Impact of IL-2 and IL-2R SNPs on proliferation and tumor- killing activity of lymphokine-activated killer cells from healthy chinese blood donors.

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM- PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-γ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL- 2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.

Mycophenolic acid inhibits natural killer cell proliferation and cytotoxic function: a possible disadvantage of including mycophenolate mofetil in the graft-versus-host disease prophylaxis regimen.

To determine how immunosuppressant agents used for graft-versus-host disease (GVHD) prophylaxis affect natural killer (NK) cells, we examined the effects of cyclosporine (CSP), tacrolimus (TAC), mycophenolic acid (MPA, an active form of mycophenolate mofetil), and methotrexate (MTX) on the proliferation and cytotoxicity of NK cells. The proliferation of NK cells from healthy individuals in the presence of interleukin (IL)-2 and IL-15 was suppressed to 51% ± 16% of that of the controls with CSP, to 31% ± 19% with TAC, to 14% ± 6% with MPA, and to 87% ± 18% with MTX. Both CSP and TAC increased the proportion of CD16(-)CD56(bright) cells, a NK cell subset capable of secreting high amount of cytokines, and also enhanced NKp30 expression, whereas MPA markedly decreased the proportion of CD16(-)CD56(bright) cells and reduced the expression of all activating NK cell receptors, including NKG2D, NKp30, NKp44, and NKp46. MPA also reduced the cytotoxicity against K562 cells from 61% ± 15% to 17% ± 7% and that against Daudi cells from 44% ± 4% to 4% ± 4%, whereas the other 3 drugs did not diminish these cytotoxicities. The inhibition of NK cell proliferation and cytotoxicity against leukemic cell lines by MPA was partially abolished by the inclusion of guanosine in the culture. Similar to the effect of MPA on T cells, MPA inhibited the down-regulation of p27 on NK cells induced by the incubation of NK cells in the presence of IL-2. These results suggest that MPA is a potent inhibitor of NK cells, and that its inclusion in the GVHD prophylaxis regimen might diminish the graft-versus-leukemia effect of NK cells.

Impaired differentiation and cytotoxicity of natural killer cells in systemic lupus erythematosus.

OBJECTIVE: To determine the cytotoxicity of natural killer (NK) cells and the level of differentiation of hematopoietic stem cells (HSCs) into NK cells in systemic lupus erythematosus (SLE). METHODS: Patients with SLE (n=108), rheumatoid arthritis (RA; n=90), Behçet's disease (n=39), or ankylosing spondylitis (n=41) and healthy control subjects (n=173) were enrolled in the study. NK cell levels, NK cell cytotoxicities, and lymphokine-activated killer (LAK) activities against K562 cells were measured by flow cytometry. Gene expression was assessed by reverse transcription-polymerase chain reaction. NK cells were differentiated from peripheral blood and bone marrow HSCs in vitro. RESULTS: Percentages and absolute numbers of NK cells, cytotoxicities, and LAK activities were significantly lower in the peripheral blood of SLE and RA patients than in that of healthy controls. In particular, this NK cell deficiency was more prominent in patients with lupus nephritis and those with thrombocytopenia. Notably, purified NK cells derived from SLE patients, but not RA patients, were found to have lower cytotoxicities and LAK activities than those from healthy controls. This defect of NK cells in SLE patients was found to be related to lower numbers of NK precursors and to the down-regulation of perforin and granzyme in NK cells. The proliferative capacity of HSCs, the percentages of NK cells differentiated from HSCs, and NK cell cytotoxicities were significantly lower in SLE patients. CONCLUSION: In SLE patients, circulating levels of NK cells were diminished and their cytotoxicities were impaired. Furthermore, the differentiation of HSCs into NK cells was found to be defective. These abnormalities possibly contribute to immune system dysregulation in SLE.

[Expression of CD16zeta in NK cells of B-cell non-Hodgkin's lymphoma patients and in vitro killing effect of rituximab combined lymphokine-activated killer cells on B-NHL cells].

