This study was aimed to explore the effect of bone marrow-derived mesenchymal stem cells (MSC) on proliferation and activity of natural killer (NK) and NK-T cells. MSC was co-cultured with peripheral mononuclear cells from healthy donors in presence of IL-2, phytohemagglutinin (PHA) and mouse anti-human CD3 McAb (culture condition known to expand NK cells). The ratio of NK cells and NK-T cells was measured by flow cytometry and the effect of MSC on killing activity of NK cells against K562 cells was detected by MTT method after co-cultured with different densities of MSC. The results showed that MSC inhibited the production of NK cells in a dose-dependent manner generally. At the densities of 0, 1 × 10(5) and 5 × 10(5)/ml, the ratios of NK cells in the co-culture conditions were (16.9 ± 4.6), (14.0 ± 8.6) and (6.4 ± 4.6), respectively (P < 0.05). However, MSC could promote the formation of NK cells at lower MSC density (1 × 10(4)/ml), the ratio of NK cells reached to (20.9 ± 7.1), which was higher than that of culture condition without MSC (P < 0.05). The different densities of MSC in the co-culture conditions had no much influence on the ratio of NK-T cells (P > 0.05). MTT assay showed that the killing activity of suspended cells in co-culture system against K562 cells was parallel with the ratio of NK cells. Different densities of MSC regulated bidirectionally killing activity of NK to K562 cells by regulating bidirectionally ratio of NK cells. It is concluded that the MSC can promote the formation of NK cells and enhance its activity against tumor cells in the lower doses, while suppress the formation of NK cells and attenuate its tumor-killing effect in higher dose condition.
Bone Marrow Cells ; cytology ; Cell Proliferation ; Coculture Techniques ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; Mesenchymal Stromal Cells ; cytology ; Natural Killer T-Cells ; cytology

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

OBJECTIVETo investigate the apoptosis of NK cells induced by the erythroleukemia cell line K562 in vitro.

METHODSPrimary NK cells isolated from the peripheral blood of healthy donors by magnetic-activated cell sorting were cultured with stem cell medium containing recombinant human interleukin-2 (rhIL-2). The NK cells and K562 cells were mixed and co-cultured at different E:T ratios for different time lengths. The apoptosis of NK cells and K562 cells were detected using PE-AnnexinV/7-AAD labeling and flow cytometry.

RESULTSThe purity of isolated NK cells reached (93.99∓4.22)%. At the same E: T ratio, the apoptotic rate of NK cells induced by K562 cells increased significantly with time. As the E:T ratio reduced, the apoptotic rate of the NK cells increased and their cytotoxic activity against K562 cells was attenuated.

CONCLUSIONK562 cells can induce the apoptosis of activated NK cells, which is one of the probable mechanisms of immune escape of tumors.

Apoptosis ; Cytotoxicity, Immunologic ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; Tumor Escape

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

AIMTo investigate the effect of acetylcholine (ACh) on the cytotoxicity of natural killer (NK) cells and to explore the receptor mechanisms involved in the effect.

METHODSThe effector cells (i. e. NK cells) from the spleens of rats were collected and cultured with the target cells (Yac-1 cells). The various concentrations of ACh, cholinergic receptor agonists or antagonists were added to the cultures, respectively according to distinct experimental purposes. Lactate dehydrogenase (LDH) release assay was used to evaluate NK cell cytotoxicity.

RESULTSNK-cell-mediated lysis of Yac-1 lymphoma cells was reduced by 10(-10) - 10(-6) mol/L ACh. The inhibitory effect of ACh on NK cell cytotoxicity was mimicked by pilocarpine, an agonist of muscarinic receptor, and by nicotine, an agonist of nicotinic receptor, at all applied concentrations (10(-10) - 10(-6) mol/L). Muscarinic receptor antagonist atropine blocked the inhibitory effect of ACh on the cytotoxicity of NK cells. Nevertheless, tubocurarine, an antagonist of nicotinic receptor, had no blocking effect on the suppression of NK cell cytotoxicity by ACh.

CONCLUSIONACh results in an inhibition of the cytotoxicity of NK cells, and this inhibition is realized mainly through M and N1 cholinergic receptor.

