examined the expression of ULBP1-3 that are ligands for the activating NK receptor, NKG2D, on leukemic blasts before and after HLA loss. We found upregulated ULBP-2 expression on leukemic cells after the loss of HLA. Conclusions: Donor NK cells efficiently killed patients' AML blasts at relapse but NK cell activity in patients against their own leukemic blasts was impaired after HSCT. These findings indicate a rationale for donor NK cell infusions after HLA haplo-identical HSCT to avoid decreasing NK cell alloreactivity and to prevent the escape of leukemic cells from allo-immune surveillance by donor cytotoxic T lymphocytes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Differential Cytotoxicity of Cord Blood and Bone Marrow–Derived Natural Killer Cells

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 207-213
Author(s):  
Clair M. Gardiner ◽  
Anne O' Meara ◽  
Denis J. Reen
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Cytotoxic Activity ◽  
Natural Killer ◽  
Nk Cell ◽  
Granzyme B ◽  
Cell Activity ◽  
Lak Cells ◽  
Target Cells ◽  
Nk Cell Activity

Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell–associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.

A Novel Mechanism of Action of Bortezomib: Proteasome Inhibition Down-Regulates HLA-Class I on Myeloma and Enhances NK Cell-Mediated Lysis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1525-1525
Author(s):  
Jumei Shi ◽  
Guido J. Tricot ◽  
Priyangi A. Malaviarachchi ◽  
Tarun K. Garg ◽  
Susann M. Szmania ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Cell Activity ◽  
Hla Class I ◽  
Class I ◽  
Target Cells ◽  
Down Regulation

Abstract Introduction: Natural killer (NK) cell activity is regulated by a dynamic balance between inhibitory and activating receptors that recognize ligands on target cells. Human leukocyte antigen (HLA)-class I, particularly HLA-C and -Bw4 molecules, are key ligands transmitting inhibitory signals to NK cells. NK cells avidly lyse tumor cells that do not display such inhibitory KIR-ligands. The proteasome is responsible for the generation of peptides that bind to and stabilize class I molecules at the cell surface. We hypothesized that bortezomib, a partial proteasome inhibitor that is clinically approved for the treatment of refractory/relapsed myeloma (MM), could reduce HLA expression on MM cells and thus enhance NK cell-mediated cytotoxicity. Methods: HLA-class I or HLA-C expression was assessed using flow cytometry, after gating on AnnexinV/PI double negative cells, and/or confocal microscopy. Expression of other proteins was measured by flow cytometry using specific mAb. NK cell-mediated lysis of myeloma cells was measured by 51Cr-release. Results: Bortezomib at clinically attainable concentrations down-regulated HLA-class I expression on MM cells in a time- and dose-dependent fashion. Reduction of class I post-10 nM bortezomib treatment was observed in all myeloma cell lines tested (n=10), by a median of 49% (range: 19–66%). A similar decrease of HLA-class I was obtained in 10–50 nM bortezomib treated primary MM cells (n=6). Bortezomib significantly enhanced the sensitivity of MM cells to allogeneic and autologous NK cell-mediated lysis. Further, the level of reduction in HLA-class I expression correlated well with increased susceptibility to lysis by NK cells. The level of down-regulation of HLA-class I induced by bortezomib was reproduced by incubating MM cells with HLA-blocking antibody and resulted in equipotent enhancement of NK cell-mediated lysis (Figure 1). The extent of HLA-class I down-regulation by bortezomib was therefore biologically relevant. Down-regulation of HLA-class I was also observed in vivo on purified MM cells 48 hours after a single dose of bortezomib, by a median of 47% (range: 16–63%, n=6, P= .002). HLA-C expression (the principal NK cell inhibitory ligand) was rescued by exogenous provision of HLA-C binding peptides providing a mechanistic explanation for the effect of bortezomib on HLA-class I expression. Finally, we did not observe bortezomib-mediated enhancement of NK cell-mediated lysis of myeloma through receptors other than the KIR receptor family, including tumor necrosis factor related apoptosis-inducing ligand, NKG2D and natural cytotoxicity receptors. HLA-class I down-regulation was not observed in renal cell and breast carcinoma cell lines, which is in keeping with the remarkable activity of bortezomib in myeloma. Our findings have clear therapeutic implications for MM and other NK cell-sensitive malignancies in the context of both allogeneic and autologous adoptively transferred NK cells. Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis Figure 1. Reduced class I on MM cell surface results in NK cell-mediated recognition and lysis

NOVEL Pathways Modulate TUMOR CELL Susceptibility to NK CELLS.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 277-277
Author(s):  
Roberto Bellucci ◽  
Hong-Nam Nguyen ◽  
Allison Martin ◽  
Anna C. Schinzel ◽  
Stefan Heinrichs ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cell Activity ◽  
Tumor Cell Lines ◽  
Nk Cell Activity ◽  
Myeloma Cells ◽  
Increased Sensitivity

