Wnt pathway activator TWS119 enhances the proliferation and cytolytic activity of human γδT cells against colon cancer.

γδT cells are a distinct T-cell subset that display unique characteristics regarding T-cell receptor gene usage, tissue tropism and antigen recognition. Adoptive γδT cell transfer therapy has recently been gaining importance as an efficient approach in cancer immunotherapy. However, exploiting γδT cell response for tumour immunotherapy is a challenge due to cell numbers, activities and differentiation states that minimize the clinical therapeutic effects. Previous studies have indicated that the wnt/ß-catenin signalling pathway plays a crucial role in the differentiation, survival and enhancement of the immune response of T lymphocytes. In this study, we sought to evaluate whether the activation of the wnt/ß-catenin pathway through inhibition of glycogen synthase kinase-3ß (GSK-3ß) using 4,6-disubstituted pyrrolopyrimidine (TWS119) could be an efficient strategy to improve the proliferation, differentiation and cytolytic activity of γδT cells against colon cancer cells. Remarkably, we found that TWS119 significantly enhanced the proliferation and survival of γδT cells via activation of the mammalian target of rapamycin (mTOR) pathway, upregulation of the expression of the anti-apoptotic protein Bcl-2 and inhibition of cleaved caspase-3 in addition to the Wnt pathway. Our results also showed that enhancement of the cytolytic activity of γδT cells against human colon cancer cells by TWS119 was chiefly associated with upregulation of the expression of perforin and granzyme B in vitro and in vivo. Additionally, TWS119 can induce the expression of CD62L or CCR5 to generate a population of CD62L+γδT or CCR5+γδT cells in a dose-dependent manner. These findings suggested that TWS119 could be a useful complementary agent for improving γδT cell-based immunotherapy.

Artesunate enhances γδ T-cell-mediated antitumor activity through augmenting γδ T-cell function and reversing immune escape of HepG2 cells.

OBJECTIVE: To explore the effect and mechanism of artesunate on γδ T cell-mediated antitumor immune responses against hepatoma carcinoma cells (HepG2) in vitro. METHODS: Human γδ T cells or HepG2 were respectively treated with artesunate, subjected to co-culture as appropriate, and the following assays were subsequently conducted: CCK8 to examine cell viability; LDH release assay to detect the killing effect of γδ T cells on HepG2 cells; flow cytometry to examine the expression of perforin (PFP) and granzyme B (GraB) of γδ T cells; ELISA to evaluate the levels of TGF-ß1 and IL-10 in the collected supernatant of HepG2 cells pretreated with artesunate; and Western blot analysis to examine Fas, FasL, STAT3, p-STAT3 expression of HepG2 cells induced by artesunate. Results: The results showed that the cytotoxicity effect of γδ T cells pretreated with artesunate on HepG2 cells was augmented via elevating the expression of GraB in γδ T cells. Furthermore, treatment with artesunate reversed the inhibition of HepG2 cells on γδ T cells by reducing the secretion of TGF-ß1 in HepG2 cells supernatant and enhanced the antitumor effect of γδ T cells against HepG2 cells through increasing the expression of Fas on HepG2 cells, which may be attributed to the inhibition of STAT3 signaling protein. CONCLUSION: Artesunate has several mechanisms for augmenting the antitumor immune responses mediated by γδ T cells. These results suggested artesunate may be an efficacious agent in the treatment of hepatocellular carcinoma.

Immunopotentiation effects of apigenin on NK cell proliferation and killing pancreatic cancer cells.

Apigenin is a kind of flavonoid with many beneficial biological effects. It not only has direct cytotoxicity to tumor cells, but also can boost the antitumor effect of immune cells by modulating immune system. The purpose of this study was to investigate the proliferation of NK cells treated with apigenin and its cytotoxicity to pancreatic cancer cells in vitro, and explore its potential molecular mechanism. In this study, the effect of apigenin on NK cell proliferation and killing pancreatic cancer cells were measured by CCK-8 assay. Perforin, granzyme B (Gran B), CD107a, and NKG2D expressions of NK cells induced with apigenin were detected by flow cytometry (FCM). The mRNA expression of Bcl-2, Bax and protein expression of Bcl-2, Bax, p-ERK, and p-JNK in NK cells were evaluated by qRT-PCR and western blotting analysis, respectively. The results showed that appropriate concentration of apigenin could significantly promote the proliferation of NK cells in vitro and enhance the killing activity of NK cells against pancreatic cancer cells. The expressions of surface antigen NKG2D and intracellular antigen perforin and Gran B of NK cells were upregulated after treating with apigenin. Bcl-2 mRNA expression was increased, while Bax mRNA expression was decreased. Similarly, the expression of Bcl-2, p-JNK, and p-ERK protein was upregulated, and the expression of Bax protein was downregulated. The molecular mechanism of the immunopotentiation effects of apigenin may be that it up-regulates Bcl-2 and down-regulates Bax expression at the gene and protein levels to facilitate NK cell proliferation, and up-regulates the expression of perforin, Gran B, and NKG2D through the activation of JNK and ERK pathways to enhance NK cell cytotoxicity.

