Antigen-loaded dendritic cells and CD40L triggers the killing effects of cytotoxic T lymphocytes on K562 cells in vitro

AIM:To investigate the anti-tumor effects of special cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) loaded with antigens and CD40L in vitro.METHODS:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood.The adherent cells were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-4 (IL-4),alpha tumor necrosis factor (TNF-α),DCs were co-cultured with frozen-thawed antigen of K562 cells and CD40L,then triggered T cells into specific CTLs.RESULTS:Most suspended cells exhibited distinctive morphological features of DCs which expressed CD40 96%,CD86 97%,CD80 77%,CD1a 69%,and gained the powerful capacity to stimulate proliferation of allogenic lymphocytes.Under the effector∶target ratio of 20∶1,CTLs derived from cultures with DCs and frozen-thawed antigen of K562 cells were showed 71.3% cytotoxicities against K562 cells.CTLs derived from cultures with DCs loaded with frozen-thawed antigen and CD40L were showed 86.9% cytotoxicities against K562 cells.Cytotoxicities by CTLs derived from cultures with unloaded DCs against K562 cells were 37.6% and cytotoxicities by monocytes were 21.1%.Cytotoxicities by CTLs derived from experiment groups were stronger than control groups (P<0.05).CONCLUSIONS:The tumor antigen-pulsed DCs induces efficient and specific anti-tumor immunity,CTLs derived from cultures containing DCs pulsed with CD40L show the strongest cytolytic activities on K562 cells.     

WT1 peptide-loaded DC triggers cytotoxic T lymphocytes and killing effects on K562 cells in vitro

AIM: To study the effects of WT1 peptide-loaded dendritic cells (DC) stimulating the cytotoxic T lymphocytes (CTL) on K562 cells in vitro. METHODS: DC were generated from normal human peripheral blood mononuclear cells (PBMC) in the presence of granulocyte-macrophage colony stimulating factor(GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) , DC were cultured with WT1 peptides , and then triggered T cells into specific CTL. RESULTS: Most suspended cells exhibited distinctive morphological features of DCs and they stimulated proliferation of allogenic lymphocytes. Under the effector : target ratio of 20∶1, CTLs derived from cultures with DC and WT1 peptides were showed 86.1%±26.8% cytotoxicity against K562 cells, cytotoxicity by CTLs derived from cultures with unloaded DC against K562 cells were 47.1%±20.8% and cytotoxicity by lymphocytes were 27.7%±15.3%. Cytotoxicity by CTLs derived from culture with WT1 peptides-loaded DC were the strongest among three groups （P＜0.05）. CONCLUSION: CTLs derived from cultures containing DC pulsed with WT1 peptides show the strongest cytolytic activities on K562 cells.     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

Anti-cancer effect of cytotoxic T lymphocytes activated by sensitized dendritic cells

AIM: To investigate the anti-cancer effect of cytotoxic T lymphocytes (CTL) activated by sensitized dendritic cells (DCs). METHODS: Immature DCs were induced in vitro from peripheral blood monocytic cells (PBMC) and sensitized by adding tumor cells antigen extract. DCs were identified by their morphology and surface markers. MTT assay was used to evaluate the killing activity of CTL activated by sensitized DCs. The effects of specific CTL cells on inhibiting transplanted tumor HT-29 growth and on preventing HT-29 tumor generation were evaluated by injecting CTL into nude mice. RESULTS: After cultured for seven days, a large number of activated DCs were obtained with typical morphology, extensive stimulatory proliferation capacity and high CD80 (63.5%), CD83 (67.6%) and CD3/HLA-DR (83.2%) expressions. The killing activity of CTL at 20∶1 ratio of effective cells to target cells was more than 75% to tumor cells, 35%-45% to homologous cell line and weaker to other germ cell line (P<0.01). Injection of CTLs activated by HT-29 cell antigen sensitized DCs inhibited HT-29 transplanted tumor growth and prevented HT-29 tumor occurring in nude mice (P<0.05). PCNA expression level in tumor cells in CTL therapy group was dramatically lower than that in control (P<0.05). CONCLUSION: CTL activated by sensitized DCs kill tumor cells specifically, inhibit transplanted tumor growth and prevent tumor transplantation in nude mice.     