Abstract Abstract 277 NK cells play an important role in innate immune responses directed against autologous cells that have undergone viral or malignant transformation. The ability of NK cells to lyse targets is primarily dependent on the expression of various inhibitory or activating receptors. However, transformed cells have also developed mechanisms to evade immune surveillance and the molecular basis for target resistance to immune-mediated lysis is not well understood. To address this issue we undertook a genetic screening approach to identify novel pathways that modulate tumor cell susceptibility to NK cell lysis. Our genetic screen utilized a subset of the TRC1 lentiviral shRNA library developed at the Broad Institute of Harvard and MIT. The library subset targeted 476 protein kinases and 180 phosphatases that represent 88% and 80%, respectively, of known NCBI sequences with these functions. The library also targeted 372 genes representing tumor suppressors, DNA binding proteins as well as irrelevant shRNAs as controls. Each gene was targeted by 5 or more independent shRNAs tested individually in a 384 well format using robotic manipulations. A total of 6,144 individual shRNAs were incubated with IM-9 myeloma cells and subsequently tested for their ability to modulate response by NKL effector cells (an IL-2 dependent human NK cell line). The top 10 percentile of shRNAs inducing increased secretion of interferon-gamma (INF-γ) from NKL cells was identified. To reduce the likelihood of off-target effects, only genes that were positive for 2 or more independent shRNAs were selected for further analysis. Among the genes that increased target cell susceptibility to NK activity we found 2 members of the Jak family (Jak1 and Jak2) with Jak1 being one of the strongest hits in our screen. IM-9 myeloma cells with stable expression of at least 2 independent shRNAs targeting Jak1 and Jak2 were established and tested for their sensitivity to NKL, NK92 or primary NK cells using INF-γrelease and 51Cr release cytotoxicity assays. Stable suppression of both Jak1 and Jak2 in IM-9 cells induced a significant increase of INF-γsecretion from NK cells and increased sensitivity in cytotoxicity assay when compared to parental IM-9 or cells expressing irrelevant shRNAs. Western blot analysis showed a selective decrease of Jak1 and Jak2 protein in IM-9 cells expressing specific shRNAs but not irrelevant shRNAs. While target cells with reduced expression of Jak1 and Jak2 were more susceptible to NK cell activity, no effect was observed when Jak3 and TYK2 were silenced. We then tested the NK susceptibility of different tumor cell lines with reduced expression of Jak1 and Jak2. Seven additional tumor cell lines representing other hematologic malignancies expressing Jak1 and Jak2 shRNAs were established: myeloma (KM12BM), chronic myeloid leukemia (K562), Burkitt's lymphoma (Daudi), acute myeloid leukemia (U937, ML2 and KG1) and acute T cell leukemia (Jurkat). These experiments confirmed that Jak1 silencing can induce increased susceptibility to NK cell activity. However, this effect was more pronounced in some cases (IM-9, KM12BM, U937, KG1) compared to other cell lines where this effect was limited (K562, ML-2, Jurkat) or absent (Daudi). To investigate the mechanism for modulation of target cell susceptibility to NK cells by Jak1, we compared gene expression profiles of IM-9-Jak1-KO with IM-9 parental cells and IM-9 cells infected with an irrelevant shRNA. No difference in expression was found for ligands of activating NKG2D receptors (MICA, MICB, ULPB1, 2, 3) or ligands for NK inhibitory receptors (HLA Class I genes A, B, C, E). One of the most up-regulated genes in IM-9-Jak1-KO cells was TNFSFR10A (TRAIL-R1), a gene that is known to induce apoptotic signals upon TRAIL engagement. In contrast, FAIM3, an inhibitor of FAS (CD95) signaling, was significantly down-regulated. IM-9-Jak1-KO cells also over-expressed several GALNT genes, recently shown to be markers of TRAIL sensitivity. These results suggest that Jak1 and possibly Jak2 can modulate susceptibility of some tumor cells to NK cell lysis. The mechanism for this effect appears to be at least partly through increased sensitivity to engagement of the TRAIL/FAS extrinsic apoptotic pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Assessment of NK Cell Activity Based on NK Cell-Specific Receptor Synergy in Peripheral Blood Mononuclear Cells and Whole Blood

2020 ◽  
Vol 21 (21) ◽  
pp. 8112
Author(s):  
Jung Min Kim ◽  
Eunbi Yi ◽  
Hyungwoo Cho ◽  
Woo Seon Choi ◽  
Dae-Hyun Ko ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Natural killer (NK) cells are cytotoxic innate lymphocytes endowed with a unique ability to kill a broad spectrum of cancer and virus-infected cells. Given their key contribution to diverse diseases, the measurement of NK cell activity (NKA) has been used to estimate disease prognosis or the effect of therapeutic treatment. Currently, NKA assays are primarily based on cumbersome procedures related to careful labeling and handling of target cells and/or NK cells, and they require a rapid isolation of peripheral blood mononuclear cells (PBMCs) which often necessitates a large amount of blood. Here, we developed an ELISA-based whole blood (WB) NKA assay involving engineered target cells (P815-ULBP1+CD48) providing defined and synergistic stimulation for NK cells via NKG2D and 2B4. WB collected from healthy donors (HDs) and patients with multiple myeloma (MM) was stimulated with P815-ULBP1+CD48 cells combined with IL-2. Thereafter, it utilized the serum concentrations of granzyme B and IFN-γ originating in NK cells as independent and complementary indicators of NKA. This WB NKA assay demonstrated that MM patients exhibit a significantly lower NKA than HDs following stimulation with P815-ULBP1+CD48 cells and had a good correlation with the commonly used flow cytometry-based PBMC NKA assay. Moreover, the use of P815-ULBP1+CD48 cells in relation to assessing the levels of NKG2D and 2B4 receptors on NK cells facilitated the mechanistic study and led to the identification of TGF-β1 as a potential mediator of compromised NKA in MM. Thus, our proposed WB NKA assay facilitates the reliable measurement of NKA and holds promise for further development as both a clinical and research tool.