BACKGROUND & OBJECTIVE: Natural killer (NK) cells are the main effector of antibody-dependent cellular cytotoxicity (ADCC). The activation disorder of NK cells in cancer patients may affect the treatment effect of monoclonal antibody. Reversing the dysfunction of signal transduction of CD16zeta chain in NK cells and combining lymphokine-activated killer (LAK) cells with rituximab may give rise to synergistic effect. This study was to find out whether the activation disorder of NK cells exist in B-cell non-Hodgkin's lymphoma (B-NHL) patients, whether interleukin-2 (IL-2) can reverse the activation disorder in vitro, and whether the combination of rituximab and LAK cells can produce synergistic antitumor effect. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 69 B-NHL patients and 30 healthy donors by density gradient centrifugation method, and cultured with IL-2 (1 000 U/ml) to prepare LAK cells. The positive rate and median fluorescence intensity (MFI) of CD16zeta chain in PBMCs and LAK cells were detected by flow cytometry (FCM). The expression of CD20 on Raji cells was also detected by FCM. The apoptosis of Raji cells after treatment of rituximab was detected by FCM with Annexin V/PI staining. The cytotoxicity was assessed by lactate dehydrogenase (LDH) release experiment. RESULTS: The positive rate and MFI value of CD16zeta chain on CD56+ cells were significantly lower in B-NHL group than in control group [(63.3+/-16.4)% vs. (97.8+/-3.1)%, P<0.001; 1.3+/-1.3 vs. 3.6+/-1.7, P<0.001]. There was no significant difference in the positive rate and MFI value of CD16zeta on LAK cells between the 2 groups [(99.3+/-4.1)% vs. (99.7+/-3.9)%, P=0.145; 29.2+/-12.5 vs. 31.4+/-13.8, P=0.44]. At the concentration of 40 mug/ml, rituximab completely combined CD20 antigens on cell membrane. The obvious enhancive effect of rituximab on cell apoptosis appeared at 24 h after treatment. The killing rate of Raji cells was significantly higher in rituximab combined LAK group than in LAK group (P<0.05). While the combination of LAK cells and Herceptin (40 mug/ml) didn't make a significant increase as compared with Herceptin alone (P>0.05). There was no significant difference in killing rate of Jurket cells between rituximab combined LAK group and LAK group (P>0.05). CONCLUSIONS: The down-regulation of CD16zeta chain expression widely exists in B-NHL patients, while high dose of IL-2 could enhance the expression of CD16zeta chain greatly in vitro. The combination of rituximab and LAK cells shows strong killing effect on Raji cells.

Cytolytic cells induce HMGB1 release from melanoma cell lines.

High mobility group box 1 (HMGB1) is one of the recently defined damage-associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis. We induced melanoma cell death with cytolytic lymphokine-activated killing (LAK) cells, tumor-specific cytolytic T lymphocytes, TRAIL, or granzyme B delivery and assessed intracellular HMGB1 retention or release to investigate the mechanism of HMGB1 release by cytolytic cells. HMGB1 release from melanoma cells (451Lu, WM9) was detected within 4 h and 24 h following incubation with IL-2-activated PBMC (LAK activity). HLA-A2 and MART1 or gp100-specific cytolytic T lymphocytes induced HMGB1 release from HLA-A2-positive and MART1-positive melanoma cells (FEM X) or T2 cell-loaded, gp100-specific peptides. TRAIL treatment, however, induced HMGB1 release, and it is interesting that this extrinsic pathway-mediated cell death was blocked with the pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Conversely, granzyme B delivery did not induce HMGB1 release. HMGB1, along with other intracellular factors released from tumor cells induced by cytolysis, may be important components of the disordered tumor microenvironment. This has important implications for the immunotherapy of patients with cancer. Specifically, HMGB1 may promote healing or immune reactivity, depending on the nature of the local inflammatory response and the presence (or absence) of immune effectors.

The effect of silencing NKG2D through RNA interference on receptor functions in interleukin-2-activated human natural killer cells.

Natural killer (NK) cells are effectors of the innate immunity involved in tumor surveillance. NKG2D is a potent activating receptor eliciting cytokine and cytolytic NK responses upon recognition of tumor-associated ligands. We engineered primary interleukin (IL)-2-activated human NK cells to express constitutively low levels of NKG2D by lentiviral delivery of small interfering RNA. NKG2D-mediated effector functions were strongly impaired in NKG2D(low) NK cells. Reduction of NKG2D surface expression to 15%, corresponding to receptor levels in resting NK cells, rendered cells fully insensitive to NKG2D triggering. These data underscore the importance of NKG2D receptor cell surface density and suggest a threshold of expression for optimal reactivity of human NK cells.

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms.

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2-activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2-activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.

Cytoskeleton actin changes in IL-2 activated cells.

In the present study we analysed the changes in cytoskeleton actin in lymphoid cells following IL-2 activation and during cell interactions by means of light and electron microscopy, immunofluorescence and molecular analysis. By morphological analysis we observed a higher fluorescence in the activated cells than in the quiescent ones with no modifications in the cytoskeleton pattern comparing activated to resting cells. The results of molecular analysis indicate that, after IL-2 activation, there is a reorganisation of the actin component of the cell cytoskeleton accompanied by the differential expression of the corresponding genes. A future study will be extended to the analysis of others components of the cytoskeleton network.

