OBJECTIVETo investigate the quantitative and qualitative changes of TCRVα24(+)Vβ11(+) natural killer T (NKT) cells from bone marrow (BM) of aplastic anemia (AA) after in vitro stimulation of α-galactosylceramide (α-Galcer).

METHODSNKT cells in the bone marrow mononuclear cells (BMMNCs) from either AA patients or healthy controls were enumerated with flow cytometry. BMMNCs were cultured in RPMI1640 medium supplemented with either α-Galcer and rhIL-2 or α-Galcer, rhIL-2 and rhG-CSF. The proliferative capacity of NKT cells was determined by NKT cell numbers before and after in vitro culture. Expression of intracellular IFNγ and IL-4 in activated NKT cells was analyzed with flow cytometry.

RESULTSIn AA group, the percentage of NKT cells in BMMNCs was (0.19 ± 0.09)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium resulted in significantly reduced expansion of NKT cells (67.45 ± 29.42-fold vs 79.91 ± 40.56 fold, P < 0.05). Meanwhile, addition of rhG-CSF reduced IFNγ positive NKT cells \[(37.45 ± 7.89)% vs (62.31 ± 14.67)%, P < 0.01\] and increased IL-4 positive NKT cells \[(55.11 ± 12.13)% vs (27.03 ± 9.88)%, P < 0.01\]. In healthy control group, the percentage of NKT cells in BMMNCs was (0.25 ± 0.12)%. Addition of rhG-CSF into the α-Galcer/rhIL-2 culture medium also significantly reduced expansion of NKT cells (97.91 ± 53.22-fold vs 119.58 ± 60.49-fold, P < 0.05), reduced IFNγ positive NKT cells \[(28.65 ± 10.63)% vs (50.87 ± 12.66)%, P < 0.01\], and increased IL-4 positive NKT cells \[(66.53 ± 14.96)% vs (31.11 ± 10.07)%, P < 0.01\].

CONCLUSIONCompared to those from healthy controls, BMMNCs from AA patiants have a reduced fraction of NKT cells, which possesses a decreased potential to expand in vitro in response to α-Galcer stimulation, and produce more IFNγ(+) NKT1 cells. rhG-CSF, in combination with α-Galcer, confers polarization of NKT cells towards IL-4(+) NKT2 subpopulation.

Anemia, Aplastic ; metabolism ; Bone Marrow ; metabolism ; Humans ; Interleukin-4 ; metabolism ; Killer Cells, Natural ; cytology ; Natural Killer T-Cells

Endometriosis, a heterogeneous, inflammatory, and estrogen-dependent gynecological disease defined by the presence and growth of endometrial tissues outside the lining of the uterus, affects approximately 5-10% of reproductive-age women, causing chronic pelvic pain and reduced fertility. Although the etiology of endometriosis is still elusive, emerging evidence supports the idea that immune dysregulation can promote the survival and growth of retrograde endometrial debris. Peritoneal macrophages and natural killer (NK) cells exhibit deficient cytotoxicity in the endometriotic microenvironment, leading to inefficient eradication of refluxed endometrial fragments. In addition, the imbalance of T-cell subtypes results in aberrant cytokine production and chronic inflammation, which contribute to endometriosis development. Although it remains uncertain whether immune dysregulation represents an initial cause or merely a secondary enhancer of endometriosis, therapies targeting altered immune pathways exhibit satisfactory effects in preventing disease onset and progression. Here, we summarize the phenotypic and functional alterations of immune cells in the endometriotic microenvironment, focusing on their interactions with microbiota and endocrine and nervous systems, and how these interactions contribute to the etiology and symptomology of endometriosis.
Female ; Humans ; Endometriosis/metabolism* ; Killer Cells, Natural/metabolism* ; T-Lymphocytes/metabolism* ; Estrogens ; Endometrium/metabolism*

B7-H6 is a co-stimulatory molecule discoveried recently. B7-H6 is not expressed on normal cells, but specially expressed on tumor cells. It can also be expressed on antigen presenting cells (APC) by the induction. The B7-H6 expression can be downregulated by HDACi. NK cells can be activated to release TNFα and IFNγ through B7H6-NKp30 pathway. The B7-H6 molecules expressed on the cell surface can be shedded to form soluble molecules. In the meantime, the B7-H6/NKp30 pathway may be involved in the pathogenesis of primary Sjogren syndrome. B7-H6/NKp30 may become a new therapeutic target for tumor, inflammation and autoimmune diseases. This review discusses the B7-H6 and receptor sructure, the expression and significance of B7-H6, the function and regulating mechanism of B7-H6 and the soluble molecules of B7-H6.
Autoimmune Diseases ; B7 Antigens ; metabolism ; Down-Regulation ; Humans ; Inflammation ; Killer Cells, Natural ; metabolism ; Natural Cytotoxicity Triggering Receptor 3 ; metabolism ; Neoplasms

