Cancer cells and the immune system are closely related and thus influence each other. Although immune cells can suppress cancer cell growth, cancer cells can evade immune cell attack via immune escape mechanisms. Natural killer (NK) cells kill cancer cells by secreting perforins and granzymes. Upon contact with cancer cells, NK cells form immune synapses to deliver the lethal hit. Mature NK cells are differentiated from hematopoietic stem cells in the bone marrow. They move to lymph nodes, where they are activated through interactions with dendritic cells. Interleukin-15 (IL-15) is a key molecule that activates mature NK cells. The adoptive transfer of NK cells to treat incurable cancer is an attractive approach. A certain number of activated NK cells are required for adoptive NK cell therapy. To prepare these NK cells, mature NK cells can be amplified to obtain sufficient numbers of NK cells. Alternatively, NK cells can be differentiated and amplified from hematopoietic stem cells. In addition, the selection of donors is important to achieve maximal efficacy. In this review, we discuss the overall procedures and strategies of NK cell therapy against cancer.
Cell Differentiation ; *Cell- and Tissue-Based Therapy ; Gene Expression Regulation ; Hematopoietic Stem Cells/cytology/metabolism ; Humans ; *Immunotherapy, Adoptive ; Killer Cells, Natural/cytology/*immunology/*metabolism ; Lymphocyte Activation/immunology ; Signal Transduction

PURPOSE: This study was done to evaluate the effects of psychosocial interventions on cortisol and immune response in adult patients with cancer. METHODS: MEDLINE via PubMed, Cochrane Library CENTRAL, EMBASE, CINAHL and domestic electronic databases were searched. Twenty controlled trials (11 randomized and 9 non-randomized trials) met the inclusion criteria with a total of 862 participants. Methodological quality was assessed using the Cochrane's Risk of Bias for randomized studies and the Risk of Bias Assessment tool for non randomized studies. Data were analyzed using the RevMan 5.2.11 program of Cochrane library. RESULTS: Overall, study quality was moderate to high. The weighted average effect size across studies was -0.32 (95% CI [-0.56, -0.07], p=.010, I2=45%) for cortisol concentration, -0.62 (95%CI [-0.96,-0.29], p<.001, I2=0%) for T lymphocyte (CD3) and -0.45 (95%CI [-0.74, -0.16], p=.003, I2=0%) for Th lymphocyte (CD4) numbers. Psychosocial interventions were not effective for Tc lymphocyte (CD4), NK cell, monocyte, and cytokine response. CONCLUSION: Although these results provide only small evidence of successful immune modulation, they support the conclusion that psychosocial interventions can assist cancer patients in reducing emotional distress and improving immune response.
CD4-Positive T-Lymphocytes/cytology/immunology ; Cytokines/metabolism ; Databases, Factual ; Humans ; Hydrocortisone/*analysis ; Killer Cells, Natural/cytology/immunology ; Monocytes/cytology/immunology ; Neoplasms/metabolism/pathology/*therapy ; Psychotherapy ; T-Lymphocytes/cytology/*immunology

OBJECTIVETo explore the effects of the exogenous and endogenous reactive nitrogen metabolites (RNM) as NK cell inhibitors on NK cell-mediated killing of K562 cells and the influence of Tiopronin (TIP), glutamylcysteinylglycine (GSH) and histamine dihydrochloride (DHT) as RNM scavengers on reversing the suppressing effect of RNM.

METHODSThe exogenous ONOO(-) was administered in the NK+K562 culture system, then the RNM scavengers were added in the NK+K562+ONOO(-) culture system, respectively. The concentrations of RNM, TNF-beta and IFN-gamma, K562 cell inhibition rate (KIR) and the percentage of living NK cells were examined. IL-2+PHA were used as monocyte (MO) activators in the culture system of MO+NK+K562. Then TIP, GSH and DHT were administered and the parameters of NK cell activity were analyzed.