Dendritic cells transfected with recombinant adenovirus carrying IL-24 gene induces CTLs to promote apoptosis of cervical cancer cell line CaSki

AIM：To investigate the therapeutic potential of interleukin-24 (IL-24) in cancer treatment, we observed the inductive effect of cytotoxic T-lymphocytes (CTLs) against human cervical cancer cell line CaSki using dendritic cells (DCs) transfected with recombinant adenovirus carrying IL-24 gene (Ad-IL-24-DCs). METHODS：Immature mouse DCs were isolated and cultured. The DCs were infected with recombinant adenovirus carrying IL-24 gene. The CaSki cell lysate-loaded autologous DCs vaccine was also prepared. The cell proliferation was analyzed by MTT assay. The cell apoptosis was determined by flow cytometry with PE-Annexin V staining. The cleaved caspase-3 in the Ad-IL-24-DCs was measured by Western blotting. Colony-forming assay was used to detect the colony number of Ad-IL-24-DCs. The tumorigenic capacity of CaSki cells in vivo in the presence of Ad-IL-24-DCs was observed by tumor-burdened model. RESULTS：IL-24 was highly expressed in the Ad-IL-24-DCs. In the presence of Ad-IL-24-DCs, the apoptosis of CaSki cells was significant increased. Additionally, the protein level of cleaved caspase-3 was increased in CaSki cells in the presence of Ad-IL-24-DCs, suggesting that the CaSki cells with Ad-IL-24-DCs induced apoptosis. CTLs induced by Ad-IL-24-DCs inhibited the tumorigenic capacity of CaSKi cells in vivo. CONCLUSION：Genetically modified DCs are successfully prepared by infection with Ad-IL-24 and show a significant effect on triggering specific CTLs against CaSki cells.     

OK-432 enhances the effect of umbilical cord blood dendritic-cells treated with gastric cancer tumor vaccine

AIM: To evaluate the effect of replacing fetal cattle serum with umbilical cord plasma and application of OK-432 on dendritic cells from umbilical cord blood in vitro so as to offer a new technical way of preparation high-powered dendritic-cell-based vaccines for the cancer immunotherapy.METHODS: Human cord blood mononuclear cells (CBMCs) were cultured in RPMI-1640 containing 10% autologous plasma,GM-CSF and IL-4,some of which were supplemented with tumor lysates and/ or OK-432.MTT assay was applied to measure the antigen presenting ability of DCs in allo-MLR.Killing rates of autologous T lymphocytes induced by DCs on different target cells were measured by LDH method.RESULTS: Cells appeared typical morphology of DCs after culture and the allo-stimulate capacity of DCs and the CTL response in vitro were enhanced by treating with tumor lysates and OK-432.CONCLUSION: Mature DCs can be induced from human CBMCs by this means with fewer cytokines and less time.The tumor lysate antigens are captured by DCs treated with tumor lysates and OK-432,and presented to lymphocytes successfully,indicating a new way to develop dendritic-cell-based vaccines for clinical immunotherapy of gastric cancer and other tumors.     