NK cell immunotherapy is a promising antitumor therapeutic modality after the development of T cell immunotherapy. Structural modification of NK cells with biomaterials may provide a precise, efficient, and low-cost strategy to enhance NK cell immunotherapy. The biomaterial modification of NK cells can be divided into two strategies: surface engineering with biomaterials and intracellular modification. The surface engineering strategies include hydrophobic interaction of lipids, receptor-ligand interaction between membrane proteins, covalent binding to amino acid residues, click reaction and electrostatic interaction. The intracellular modification strategies are based on manipulation by nanotechnology using membranous materials from various sources of NK cells (such as exosome, vesicle and cytomembranes). Finally, the biomaterials-based strategies regulate the recruitment, recognition and cytotoxicity of NK cells in the solid tumor site in situ to boost the activity of NK cells in the tumor. This article reviews the recent research progress in enhancing NK cell therapy based on biomaterial modification, to provide a reference for further researches on engineering NK cell therapy with biomaterials.
Humans ; Biocompatible Materials/metabolism* ; Immunotherapy ; Killer Cells, Natural/metabolism* ; Immunotherapy, Adoptive ; Neoplasms/therapy*

OBJECTIVE: To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. METHODS: Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. RESULTS: After a 14-day's culture, the contents of CD3^-CD56⁺ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3⁺CD4⁺ T cells and CD3⁺CD56⁺ NKT cells in M-NK group decreased significantly. The percentages of CD16⁺, NKG2D⁺, NKp44⁺, CD25⁺ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a⁺ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05). CONCLUSION The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.
Humans ; Fetal Blood ; Killer Cells, Natural ; T-Lymphocytes ; Leukocytes, Mononuclear/metabolism* ; Cell Proliferation ; CD56 Antigen/metabolism*

Objective: To investigate the expression characteristics of Tim-3 on natural killer (NK) cells of peripheral blood in patients with acute myeloid leukemia (AML) and its clinical significance. Methods: Peripheral blood was obtained from 39 patients with newly diagnosed AML before intervention, with peripheral blood from 28 cases of healthy volunteers collected as normal control. Using CD3, CD56 and Tim-3 as markers, expression levels of Tim-3 on the peripheral blood NK cells were detected by immune fluorescence labeling and flow cytometry. Results: The ratio of the peripheral blood CD3(-)CD56(+) NK cells in newly diagnosed AML patients (5.74±5.31) %decreased significantly, compared with the normal control (12.55±6.33) % (t=4.596, P<0.001) . Tim-3 expression on the peripheral blood NK cells in newly diagnosed AML patients (42.67±19.08) % decreased significantly, compared with the normal control group (60.99±20.69) % (t=3.781, P<0.001) . CD3(-)CD56(+)NK cell ratio of peripheral blood in AML patients was significantly correlated with Chromosome karyotype (t=2.915, P<0.005) . Expression level of Tim-3 on NK cells in the peripheral blood of AML patients had significant correlation with ratio of CR and NCCN high risk group (P<0.05) . Conclusion: The rate of NK cells in peripheral blood and the expression level of Tim-3 on NK cells in AML patients decreased significantly.The lower expression level of Tim-3 on NK cells correlate with prognosis of AML.
Flow Cytometry ; Hepatitis A Virus Cellular Receptor 2/metabolism* ; Humans ; Killer Cells, Natural ; Leukemia, Myeloid, Acute ; Prognosis

The aim of study was to establish an accurate and stable technique for the detection of NK cell activity in the diagnosis of hemophagocytic lymphohistiocytosis (HLH). 21 suspected acquired HLH patients and 20 healthy subjects as controls were enrolled in the study, and the suspected HLH patients were divided into confirmedly diagnosed group and excluded group according to HLH-2004 diagnostic criteria. The plasmid pEGFP-N1 was transfected into K562 cells. After scanned with G4l8 and monoclone, the EGFP-K562 cell line stably expressing enhanced green fluorescent protein was obtained. PBMNC and EGFP-K562 were mixed at the effector to target ratio of 10:1. After incubation for of 2 hours, propidium iodine (PI) was added to stain dead cells, and then cytotoxic activity was analyzed by using flow cytometry. Meanwhile, the cytotoxic activity of NK cells in peripheral blood on K562 cells was detected by LDH release assay, and was compared with results detected by flow cytometry. The results showed that a K562 cell line stably expressing EGFP was constructed and were used to measure NK cytotoxicity against the target cells by flow cytometry without pre-staining or pre-labeling target cells. There was significant difference in killing rate of NK cells between the diagnostic group and the control group. This new technique correlated strongly with the results by LDH release assay. In conclusion, this study provides a novel, simple, rapid, repeatable and reproducible method to measure NK activity by flow cytometry using EGFP-K562 without pre-staining or pre-labeling target cells. It may be widely used in the diagnosis of hemophagocytic lymphohistiocytosis.
Case-Control Studies ; Flow Cytometry ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; immunology ; metabolism

OBJECTIVETo investigate the enhanced cytotoxicity against leukemia cells of natural Killer (NK) cells from cord blood (CB) after expansion in vitro.