RESULTSAfter exogenous ONOO(-) was administered in NK+K562 culture system, the percentage of living NK cells was decreased from (93.17 +/- 2.57)% to (71.87 +/- 1.02)% (P < 0.01) and KIR was decreased from (67.47 +/- 2.64)% to (43.44 +/- 2.87)% (P < 0.01). When TIP, GSH and DHT were administered into the systems, the percentage of living NK cells was increased to (91.13 +/- 3.67)% (P < 0.05), (88.03 +/- 1.46)% (P < 0.05), (73.60 +/- 2.76)% (P > 0.05), respectively; KIR was increased to (61.58 +/- 1.89)% (P < 0.05), (60.68 +/- 2.07)% (P < 0.05) and (45.26 +/- 3.31)% (P > 0.05), respectively. When IL-2/PHA were administered in the NK+K562+MO culture system, RNM products was increased from (82.10 +/- 6.60) micromom/L to (193.65 +/- 5.95) micromom/L(P < 0.01);KIR was decreased from (90.64 +/- 3.06)% to (61.29 +/- 2.22)% (P < 0.01). When the TIP, GSH and DHT were administered in the systems, RNM products were decreased to (91.32 +/- 6.81) micromom/L (P < 0.05), (84.66 +/- 5.99) micromom/L (P < 0.05) and (188.92 +/- 5.00) micromom/L (P > 0.05), respectively; KIR was increased to (84.31 +/- 4.56)%(P < 0.05), (81.65 +/- 3.09)% (P < 0.05) and (72.20 +/- 4.10)% (P < 0.05), respectively.

CONCLUSIONNK Cell-mediated killing of K562 cells can be suppressed by exogenous and endogenous RNM administration. Both of TIP and GSH can protect NK cells by scavenging RNM and enhance the antineoplasmic activity of NK cells.

Cells, Cultured ; Coculture Techniques ; Glutathione ; pharmacology ; Histamine ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 ; immunology ; pharmacology ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; Lymphotoxin-alpha ; metabolism ; Monocytes ; cytology ; Peroxynitrous Acid ; pharmacology ; Reactive Nitrogen Species ; antagonists & inhibitors ; metabolism ; Tiopronin ; pharmacology

To investigate the reversal effect of reduced glutathione (GSH) on suppression of NK cells by reactive oxygen metabolites (ROM) in K562 cells, interleukin-2 (IL-2) or mononuclear cell (Mo) was added in cultured cell line of K562 cells and NK cells, the yield of ROM and K562 cell suppression rate were observed. Then the histamine dihydrochloride (DHT) or GSH was added in the mixed cultured cell lines, the ROM production and K562 cell suppression rate were observed. The results showed that the ROM yield increased from 33.17 +/- 5.08 U/L to 223.59 +/- 9.41 U/L by IL-2, and K562 cell suppression rate increased from 65.56% to 85.89% by IL-2 (P < 0.01). The ROM yields were 389.79 +/- 43.83 U/ml, 456.74 +/- 42.77 U/ml and 601.42 +/- 21.92 U/ml respectively, and K562 cell suppression rates were 82.36%, 81.36% and 48.09% respectively, when Mo was added in the mixed cultured cell lines under ratios of E/Mo being 10/2, 10/5 and 10/10. When E/Mo was 10/2, DHT or GSH was added in the mixed cultured cell line ROM yield decreased from 389.79 +/- 3.83 U/L to 50.21 +/- 2.4 U/L or -3.58 +/- 9.49 U/L (P < 0.05) respectively. With increase of concentration of DHT or GSH, the ROM yield in the mixed cultured cell line decreased (P < 0.05), the K562 cell suppression rate increased from 82.53% to 94.64% or 96.39% (P < 0.05), the more ROM yield, the less K562 suppression rate (P < 0.05). When E/Mo is 10/5 or 10/10, the ROM yield decreased by the high concentration of DHT or GSH (P < 0.05), but the K562 cell suppression rate not increased by every concentration of DHT or GSH. GSH was as effective as DHT in the reversing ROM and increasing K562 cell suppression rate. It is concluded that GSH may reverse ROM and increase K562 cell suppression rate, and GSH is as effective as DHT, but GSH has less side-effect than DHT. Therefore, GSH would be better antileukemia immune adjuvant.
Adjuvants, Immunologic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Cell Proliferation ; Coculture Techniques ; Glutathione ; pharmacology ; Histamine ; pharmacology ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; Reactive Oxygen Species ; metabolism