Enhancement of CD3AK cell proliferation and killing ability by α-Thujone.

Thujone is a monoterpene ketone natural substance found mainly in wormwood and sage. Previous studies have shown that Thujone has various pharmacological effects, such as anti-tumor, analgesic, and insecticide. The effect of α-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of α-Thujone to CD3AK (anti- CD3 antibody induced activated killer) cells proliferation and cytotoxicity to colon cancer cell lines. With cell proliferation and FCM assay, it is found that α-Thujone could significantly enhance CD3AK cell proliferation and expression of CD107a in a dose-dependent manner. The cytotoxicity to colon cancer cells detected by CCK-8 assay is also improved. The expressions of TNF-α and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that α-Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, α-Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, α-Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2.

The effect of phloretin on human γδ T cells killing colon cancer SW-1116 cells.

OBJECTIVE: To explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells. METHODS: γδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells. RESULTS: After cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31±3.00% to 78.40±10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35µg/ml to 18.75µg/ml, it could significantly proliferate the γδ T cell growth (P<0.05) and inhibit the growth of SW-1116 cells in dose-response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P<0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P<0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group. CONCLUSION: Phloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity.

The enhanced effect of lupeol on the destruction of gastric cancer cells by NK cells.

Lupeol, a triterpene, was reported to possess beneficial effects as a therapeutic and preventive agent for a range of disorders. Many studies have confirmed that lupeol possesses strong activities such as antioxidative, antiinflammatory, antiarthritic, antimutagenic, and antimalarial, both in vitro and in vivo, and at its effective therapeutic doses exhibit no toxicity to normal cells and tissues. Lupeol was observed to inhibit the proliferation of gastric tumour cells in a dose-dependent manner, as assessed by MTT assay, and induce the proliferation of NK cells, as assessed by flow cytometry and Western blotting. The killing effect of NK cells on gastric tumour cells was assessed by LDH. Our experiment demonstrated that lupeol at appropriate concentrations could promote the proliferation of NK cells, inhibit the proliferation of gastric cancer cell lines BGC823, N87 and HGC27, and increase the killing effect of NK cells on gastric cancer cells. We speculated that lupeol might increase the expression of PFP, IFN-γ, and CD107a via the activation of PI3K/Akt and Wnt/ß-catenin signalling pathways. Lupeol could serve as a potential agent against gastric cancer; however, further in-depth in vivo studies are still required.

[Association of traumatic severity with change in lymphocyte subsets in the early stage after trauma].

OBJECTIVE: To investigate the association of traumatic severity with changes in lymphocyte subsets in the early stage after trauma. METHODS: Sixty-three male patients admitted within 4 hours after trauma were enrolled. According to injury severity score (ISS), the patients were divided into two groups: mild trauma group (ISS<16, n=35) and severe trauma group (ISS≥16, n=28). At admission, the patients peripheral blood were extracted to detect T lymphocytes subsets, blood routine test, blood biochemical and arterial blood gas analysis which were used to calculate the acute physiology and chronic health evaluation II (APACHEII) scores. The correlation of lymphocyte subsets and ISS score, and the correlation of lymphocyte subsets and APACHEII score were both analyzed statistically. Another 20 cases of healthy male adults were enrolled as the control group. RESULTS: Compared with the healthy control group, CD3(+) T cell contents in blood were decreased obviously in mild trauma group and severe trauma group (0.648±0.112, 0.647±0.110 vs. 0.708±0.082, both P<0.05); CD4(+) T cells contents in severe group were decreased significantly (0.317±0.086 vs. 0.389±0.064, P<0.05), and natural killer (NK) cells were significantly increased (0.217±0.107 vs. 0.158±0.068, P<0.05). B cells content in severe group was decreased significantly than that of mild group (0.114±0.060 vs. 0.155±0.075, P<0.05). There were no significant difference in CD8(+) and CD4/CD8 ratio among the healthy control group, mild trauma group and severe trauma group (CD8(+): 0.260±0.074, 0.260±0.091, 0.271±0.105; CD4/CD8 ratio: 1.69±0.75, 1.56±0.83, 1.34±0.65, all P>0.05). Except that there were negative correlation between CD3(+) T cells and the ISS scores (r=-0.42, P=0.03), the other lymphocyte subsets showed no correlation with the ISS scores and the APACHEII scores (mild trauma group with ISS scores: CD3(+) r=-0.10, CD4(+) r=-0.31, CD8(+) r=0.18, B cells r=0.20, NK cells r=-0.04; mild trauma group with APACHEII scores: CD3(+) r=0.04, CD4(+) r=-0.07, CD8(+) r=0.06, B cells r=-0.10, NK cells r=0.05, severe trauma group with ISS scores: CD4(+) r=-0.12, CD8(+) r=-0.17, B cells r=0.02, NK cells r=0.31,all P>0.05;severe trauma group with APACHEII scores:CD3(+) r=-0.24, CD4(+) r=0.11, CD8(+) r=-0.26, B cells r=0.15, NK cells r=0.08, all P>0.05). CONCLUSIONS: CD3(+) and CD4(+) T cells decreased and NK cells increased significantly in blood in the early stage after severe trauma. CD3(+) T cells are independent indexes which reflect body injury. Therefore, it is necessary to monitor the changes of immune cells dynamically after severe trauma.