Acetylcholine ; pharmacology ; Animals ; Cells, Cultured ; Female ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Male ; Rats ; Receptors, Natural Killer Cell ; drug effects

The activity of the NK cells in patients with preeclampsia was studied to investigate the pathogenesis of preeclampsia. By using MTT and 51Cr releasing technique, the proliferation and killing ability of the NK cells in maternal and umbilical blood from preeclampsia patients (n = 18) and normal third trimester pregnant women (n = 18) were detected. The NK-92 cell line was as the positive control. The results showed that the NK cell counts of umbilical blood in preeclampsia patients and normal third trimester pregnant women were significantly greater than those of maternal blood (both P<0.05). Compared with that in normal third trimester pregnant women, the proliferative ability of the NK cells in preeclampsia patients was apparently increased (P<0.05). Compared with that in maternal blood, the proliferative ability of the NK cells in umbilical blood from both preeclampsia patients and normal third trimester pregnant women was dramatically increased. The killing ability of the NK cells in preeclampsia patients was significantly higher than that in normal third trimester pregnant women (P <0.05). It was suggested that both number and function of the NK cells in preeclampsia women were increased, and that in umbilical blood was greater than that in maternal blood, speculating that the function of the NK cells may affect the maintenance of the maternal and fetal immune tolerance during pregnancy.
Cytotoxicity, Immunologic/*immunology ; Fetal Blood/cytology ; Immune Tolerance ; Killer Cells, Natural/*immunology ; Killer Cells, Natural/pathology ; Pre-Eclampsia/blood ; Pre-Eclampsia/*immunology ; Pregnancy Trimester, Third

OBJECTIVETo investigate the frequency distribution features of innate-like lymphocytes (iNKT cells, γΔT cells and B1 cells) in peripheral blood of normal adults.

METHODSThe flow cytometry with 6 fluorescence staining was used to detect the percentages of iNKT lymphocytes, γΔT lymphocytes, B1 lymphocytes and adaptive T lymphocyte, B2 lymphocytes in peripheral blood lymphocytes of 50 normal adults. The difference and correlation between these lymphocyte subsets were analyzed by statistical software.

RESULTSThe percentage of iNKT cells in peripheral blood of 50 normal adults was 0.18% (0.01%-2.01%), the percentage of γΔT cells was 4.90% (1.45%-20.14%), the percentage of B1 lymphocytes was 1.62% (0.20%-3.77%), the percentage of adaptive T cells was 63.52% (33.20%-83.22%), the percentage of B2 cells was 6.64% (3.07%-13.80%). B1 and B2 were two subsets of B lymphocyte, the percentage of B2 in B lymphocyte was 81.43% (57.90%-94.12%) and more than that of B1 lymphocyte; the percentage of B1 lymphocytes was 17.28% (5.28%-41.13%). In T lymphocyte group the percentage of iNKT cell was 0.32% (0.01%-3.6%), the percentages of γΔT cells and adaptive T cells were 7.55% (3.04%-27.66%) and 91.98% (72.22%-96.86%) respectively. Spearman correlation analysis was used to analyze the correlation between the percentages of several lymphocyte subsets. There was a positive correlation between iNK T cells and γΔT cells, γΔT cells and adaptive T cells, B1 cells and B2 cells (r=0.39, P=0.0056; r=0.6028, P<0.0001; r=0.4791, P=0.0004). It was also found that the percentage of iNKT cells in female peripheral blood lymphocytes was 0.29% (0.06%-2.01%), and significantly higher than that in male peripheral blood lymphocytes 0.12% (0.01%-1.37%) (P<0.05).

CONCLUSIONThe percentages of γΔT cells, B1 cells and iNKT cells in peripheral blood lymphocytes of normal adults are significantly lower than that of adaptive lymphocytes, and their contents in peripheral blood decrease in turn. There are no sex differences in the percentages of these lymphocyte subsets except iNKT cells.

Adult ; B-Lymphocytes ; cytology ; Female ; Flow Cytometry ; Humans ; Male ; Natural Killer T-Cells ; cytology ; T-Lymphocyte Subsets ; cytology

OBJECTIVESTo evaluate the effects of K562-dendritic cell (DC) and Raji-DC fusion vaccines on the cytotoxicity of cord blood (CB) derived cytokine-induced killer/natural killer (CIK/NK) cells.

METHODSDC and CIK/NK cells were derived from CB mononuclear cells. CB-DC were fused with inactivated K562 or Raji cells by PEG to form K562 or Raji-DC fusion vaccine. The CIK/NK cells stimulated by different co-culture antigens were three groups: K562-DC or Raji-DC fusion vaccine group, inactivated K562 or Raji plus DC group, and CB-DC alone group. The cytotoxicity of CIK/NK cells stimulated by different co-culture antigens was measured by MTT test.