Effects of dendritic cells co-cultured with CIK cells on renal carcinoma cells

AIM: To study the effects of CIK cocultured with DC that pulsed with RCC antigen on renal carcinoma cells.METHODS: DC and CIK cells were generated respectively by cytokines from PBMC of healthy blood donor.Cell surface markers were analyzed by flow cytometry.Then CIK were cocultured with autologous DC that was (or not) pulsed with RCC antigen (786-0 cells).Cytotoxic activity against 786-0 or PC3 cells was measured by MTT assay under three different conditions: CIK cocultured with DC which was pulsed with 786-0 antigen (group A);CIK cocultured with DC which is not pulsed with 786-0 antigen (group B);CIK without DC (group C).RESULTS: The cytotoxic activity of three groups against 786-0 cells was (70.64±8.26)%, (53.40±7.33)%, (46.64±6.01)%, respectively (E/T=20∶〖KG-*2〗1).Significant differences between group A and group B or between group A and group C were observed (P<0.05).There was a significant difference in cytotoxic effects of group A against 786-0 and PC3 cells (P<0.05).CONCLUSION: Coculture of CIK with autologous tumor-pulsed DC leads to a significant increase in cytotoxic activity against renal carcinoma cells, and cytotoxicity mediated by RCC lysate antigen possess stronger specificity.     

The immunity effect of B7-H1 blockade on immature dendritic cells

AIM: To investigate the immune stimulation capacity of B7-H1 blockade on immature dendritic cells (DCs) in vitro. METHODS: The human monocyte-derived dendritic cells were induced in the presence of cytokine GM-CSF and IL-4. The expression of B7-H1 was detected by FCM. On blockade of B7-H1, the maturation and endocytic activity, T cells stimulatory proliferation capacity, IL-12 production, T cell differentiation effect of DCs were detected by FCM, MTT assay, ELISA and ELISPOT, respectively. RESULTS: The expression of B7-H1 was increased with the induction of DCs. On day 7, the positive expression was 54.12%, and the TNF-α induced mature DCs had the positive expression rate of 83.64%. The blockade of B7-H1 on immature DCs had sharply increased their T cells stimulatory proliferation capacity and IL-12 production, and efficiently induced the development of Th1/Tc1 cells, but had no effect on their maturation and endocytic activity. CONCLUSION: The blockade of B7-H1 on immature DCs increases its immune stimulation activity. It is valuable to investigate the antitumor immune responses of DCs vaccine with B7-H1 blockade.     

Renal tumor cell lysate antigen induces the activation of dendritic cells

AIM:To investigate the effects of renal tumor cell lysates and other cytokines on the activation of immature dendritic cells (iDCs), and to develop DCs vaccines for stimulation of renal tumour-specific immunity.METHODS:DCs induced from peripheral blood mononuclear cells of healthy volunteers were cultured and propagated in vitro using rhGM-CSF and rhIL-4, and then were cocultured with renal tumor cell lysates and different cytokines. A group: only with renal tumor cell lysates; B group: with renal tumor cell lysates and TNF-α; C group: with renal tumor cell lysates and IL-1β; D group: renal tumor cell lysates and TNF-α+IL-1β. RESULTS:iDCs were induced to mature in all four groups, and high level expressions of CD86, CD80 and HLA-DR were observed. Compared to other groups, DCs in D group expressed CD83 and CD54 at higher level (P<0.05), secreted higher quantity of IL-12 (P<0.01). Moreover, mDCs in D group induced multiplication of lymphopoiesis more effectively (P<0.05).CONCLUSION:Renal tumor cell lysates and TNF-α+IL-1β induce the mature phenotype and IL-12 production in DCs and synergistically promote the stimulatory effects of lymphopoiesis.     

Allogeneic antigen-pulsed murine immature CD8α+ dendritic cells prevent T-cells proliferation in vitro

AIM: To explore the optimal condition of predominant immature CD8α⁺ dendritic cells(predo-iDCs) preventing T-cell proliferation pulsed by allogeneic antigen. METHODS: Predo-iDCs, induced with GM-CSF +IL-4 +SCF +Flt3L from SPF healthy C57BL/6 murine bone marrow cells, were pulsed by different doses (0, 2.5, 5, 10 and 20 mg/L)of allogeneic murine splenocyte antigen. Syngeneic T-cells were co-incubated with Ag-pulsed DCs (DCs/T=1∶ 1, 2∶ 1, 4∶ 1) and the T-cell proliferation was measured by MTT. The secretion of cytokines (IFN-γ and IL-10) in the co-incubated supernatants was detected by ELISA. The effect of prevention of T-cell proliferation generated by murine predo-iDCs pulsed by allogeneic antigen was detected. The control derived from mature dendritic cells(mDCs)induced by GM-CSF +IL-4 +TNF-α. RESULTS: The effect of Ag-pulsed predo-iDCs for stimulating T-cell proliferation was the slightest in predo-iDCs/T 1∶ 1 group, compared with that in predo-iDCs/T 2∶ 1 and 4∶ 1 group (P<0.05). The secretion of IFN-γ in mixed lymphocyte reaction(MLR) was significantly lower than the one in mDCs control group, while the secretion of IL-10 was higher than that in control group when low dose of antigen (<2.5 mg/L) was added into MLR. CONCLUSION: Predominant iDCs pulsed by low dose of allogeneic antigen (2.5 mg/L) mixed 1∶ 1 with T-cells is the optimal condition for the prevention of T-cells proliferation.     