Enhancement effect of dihydroartemisinin on human γδ T cell proliferation and killing pancreatic cancer cells.

γδ T cells play important roles in innate immunity against tumors and infections. Inhibitory effect of dihydroartemisinin on growth of cancer cells has been found in recent years. In this study, we investigated the effect of dihydroartemisinin on human γδ T cell proliferation by MTT assay and killing activity against pancreatic cancer cells SW1990, BxPC-3 and PANC-1 by LDH release assay in vitro. Intracellular molecule alterations were verified by flow cytometry. The results suggested that appropriate concentration of dihydroartemisinin favored the expansion of γδ T cells and enhanced γδ T cell mediated killing activity against pancreatic cancer cells. Up-regulation of intracellular perforin, granzyme B expression and IFN-γ production may be the important mechanism of dihydroartemisinin on increased antitumor activity of γδ T cells.

Effects of pituitary adenylate cyclase activating polypeptide on CD4(+)/CD8(+) T cell levels after traumatic brain injury in a rat model.

BACKGROUND: The effect of pituitary adenylate cyclase activating polypeptide (PACAP) during traumatic brain injury (TBI) and whether it can modulate secondary injury has not been reported previously. The present study evaluated the potential protective effects of ventricular infusion of PACAP in a rat model of TBI. METHODS: Male Sprague Dawley rats were randomly divided into 3 treatment groups (n=6, each): sham-operated, vehicle (normal saline)+TBI, and PACAP+TBI. Normal saline or PACAP (1 µg/5 µL) was administered intracerebroventricularly 20 minutes before TBI. Right parietal cortical contusion was produced via a weight-dropping method. Brains were extracted 24 hours after trauma. Histological changes in brains were examined by HE staining. The numbers of CD4(+) and CD8(+) T cells in blood and the spleen were detected via flow cytometry. RESULTS: In injured brain regions, edema, hemorrhage, inflammatory cell infiltration, and swollen and degenerated neurons were observed under a light microscope, and the neurons were disorderly arrayed in the hippocampi. Compared to the sham group, average CD4(+) CD8(-) lymphocyte counts in blood and the spleen were significantly decreased in rats that received TBI+vehicle, and CD4(-) CD8(+) were increased. In rats administered PACAP prior to TBI, damage was attenuated as evidenced by significantly increased CD4(+), and decreased CD8(+), T lymphocytes in blood and the spleen. CONCLUSION: Pretreatment with PACAP may protect against TBI by influencing periphery T cellular immune function.

[Inhibitory effect of toll-like receptor 7 agonist on K562 cells].

The aim of this study was to investigate the inhibitory effect of Toll-like receptor 7 (TLR7) agonist gardiquimod on K562 cells. Human γδT cells from peripheral blood cells were amplified by isopentenyl pyrophosphate. The proliferation capacity of γδT cells and K562 cells were measured with MTT assay after treatment with different concentrations of gardiquimod. Cytotoxicity of γδT cells on K562 cells was detected by CCK-8 kit, and the intracellular expression of TLR7, cell cycle and apoptosis of K562 cells before and after treatment with gardiquimod were measured by flow cytometry. The results demonstrated that gardiquimod could significantly stimulate the proliferation of γδT cells, and inhibit proliferation of K562 cells under the concentration of 11.0 µg/ml for 48 h. The expression of TLR7 increased after treatment with gardiquimod. No apoptosis was observed, but there were significant changes in cell cycle, moreover the K562 cells treated with gardiquimod were more killed by γδT cells. It is concluded that the gardiquimod can inhibit the proliferation of K562 cells and enhance their sensitivity to killing activity of human γδT cells.