OBJECTIVETo analyze the changes of inhibitory killer cell immunoglobulin-like receptors (KIRs), NKG2D receptor and the cytotoxicity of natural killer (NK) cells induced by persistent exposure to CNE2 cells.

METHODSThe HLA-class I genotypes of CNE2 cells and KIR genotypes were determined by PCR with sequence-specific primers (PCR-SSP). The expressions of KIR2DL1, KIR2DL3, KIR3DL1, and NKG2D by the NK cells (freshly isolated NK cells, NK cells cocultured with 100 U/ml IL2 or with 100 U/ml IL2 and CNE2 cells as the control, IL2 and CNE2 groups, respectively) were analyzed by flow cytometry. Cytotoxicity of NK cells against CNE2 cells were detected by LDH releasing assay.

RESULTSThe HLA genotypes of CNE2 cells were A2, 24, B18, 35, Cw4, 7. NK cells isolated from 3 healthy donors expressed KIR2DL1, KIR2DL3, and KIR3DL1. After 4, 24 and 48 h of culture, NK cells in CNE2 group displayed higher KIR2DL1, KIR2DL3 but lower NKG2D expression than those in the control and IL2 groups (P<0.01), whereas the latter two groups showed no significant difference in KIR2DL1, KIR2DL3, and NKG2D expressions (P>0.05), and no difference in KIR3DL1 expression was found between the 3 groups (P>0.05). After 24 h of culture, the cytotoxicity against CNE2 cells mediated by the NK cells in IL2 and CNE2 groups were (26.96-/+1.47) % and (2.74-/+1.64) % at E:T ratios of 10:1, and (35.74-/+3.59)% and (4.57-/+2.41) % at E:T ratio of 20:1, respectively. NK cells in CNE2 group displayed lower cytotoxicity than those in IL2 group (P<0.01).

CONCLUSIONSPersistent exposure to tumor cells expressing NKG2D ligands can lead to downregulated expression of NKG2D receptor, increased expression of KIRs and reduction of NK-mediated cytolysis. These results elucidate the molecular mechanism of reduced cytotoxicity mediated by the edited NK cells and indicate that blocking HLA-class I-bound KIRs or enhancing the expression of NKG2D may promote NK cell-mediated cytolysis.

Cell Line, Tumor ; Cell Survival ; immunology ; Cytotoxicity, Immunologic ; immunology ; Flow Cytometry ; Humans ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; NK Cell Lectin-Like Receptor Subfamily K ; Nasopharyngeal Neoplasms ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; metabolism ; Receptors, KIR ; metabolism ; Receptors, KIR2DL1 ; metabolism ; Receptors, KIR2DL3 ; metabolism ; Receptors, Natural Killer Cell