Enhanced apoptosis of squamous cell carcinoma cells by interleukin-2-activated cytotoxic lymphocytes combined with radiation and anticancer drugs.

Induction of potent apoptosis is required in cancer therapy. We examined the combination effect of interleukin-2-activated lymphocytes (LAK cells) and anticancer drugs or gamma (gamma)-rays on the induction of apoptosis in an established oral squamous cell carcinoma cell line (OSC-3 cells). By pretreatment of OSC-3 cells with (137)Cs (5 Gy), 5-fluorouracil (5-FU) (0.5 microg/ml) or cis-dichlorodiammine-platinum (CDDP) (5 microg/ml), the activation of bid and caspase-3 by LAK cells was strongly increased and associated with an enhanced degradation of poly-(ADP-ribose) polymerase (PARP) and/or nuclear mitotic apparatus protein (NuMA) and the increased fragmentation of DNA. The LAK cell-enhanced caspase-3 activity in the pretreated OSC-3 cells was decreased to approximately 70% and 40% of the control by the addition of Z-AAD-CMK (a granzyme B inhibitor) and neutralising monoclonal antibody to Fas antigen (alphaFas-IgG), respectively. The combined treatment-induced DNA fragmentation was suppressed by approximately 20% and 30% of the control by the addition of Z-AAD-CMK and alpha Fas-IgG, respectively, in the co-culture system. While Ac-DEVD-CHO (a caspase-3 inhibitor) suppressed the DNA fragmentation levels to approximately half and this was similar to the amount of suppression that was obtained by the addition of both alpha Fas-IgG and Z-AAD-CMK. In addition, LAK cell-activated bid may have increased the intracellular reactive oxygen intermediates (ROI) level and induced a decrease of mitochondrial membrane potential. These influences by LAK cells were enhanced when OSC-3 cells were pretreated with each anticancer drug or (137)Cs. Furthermore, the increase of ROI by LAK cells was suppressed by alpha Fas-IgG and Z-AAD-CMK to approximately half the level of the control. These results indicate that anticancer drugs and gamma-rays prime squamous cell carcinoma cells to be susceptible to apoptosis by LAK cells, that LAK cell-induced apoptosis largely depends on the activation of caspase-3 by the Fas/Fas-ligand signal and granzyme B, and that LAK cells induce ROI in the target cells, which is largely mediated by Fas and granzyme B.

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals.

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.

Induction and membrane expression of heat shock proteins in heat-treated HPC-4 cells is correlated with increased resistance to LAK-mediated lysis.

In human pancreatic carcinoma cells (HPC-4), a hyperthermic treatment at 43 degrees C for 30 min resulted in the vigorous induction of Hsp72, along with a less pronounced increase in the rate of synthesis of Hsp90, Hsp60 and Hsp 27. Biotinylation of surface-exposed proteins, followed by isolation of biotin-tagged proteins by affinity chromatography, demonstrated that both Hsp72 and Hsp60 are expressed on plasma membrane. Membrane expression of these two Hsps was confirmed by immunoprecipitation of surface biotinylated proteins with anti-Hsp72 and anti-Hsp60 specific antibodies. Cytotoxic assays showed that untreated HPC-4 cells are intrinsically resistant to NK-mediated lysis, while they were efficiently killed by LAK lymphocytes, as well as by exposure to TNFalpha. Following heat-treatment, cells became much more resistant to LAK-mediated lysis, while their sensitivity to NK-mediated lysis and to TNFalpha cytotoxicity remained unmodified.

Effect of aqueous humor on apoptosis of inflammatory cell types.

PURPOSE: To determine whether aqueous humor promotes cell death in cells involved in inflammatory responses. METHODS: Multiple immune cell types, most characteristically involved in inflammatory responses, were incubated for 24, 48, and 72 hours in the presence or absence of 50% aqueous humor. Promotion of cell death was assayed by staining for an early indicator of apoptosis. The percentage of cells undergoing apoptosis was measured by flow cytometry. To identify partially the apoptosis inducing factor, aqueous humor was pretreated with proteinase K to degrade protein. In other experiments, aqueous humor was fractionated by centrifugation on filters capable of separating molecules above and below 10 kDa or 30 kDa kilodaltons in size. RESULTS: Rabbit aqueous humor promoted apoptosis in a wide variety of immune cells, including lymphokine-activated natural killer cells, resting T cells, an activated T-cell line, RAW 264.7 and J774A0.1 monocyte-macrophage cell lines, and neutrophils. As previously shown, aqueous humor did not promote apoptosis of murine corneal endothelial cells. Apoptosis was also not induced in human corneal endothelium, mouse corneal epithelium, or iris/ciliary body cell lines. Instead, aqueous humor partially protected these ocular tissues from starvation-induced cell death. Pretreatment with proteinase K inhibited the apoptosis-inducing activity. Moreover, the apoptosis-inducing activity segregated with the aqueous humor fraction containing molecules less than than 10 kDa in size. CONCLUSIONS: These data show that aqueous humor contains a factor or factors that promote death of cells that participate in inflammatory processes. By contrast, ocular tissues, such as the corneal endothelium and iris/ciliary body, are impervious to aqueous humor-induced cell death. The aqueous humor- borne factor(s) may contribute to the immune privilege of the anterior chamber by purging potential inflammatory cells.