scholarly journals Differential Cytotoxicity of Cord Blood and Bone Marrow–Derived Natural Killer Cells

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 207-213 ◽  
Author(s):  
Clair M. Gardiner ◽  
Anne O' Meara ◽  
Denis J. Reen
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Cytotoxic Activity ◽  
Natural Killer ◽  
Nk Cell ◽  
Granzyme B ◽  
Cell Activity ◽  
Lak Cells ◽  
Target Cells ◽  
Nk Cell Activity

Abstract Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell–associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.

Analysis of NK Cell Alloreactivity Against Acute Leukemia Cells with MLL Gene Rearramgement.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5169-5169
Author(s):  
Hiroko Honna ◽  
Kumiko Goi ◽  
Kinuko Hirose ◽  
Itaru Kuroda ◽  
Takeshi Inukai ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Class I ◽  
Leukemia Cells ◽  
Target Cells ◽  
Mll Gene ◽  
Concanamycin A ◽  
Group I

Abstract Acute lymphoblastic leukemia (ALL) patients with MLL gene rearramgement are associated with hyperleukocytosis, organomegaly, a high incidence of central nervous system leukemia, and poor prognosis. The optimum treatment for this type of ALL has not been defined, and the effectiveness of hematopoitic stem cell transplantation (SCT) is controversial. We have reported that MLL-rearranged leukemia cells are resistant to cytotoxic activity by death-inducing ligands TRAIL and FasL, and therefore T-cell mediating graft-versus-leukemia (GVL) effect on these leukemia cells is not fully expected. In allogeneic SCT, if donor KIR (Killer cell Ig-like Receptor) ligand class I allele is not present in the recipient cells, it is expected that donor NK cells display alloreactivity against host leukemic cells. Up to now, published data demonstrated that in patients with acute myeloid leukemia, KIR ligand incompatibility reduced the rates of relapse. Prompted by the recent report from SJCRH on the success of reduced intensity SCT from KIR ligand incompatibile donor for the infant with MLL-rearranged ALL relapsed after conventional allogeneic SCT, we extensively analyzed NK cell alloreactivity against leukemic cell lines with MLL gene rearrangement. Materials and Methods: Eleven cell lines consisting of 4 with t(4;11), 5 with t(11;19), and 2 with t(9;11) were classified into 2 groups in terms of types of HLA-C alleles; C1C1 type (n=10): both alleles belonging to group I (Ser77/Asn80, Cw1, Cw7, et. al.) and C1C2 type (n=1): each of alleles belonging to group I and group II (Asn77/Lys80, Cw2, Cw4, et. al.), respectively. K562 lacking HLA class I expression was used as a positive control target for NK cells. NK cell populations were separated from peripheral blood of 9 healthy donors classified into C1C1 type (n=5) and C1C2 type (n=4). The cytotoxic activity of NK cells was assessed by a 4h- 51Cr release assay at an effector-to-target ratio of 20 to 40. Results: Both types of donor NK cells exhibited 60–70% cytotoxicity against K562. Of interest, C1C2 type NK cells showed moderate cytotoxic activity (40–70% cytotoxicity) against C1C1 type cell lines, whereas only modest cytotoxic activity (10–30% cytotoxicity) against the C1C2 type cell line, suggesting that a loss of inhibitory signal to KIR (CD158a, KIR2DL1) from C2 type KIR ligand expressed on target cells is required for C1C2 type donor NK cells to exert their maximal cytotoxic activity. This cytotoxic activity of C1C2 type NK cells against C1C1 type targets was inhibited by greater than 70% by the addition of the perforin inhibitor concanamycin A, but not by the addition of neutralizing antibodies against TRAIL and FasL, indicating that the cytotoxic activity of C1C2 type NK cells is mediated by perforin. C1C1 type NK cells showed only modest cytotoxic activity (10–30%) against both types of cell lines. Cell lines with t(9;11) are less sensitive to NK alloreactivity compared with those with t(4;11) or t(11;19). There was no significant differences in NK alloreactivity in terms of types of HLA-A and -B alleles. Conclusion: MLL-rearranged leukemia cells are sensitive to perforin-mediating killing by KIR ligand (HLA-C) incompatible allogeneic NK cells, and the maximal GVL effect against this leukemia might be expected if donors are selected whose NK cells can exert their alloreactivity.

Sign in / Sign up

Export Citation Format

Share Document