This study was aimed to investigate the expression level of perforin in cord blood NK cells and the relation of perforin expression after IL-2, IL-15 stimulation to cytotoxicity of NK cells. NK cells were isolated from cord blood MNC by depleting CD3(+) cells and then enriching CD56(+) cells using immunomagnetic separation (CD3 and CD56 cell isolation kit, autoMACS, miltenyi). The purity was analysed by flow cytometry. According to the different combination of cytokines, there were two groups: group A (IL-2) and group B (IL-2 + IL-15). The cytotoxicity and perforin expression rate of fresh and different cultured CB-NK cells against K562/Jurkat cell lines were estimated by LDH release test (cytotoxic 96 non-radioactive cytotoxicity assay). The results showed that the purity of NK cells after separation was more than 90%. The cytotoxicity towards both tumor lines in group B was higher than that in group A (p < 0.05), and cytotoxicity in group A was higher than that of fresh NK cells (p < 0.05). Perforin expression rate of group A (84.55%) was higher than that of fresh NK cells (67.21%) (p < 0.05), and there was no significant difference between group A and B (84.55% versus 87.22%) Cytotoxic activity of CB-NK cells was positively correlated with perforin expression rate (r = 0.886, p < 0.05). It is concluded that IL-2 can enhance cytotoxicity of CB/BM-NK cells by increasing the perforin expression.
CD56 Antigen ; metabolism ; Cells, Cultured ; Cytotoxicity, Immunologic ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; Perforin ; metabolism

OBJECTIVETo evaluate the method for expanding Valpha24 natural killer T (NKT) cells with alpha-galactosylceramide (alpha-GalCer) ex vivo.

METHODSMononuclear cells (MNCs) isolated from adult peripheral blood or umbilical cord blood (UCB) were divided into three groups. In Group A1 (n = 5), CD34+ progenitorderived dendritic cells were differentiated in a cytokine-supplemented culture system from cord blood and acted as antigen presenting cells (APC) to induce the expansion of cord blood Valpha24 NKT cells in presence of alpha-GalCer; in Group A2 (n = 5), adult peripheral monocyte-derived dendritic cells (Mo-DC) were used as APC to induce the expansion of adult peripheral NKT cells in presence of alpha-GalCer; whereas in Group B (n = 16), alpha-GalCer was added into adult peripheral MNCs culture system without additional DCs. Cytokine-produce were measured by ELISA, and NKT cells' proliferation ability, cytotoxicity, and suppressive effect on mixed lymphocyte reaction (MLR) were examined by MTT assays.

RESULTSValpha24 NKT cells in Group A1, A2, and B were expanded up to 128 (95-207), 250.5 (179.6-790.6), and 326 (101-2 136) -fold by day 12, respectively. Adult NKT cells expanded in Group B were markedly better than those in Group A1 (P = 0.038). When stimulating by PMA, the NKT cells had a 3-day stimulate index of 1.80 +/- 0.41; and the secretion ratio of IL-4 to IFN-gamma of UCB or adult peripheral blood NKT cells were 0.30 +/- 0.13 and 0.28 +/- 0.18; and the ex vivo antitumor effect of expanded NKT cells were found in cell line HL60, KG1a, and Raji except for K562; and the suppressive effect of expanded NKT cells or the culture supernatant on MLR were confirmed.

CONCLUSIONSAlpha-GalCer can facilitate the rapid shorttime expansion of Valpha24 NKT cells in presence of IL-2 and IL-15. These expanded NKT cells, kill tumor cell lines, and inhibit can massively excret IL-4 and IFN-gamma allogeneic T-cell response.

Antigens, CD34 ; analysis ; immunology ; Cytotoxicity Tests, Immunologic ; Dendritic Cells ; immunology ; Galactosylceramides ; immunology ; HL-60 Cells ; pathology ; Humans ; K562 Cells ; pathology ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; Lymphocyte Activation ; drug effects ; Receptors, Antigen, T-Cell, alpha-beta ; immunology ; T-Lymphocyte Subsets ; cytology ; immunology ; metabolism

OBJECTIVETo explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.

METHODSNK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.

RESULTSCD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).

CONCLUSIONHigh purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.

Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; pharmacology ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukins ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; metabolism ; Perforin ; metabolism

To investigate the effect of a new reactive oxygen metabolites (ROM) scavenger as immune adjuvant in NK cell-mediated killing effect on K562 cell, IL-2 and PHA were used to activate monocyte to produce ROM, and different concentrations of tiopronin as ROM scavenger was used in the cultivated systems with different ratio of monocytes plus NK cells and K562 cells, while histamine dihydrochloride (DHT) with different concentrations was used as positive control. The reuslts indicated that after IL-2 and PHA were supplemented in the cultivated systems mixing with NK cells and K562 cells as the E/T ratio was 10/1, the ROM production increased from 33.17 +/- 25.02 U/ml to 223.59 +/- 59.41 U/ml (P < 0.05) while K562 cell inhibition rate (KIR) increased from 65.56% to 85.89% (P < 0.05). When the monocytes as the E/MO ratios of 10/2, 10/5 and 10/10 were supplemented respectively, ROM production increased correspondingly (ROM production was 389.79 +/- 43.83 U/ml, 456.74 +/- 42.77 U/ml, 601.42 +/- 21.92 U/ml, respectively), and KIR was on the other round (KIR was 82.36%, 81.36%, 48.09% respectively). Tiopronin, DHT were used in the K562 + NK + MO + IL-2/PHA cultivated systems as the E/MO ratio was 10/2, the ROM production also decreased from 389.79 +/- 43.83 U/ml to -1.20 +/- 60.70 U/ml, 50.21 +/- 22.4 U/ml (P < 0.05), respectively, however KIR increased from 82.53% to 96.09% and 94.64% either (P < 0.05). Higher concentrations of tiopronin and DHT were used, ROM production decreased accordingly. There showed a reverse correlation between ROM production and KIR (r = -0.518). When E/MO ratio was 10/5 or 10/10, tiopronin at any testing concentration and DHT at the higher testing concentration could reduce the ROM production (P < 0.05), but did not improve KIR significantly (P > 0.05). Tiopronin was as good as DHT in ameliorating KIR (P > 0.05) and better than DHT in scavenging ROM (P < 0.05). It is concluded that (1) Monocytes are the major resources of ROM, and the ROM derived from monocytes can disable NK cells in killing neoplasm cells (K562 cells); (2) A new ROM scavenger, tiopronin, can scavenge ROM effectively, and reverse the ROM induced inhibition of NK cell-mediated killing of K562 cell in a certain extent. And tiopronin is better than DHT in scavenging ROM, and as good as DHT in up-regulating KIR. The new ROM scavenger tiopronin with less side effect may take the place of DHT as adjuvant during the adoptive immuno-therapy in leukemia.
Coculture Techniques ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Free Radical Scavengers ; pharmacology ; Humans ; K562 Cells ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Reactive Oxygen Species ; antagonists & inhibitors ; metabolism ; Tiopronin ; pharmacology

AIMTo observe the effect of cold stress on the splenic NK cell activity and the c-fos expression in rat brain.

METHODSRats were maintained in cold chamber at 4 degrees for 4 h. The 51Cr release assay from YAC-1 cells was used to determine the splenic NK cell activity and the double staining of ABC method was employed to observe the immunoreactive expression of Fos, arginine-vasopressin and tyrosine hydroxylase.

RESULTSCold stress could induce a marked suppression of splenic NK cell activity and a significant expression of Fos protein in hypothalamic paraventricular nucleus(PVN), as well as in the brainstem locus coeruleus (LC). Double staining showed c-expression of Fos and arginine-vasopressin in some of PVN neurons, and co-expression of Fos and tyrosine hydroxylase in the majority of LC neurons.

CONCLUSIONThe vasopressinergic neurons in PVN and the catecholaminergic neurons in LC might be probably involved in the suppression of splenic NK cell activity induced by cold stress.

Animals ; Arginine Vasopressin ; metabolism ; Brain ; metabolism ; Cold Temperature ; Killer Cells, Natural ; metabolism ; Male ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Wistar ; Spleen ; cytology ; immunology ; Stress, Physiological ; Tyrosine 3-Monooxygenase ; metabolism