METHODSNK cells was expanded on a layer of trophoblast cells with irradiated K562-mb15-41BBL cell line for 21 days. The levels of receptors on NK cells were detected by flow cytometry. Cytotoxicity of expanded NK cells against leukemia cells and specific ligand of immunoglobulin like(Ig- liKe)receptors were assessed using 51Cr released assay.

RESULTSThere were no differences of inhibitory receptors expression between fresh NK cells and expanded NK cells [CD158a:(16.77±11.65)% vs(14.37±11.12)%, P>0.05; CD158b: (42.48±18.11)% vs (40.92±19.02)%, P>0.05; NKG2A: (70.20±18.43)% vs (78.90±13.69)%, P>0.05], but higher activated receptors expression on expanded NK cells [NKp30: (54.10±13.27)% vs (4.14±2.05)%, P<0.05; NKp44: (72.10±17.30)% vs (0.52±1.16)%, P<0.05; NKp46: (80.63±14.01)% vs (44.19±6.19)%, P<0.05; NKG2D: (97.50±2.55)% vs (72.25±14.35)%, P<0.05]. Expanded NK cells showed higher cytotoxicity against leuKemia cell lines than fresh NK cells [K562: (74.3±3.6)% vs (55.3±4.2)%, P<0.05; Raji: (60.6±5.0)% vs (12.0±3.6)%, P<0.05]. CD158a⁻ CD158b⁻ NK cells had higher cytotoxicity on four types of target cells, but CD158a⁺CD158b⁻ CB-NK cell had lower cytotoxicity on 221-Cw4 and 221-Cw3Cw4 cells. CD158a⁻ CD158b⁺ CB- NK cells had lower cytotoxicity on 221-Cw3 and 221-Cw3Cw4, but CD158a⁺CD158b⁺ CB-NK cells had higher cytotoxicity on 721- 221 cells.

CONCLUSIONExpression of activated receptors of expanded NK cells were up-regulated, but no changes of inhibitory receptors. Expanded NK cells showed high cytotoxicity against leukemia cells and kept the specificity of ligand of Ig-like receptors, which could be beneficial to cell-therapy for tumor.

Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; metabolism

Although the development of novel drugs has significantly improved the survival of patients with multiple myeloma (MM) over the past decades,the lack of effective therapeutic options for relapsed and refractory MM results in poor prognosis.The chimeric antigen receptor (CAR) T-cell therapy has achieved considerable progress in relapsed and refractory MM.Nevertheless,this therapy still has limitations such as cytokine release syndrome,neurotoxicity,and off-target effects.Natural killer (NK) cells,as a critical component of the innate immune system,play an essential role in tumor immunosurveillance.Therefore,CAR-modified NK (CAR-NK) cells are put forward as a therapeutic option for MM.The available studies have suggested that multiple targets can be used as specific therapeutic targets for CAR-NK cell therapy and confirmed their antitumor effects in MM cell lines and animal models.This review summarizes the anti-tumor mechanisms,biological characteristics,and dysfunction of NK cells in the MM tumor microenvironment,as well as the basic and clinical research progress of CAR-NK cells in treating MM.
Animals ; Receptors, Chimeric Antigen/metabolism* ; Multiple Myeloma/metabolism* ; Killer Cells, Natural/metabolism* ; Immunotherapy, Adoptive/methods* ; Tumor Microenvironment

Natural killer T-cell lymphoma (NKTL) is a malignant neoplasm which usually involves the nasal cavity or paranasal sinuses, while an orbit origin is extremely rare. Here we report the clinical, radiological and histopathologic features of a patient with NKTL originating from the orbit. We analyzed the clinical and radiologic records in the whole course of the disease. We also reviewed the morphology and immunohistochemistry of the neoplasm biopsy, including the presence of CD56, CD3 and cytotoxic molecules. This case demonstrated that nasal-type NKTL with a poor prognosis can originate from the orbit.
Humans ; Lymphoma, T-Cell ; etiology ; metabolism ; Male ; Middle Aged ; Natural Killer T-Cells ; metabolism ; pathology ; Orbital Neoplasms ; complications ; metabolism