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.

NK-Cell Reconstitution after HLA-Identical Blood and Marrow Transplantations for CML: The Recovery of NKG2D Is Inversely Correlated with NKG2A and It Promotes the GVL Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4879-4879
Author(s):  
Juan Tong ◽  
Huilan Liu ◽  
Liangquan Geng ◽  
Zimin Sun ◽  
Baolin Tang ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Linear Regression Analysis ◽  
Cell Activity ◽  
Target Cells ◽  
Peripheral Blood Stem Cells ◽  
Peripheral Blood Mononuclear ◽  
Blood Stem Cells

Abstract Natural killer (NK) cell alloreactivity is reported to mediate strong graft versus leukemia (GVL) effect in patients after allogeneic stem-cell transplantation. NKG2D receptors recognize human MHC class Ichain related A and B (MICA/B) and UL16-binding protein 1∼4(ULBP 1∼4) on target cells, thereby regulating NK cell activity. To examine the recovery of NKG2D, NKG2A and other receptors expression by NK cells, we used flow cytometry to evaluate samples from 11 chronic myeloid leukemia patients and their donors in the year following unmanipulated HLA completely matched peripheral blood stem cells plus bone marrow transplantation. Peripheral blood mononuclear cells from patients and their donors were tested in standard 51Cr release assays against cultured K562 targets to determine the cytotoxicity of the NK cells in the same intervals. There is no mismatched immunoglobulin-like receptor (KIR) ligand in both GVH and HVG direction. The reconstitution of KIR2DL1 (CD158a) after this transplantation protocol was very slow and these receptors didn’t reach normal value in the year and KIR2DL2 (CD158b) was much better. The NKG2D increased and the NKG2A decreased quickly at the same time after engraftment, and used linear regression analysis we demonstrated that NKG2A recovery was inversely correlated with NKG2D recovery in the year following transplantation. The ratio of NKG2D/NKG2A was directly associated with the capacity of NK-cell cytotoxicity. Thus, the reconstitution of NKG2D makes contribution to the recovery of the NK cytotoxicity. These results reveals that the NK cells generated after HLA matched blood plus bone morrow transplantation of CML patients are promoted at an immature state characterized by specific phenotypic features and enhanced functioning, having potential impact for immune responsiveness and transplantation outcome.

634 Production and testing of a novel bispecific nanobody construct targeting NK cells and EGFR expressing malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A670-A670
Author(s):  
Elisa Toffoli ◽  
Abdolkarim Sheikhi ◽  
Roeland Lameris ◽  
Lisa King ◽  
Jurriaan Tuynman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Peripheral Blood Mononuclear ◽  
Mutational Status ◽  
Tumor Cell Lysis

BackgroundThe ability to kill tumor cells with an acceptable toxicity profile, makes Natural Killer (NK) cells promising assets for cancer therapy. However, strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment would likely increase the efficacy of NK cell-based therapies.MethodsIn this study, we show a novel bispecific nanobody-based construct (biVHH) targeting both CD16A (low-affinity Fc receptor: FcRγIIIA) on NK cells and EGFR on tumors of epithelial origins.ResultsHigher levels of NK cell activity and subsequent tumor cell lysis were found in vitro in the presence of the biVHH and were dependent on the expression of both CD16A and EGFR while they were independent of the KRAS mutational status of the tumor. Increased NK cell activity was found in NK cells derived from colorectal cancer (CRC) patients when co-cultured with the biVHH and EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the biVHH and autologous peripheral blood mononuclear cells or allogeneic NK cells.ConclusionsBased on our results, the bispecific CD16A and EGFR targeting VHH construct could be a useful tool in combination with various NK cell-based therapies.