RESULTSAll the antigens used for stimulation could enhance the cytotoxicity of CB-CIK/NK cells, with no specificity difference. At 20:1 effector-target ratio, the cytolytic activities of K562-DC and Raji-DC fusion vaccine groups against Raji cells were (75.44 +/- 4.19)% and (81.33 +/- 4.18)% respectively (P < 0.05); and that of inactivated K562 + DC and Raji + DC group against Raji cells were (73.12 +/- 4.22)% and (80.49 +/- 4.27)%, respectively (P < 0.01). There was no significant difference in the cytotoxicity to K562 cells between the two fusion vaccine groups (P > 0.05). The cytotoxicity of CB-CIK/NK cells immunized by Raji cells was higher than that by K562 cells. In CIK/NK cells co-stimulated by the same tumor antigen, there was no significant difference in the cytotoxicity between DC fusion vaccine group and inactivated cells plus DC group to different tumor cells.

CONCLUSIONSThe cytotoxicity of CB-CIK/NK cells to tumor cells was not specific. There was no significant difference in the cytotoxic activity of CB-CIK/NK cells between the DC fusion vaccine group and inactivated cells plus DC group.

Cancer Vaccines ; immunology ; pharmacology ; Cells, Cultured ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Fetal Blood ; cytology ; Humans ; K562 Cells ; Killer Cells, Lymphokine-Activated ; immunology ; Killer Cells, Natural ; immunology

OBJECTIVETo observe the effect of mesenchymal stem cells derived from umbilical cord (UC-MSCs) on natural killer (NK) cells-mediated cytotoxicity against dendritic cells (DCs) and explore the mechanism.

METHODSMSCs were isolated from human umbilical cord by collagen digestion and cultured in vitro. NK cells were separated from healthy human peripheral blood by magnetic bead sorting. Mononuclear cells from healthy human peripheral blood were cultured in the presence of granulocyte and macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) to obtain the immature DCs. The DCs were then co-cultured with UC-MSCs in the presence of tumor necrosis factor α (TNFα) for 2 days, and the expressions of CD11c and CD86 on DCs and IL-12 level in the culture medium was detected using flow cytometry and ELISA, respectively. The cytotoxicity of NK cells against DCs was analyzed by LDH-releasing assay, and the expressions of ligands for killer activator receptor (MICA/B and ULBP1-3) on the DCs were detected with flow cytometry.

RESULTSCompared with the cytokine-induced DCs, the DCs induced by co-culture with UC-MSCs showed an identical CD11c expression but lowered CD86 expression and IL-12 secretion. The natural killer cells produced a stronger cytotoxicity against UC-MSCs-induced DCs than against cytokine-induced DCs. The UC-MSCs-induced DCs also showed increased expressions of MICA and MICB on the surface.

CONCLUSIONUC-MSCs can enhance NK cells-mediated cytotoxicity against DCs possibly by inhibiting DC maturation and up-regulating the ligands for killer activator receptor on the surface of the DCs.

Cells, Cultured ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; Humans ; Killer Cells, Natural ; cytology ; Mesenchymal Stromal Cells ; cytology ; Umbilical Cord ; cytology

Mesenchymal stem cells (MSCs) can inhibit T cell proliferation, the effects of MSCs on various T cell subsets have showed different immune regulatory reactions, and their mechanisms mainly include cell-cell contact and mediation by cytokines secreted from MSCs. Encouragingly, recent studies have showed that the effects of MSCs on T-cell response to pathogens is not significant, but can obviously suppress T cell response to allogeneic antigens. In addition, MSCs can regulate the proliferation, survival, antibody secretion and differentiation of B cells, inhibit the production, proliferation, migration and antigen-presentation of DCs, and modulate the differentiation and maturation of DCs, and regulate the proliferation, cell toxicity and cytokine secretion of NK cells. In this review, the research advances on immunomodulatory effects of MSCs on various immune cells including T-lymphocytes, B-lymphocytes, NK cells and DCs are summarized with emphasis on the immunoregulatory effects of MSCs on T-lymphocytes.
B-Lymphocytes ; immunology ; Dendritic Cells ; immunology ; Humans ; Killer Cells, Natural ; immunology ; Mesenchymal Stromal Cells ; cytology ; immunology ; T-Lymphocytes ; immunology