[Co-stimulation of multiple activating factors on proliferation and phenotype of T lymphocytes in peripheral blood in vitro].

AIM: To observe the costimulation of multiple activating factors effects on the proliferation and phenotype of T lymphocytes in vitro. METHODS: Peripheral blood mononuclear cells (PBMCs) were separated by fractionation on Ficoll-Hypaque gradient. According to adding different cytokines (CD3 mAb, CD28 mAb, IFN-γ, IL-1α, IL-2 and IL-15), the experiments were divided into seven groups. Effects of different cytokines on the proliferation of PBMC were counted by automated hematology analyzer five categories. The phenotypes (CD3, CD4, CD8, CD28, CD16, CD56(+);CD16, CD3(+);CD8(+);, CD3(+);CD4(+);, CD3(+); CD56(+);, CD45RO) expressing on the surface of costimulatory cells were detected by flow cytometry, and the cytotoxicity of costimulatory cells on SGC-7901, SW-1990 and SW-116 cell lines was examined by lactate dehydrogenase release method. RESULTS: The proliferation has significant difference when adding different cytokines into PBMCs culture system, the highestest proliferation multiples group is the one contains cytokines CD3, CD28, IFN-γ, IL-2, IL-1α, IL-15 and IL-21, which proliferation multiple is 255.3±6.3 at the tenth day of cell culture, obviously higher than the other culture systems which only contains CD3, IFN-γ and IL-2 (166.6±5.5) (P<0.05). Part of cells'phenotype changed when adding different activating factors. Without IL-15, the proportion of CD16(+);CD56(+);(NK) cells and CD3(+);CD56(+); cells was higher than the other groups; CD45RO(+); memory cells is most evident when delayed adding IL-15 and IL-21 for three days. The cytotoxicity of PBMCs cultured for ten days with different activating factors had significant difference, the highest was the one which delayed adding IL-15 and IL-21 for three days (76.2%, 60.3% and 70.6%, respectively.), higher than the cell culture groups containing CD3, IFN-γ and IL-2 (54.9%, 44.6% and 50.4%, respectively) (P<0.05). The cultured cells had the strongest cytotoxicity on SGC-7901 gastric adenocarcinoma cells. CONCLUSION: The PBMCs' proliferation, phenotype and cytotoxicity had significant difference after being activated by different stimulating factors, adding matching stimulating factors into the culture system have great value on cell-directed culture.

[Clinical observation on adoptive immunotherapy with autologous cytokine-induced killer cells for advanced malignant tumor].

BACKGROUND & OBJECTIVE: Cytokine-induced killer (CIK) cells have the characteristics of rapid proliferation, high efficiency, and broad spectrum in killing of tumor cells. However, there was few report about its clinical application on treatment for cancer patients. The current study was designed to evaluate the effect of adoptive transfer of autologous CIK cells on the patients with advanced malignant tumor. METHODS: Peripheral blood mononuclear cells of the patients with advanced malignant tumor were separated by fractionation on Ficoll-Hypaque gradient, then cultured in the medium containing IFN-gamma, IL-2, and CD3McAb for 7 days in vitro, and than the cultured auto-CIK cells were transfused back to the patients. The numbers of transferred CIK cells per patient were 5-15 x 10(9) in one course of treatment. Among these patients, 47 cases received chemotherapy, 3 cases received radiotherapy before CIK cells transfusion. The interval between chemoradiotherapy and immunotherapy was over 2 to 4 weeks. RESULTS: Among 63 patients receiving CIK cells immunotherapy, the total effective rate (PR + MR) was 44.46% (28). In the patients with increasing of CEA level in serum, 14 cases showed reduction of serum CEA and 1 cases remained increasing after the treatment with CIK cell. In the patients with increasing of AFP level in serum, similarly, 9 cases showed reduction of serum AFD and 1 case remained increasing. The absolute members of CD3, CD4, and CD8T cells increased to over 45% after being treated with CIK cells. Among treated patients, the appetite of 51 cases and performance and sleep of 32 cases got improved. Among 18 cases, 13 cases showed the pain relief. CONCLUSION: Adoptive immunotherapy of auto-CIK cells can significantly enhance cellular immune functions and improve subjective symptoms, but without side effects, so this is a safe and effective treatment for the patients with malignant tumor.