Lymphokine-activated killer cells induced in vivo in mice showing IL-2 toxicity have cytoplasmic granules containing perforin and its hemolytic activity.

Cytoplasmic granules of LAK cells isolated from mice showing side effects of recombinant human IL-2 (rhIL-2) display BLT-serine esterase activity and calcium-dependent hemolytic activity and seem to be involved in the mechanisms of LAK cell-mediated rhIL-2 toxicity. In this report, the hemolytically active substance was partially purified from the LAK granules and shown to be mouse perforin. Mice were implanted with miniosmotic pumps filled with a dose of rhIL-2 that induced known toxicity. The LAK granules were isolated from LAK cell-rich subcutaneous tissue around the infusion sites. The hemolytically active substance in the granule preparation was solubilized by 2 M NaCl and passed through a butyl Sepharose column and then a DEAE Toyopearl column. In immunoblotting, the hemolytically active fractions reacted with an anti-mouse perforin mAb and the reaction depended on the activity. The molecular sizes of the perforin-positive bands were 66 kDa and 51 kDa under reducing and nonreducing conditions, respectively. These results confirmed the existence of mouse perforin and its hemolytic activity in the LAK granules of rhIL-2-treated mice, as has been shown for granules of cytotoxic T or NK cell clones in vitro.

Nitric oxide exposure inhibits induction of lymphokine-activated killer cells by inducing precursor apoptosis.

Nitric oxide synthesis is strongly induced during IL-2 treatment of mice and humans. While this free radical can act as an antitumor mechanism by inhibiting cellular respiration and DNA synthesis in cancer cells, immunosuppressive effects have also been suggested. We evaluated the effects of NO exposure on the induction of murine lymphokine-activated killer (LAK) cells from splenocytes by IL-2 (6000 IU/ml). When splenocytes were exposed to pure NO gas for 30 min prior to the addition of IL-2, complete abrogation of LAK cell cytotoxicity was observed. In contrast, cytolytic activity of already activated LAK cells was only minimally affected by NO exposure. NO exposure markedly depressed cellular proliferation in response to concanavalin A or IL-2. Immunostaining of LAK cell cultures following NO exposure revealed a marked decrease in CD8+, and peanut lectin (PNA+)/CD56+ subsets (48 and 69%). Dual staining of LAK cells for DNA strand breaks and either PNA or CD8+ identified the induction of programmed cell death in these subsets 12-24 h following NO exposure. These experiments demonstrate that NO has the capacity to inhibit LAK cell induction by inducing apoptosis of cytolytic lymphocyte precursors.

Effect of glutamine supplementation on changes in the immune system induced by repeated exercise.

UNLABELLED: The ability of lymphocytes to proliferate and generate lymphokine activated killer (LAK) cell activity in vitro is dependent on glutamine. In relation to intense exercise the lymphocyte concentration, the proliferative response, the natural killer and LAK cell activity, and the plasma glutamine concentration decline. It has been hypothesized that in relation to physical activity a lack of glutamine may temporarily affect the function of the immune system. PURPOSE: The purpose of this study was to examine the influence of glutamine supplementation on exercise-induced immune changes. METHODS: In a randomized cross-over placebo-controlled study, eight healthy male subjects performed three bouts of ergometer bicycle exercise lasting 60, 45, and 30 min at 75% of their VO2max separated by 2 h of rest. RESULTS: The arterial plasma glutamine concentration declined from 508 +/- 35 (pre-exercise) to 402 +/- 38 microM (2 h after the last exercise bout) in the placebo trial and was maintained above pre-exercise levels in the glutamine supplementation trial. The numbers of circulating lymphocytes and the phytohemagglutinin-stimulated lymphocyte proliferative response declined 2 h after, respectively, during each bout of exercise, whereas the LAK cell activity declined 2 h after the third bout. Glutamine supplementation in vivo, given in the described doses at the specific times, did not influence these changes. CONCLUSION: The present study does not appear to support the hypothesis that those aspects of postexercise immune changes studied are caused by decreased plasma glutamine concentrations.