scholarly journals Non-Coated Rituximab Induces Highly Cytotoxic Natural Killer Cells From Peripheral Blood Mononuclear Cells via Autologous B Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Chao Niu ◽  
Yongchong Chen ◽  
Min Li ◽  
Shan Zhu ◽  
Lei Zhou ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
New Method ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Natural killer (NK) cells are becoming valuable tools for cancer therapy because of their cytotoxicity against tumor cells without prior sensitization and their involvement in graft-versus-host disease; however, it is difficult to obtain highly cytotoxic NK cells without adding extra feeder cells. In this study, we developed a new method for obtaining highly cytotoxic NK cells from peripheral blood mononuclear cells (PBMCs) independently of extra feeder cell addition using rituximab not coated on a flask (non-coated rituximab). We found that rituximab could promote both the activation and expansion of NK cells from PBMCs, irrespective of being coated on a flask or not. However, NK cells activated by non-coated rituximab had much greater antitumor activity against cancer cells, and these effects were dependent on autologous living B cells. The antibody-dependent cellular cytotoxicity effect of NK cells activated by non-coated rituximab was also more substantial. Furthermore, these cells expressed higher levels of CD107a, perforin, granzyme B, and IFN-γ. However, there was no difference in the percentage, apoptosis, and cell-cycle progression of NK cells induced by coated and non-coated rituximab. Non-coated rituximab activated NK cells by increasing AKT phosphorylation, further enhancing the abundance of XBP1s. In conclusion, we developed a new method for amplifying NK cells with higher antitumor functions with non-coated rituximab via autologous B cells from PBMCs, and this method more efficiently stimulated NK cell activation than by using coated rituximab.

Heat shock protein 70 vaccines activate NK cells through NKG2D

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10044-10044
Author(s):  
B. Liu ◽  
Y. Qaio ◽  
Z. Li
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Nk Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Heat Shock Protein 70 ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

10044 Background: We have previously reported that vaccination with heat shock protein 70 (HSP70) induced natural killer (NK) cell activity in patients with chronic myelogenous leukemia (CML). The underlying mechanism is unclear. Methods: HSP70 was purified from the peripheral blood mononuclear cells. CD56+ NK cells were enriched by magnetic sorting after staining PBMCs with anti-CD56 monoclonal antibody. Dendritic cells (DCs) were derived from peripheral blood monocytes after culture in the presence of IL-4 and GM-CSF. NK activation was measured by the IFNgamma ELISPOT assay. Results: Unexpectedly, HSP70 of autologous, allogeneic as well as xenogeneic origin was found to stimulate IFNgamma production from peripheral blood mononuclear cells of CML patients as well as normal subjects. Further studies demonstrated that the activity of HSP70 was dependent on both NK cells and DCs. HSP70 did not induce significant IFNgamma production from either NK cells or DCs alone. Mechanistically, we found that HSP70-mediated DC-NK cell crosstalk required cell-cell contact, which could be inhibited completely by neutralizing antibody against NK activating receptor NKG2D. The significance of NKG2D was further corroborated by the finding that HSP70 induced the expression of an NKG2D ligand, the MHC class I chain-related protein A (MICA), on DCs; HSP70-augmented IFNgamma release was abrogated by antibody against MICA. Conclusions: Thus HSP70 may serve as a critical link between NK and DCs in mounting immune responses against infections, cancers and self-antigens. Our novel findings support further studies in the development of HSP70-based vaccines against human malignancies. Acknowledgement: Z.L. is a clinical scholar of the Leukemia and Lymphoma Society. No significant financial relationships to disclose.

scholarly journals Essential Roles of Monocytes in Stimulating Human Peripheral Blood Mononuclear Cells with Lactobacillus casei To Produce Cytokines and Augment Natural Killer Cell Activity

2006 ◽  
Vol 13 (9) ◽  
pp. 997-1003 ◽  
Author(s):  
Kan Shida ◽  
Tomomi Suzuki ◽  
Junko Kiyoshima-Shibata ◽  
Shin-ichiro Shimada ◽  
Masanobu Nanno
Keyword(s):  
Lactobacillus Casei ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACT We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-α, and IL-10. IFN-γ production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-γ effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-γ through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.

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