A dynamic observation on the levels of TNF-α、IL-6 and IFN-γ produced by human dendritic cells infected with Dengue virus

AIM: To study the dynamic levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and gamma interferon （IFN-γ） produced by human dendritic cells infected with Dengue virus. METHODS: Monocytes isolated from healthy human peripheral blood were incubated in medium with GM-CSF and IL-4 for more than 7 days. DCs were then collected and identified by scanning electron microscope, immunohistochemistry and lymphocytes stimulatory ability. Human dendritic cells （DC) were infected with Dengue-2 virus (DV-2) in vitro， culture supernatants were collected in different time postinfection (6 h, 12 h, 24 h, 48 h and 72 h)， DV antigen in human dendritic cells were demonstrated by an indirect immunofluorescent assay （IFA）, production of TNF-α, IL-6 and IFN-γ in the culture supernatants were evaluated by ELISA. RESULTS: After 7 days, typical dendritic cells could be obtained. Virus antigen were detected in infected DC by IFA. Dengue virus induces TNF-α and IL-6 secretion from DC and does not induce IFN secretion. CONCLUSION: Human dendritic cells are target of dengue virus infection. TNF-α， IL-6 production from DC are increased with DV infection. Dendritic cells may play an important role in DV pathogenicity and immunity.     

Effect of glucocorticoid-treated dendritic cells on asthmatic Th2 response

AIM: To investigate the effect of dexamethasone-treated dendritic cells (DCs) on Th2 cytokine production from autologous T cells in asthmatic patients and explore the mechanisms by studying the effect of dexamethasone on differentiation, maturation and function of DCs from patients with asthma. METHODS: Human peripheral blood monocyte-derived DCs generated from asthmatic patients and healthy subjects were cultured in the absence or presence of dexamethasone. The phenotypic characterization of DCs was analyzed by flow cytometry. The mature DCs were harvested, washed, and then cocultured in vitro with autologous T cells purified by a nylon cotton column. The DC-T coculture supernatants were collected after 72 h incubation and analyzed for levels of IL-5 and IFN-γ by ELISA. RESULTS: The concentrations of IL-5 in the culture supernatants of DC-T coculture were significantly up-regulated in patients with asthma compared with that in healthy controls ［(145.13±89.76) ng/L vs (50.28±22.37) ng/L, P<0.01］. The level of IFN-γ in the DC-T coculture supernatants tended to be decreased in asthmatic patients than that in healthy controls, although this difference did not achieve statistical significance ［(197.58±76.32) ng/L vs (220.46±65.34) ng/L, P>0.05)］. There were significantly decreased levels of IL-5 by autologous T cells primed by dexamethasone-treated mature DCs from asthmatic patients ［(45.39±19.61) ng/L vs (145.13±89.76) ng/L, P<0.01］, alterations not observed from healthy controls (P>0.05). IFN-γ production was decreased by autologous T cells primed by dexamethasone-treated mature DCs from both asthmatic patients and healthy controls ［asthma group: (40.21±22.89) ng/L vs (197.58±76.32) ng/L, P<0.01; healthy controls: (56.78±20.37) ng/L vs (220.46±65.34) ng/L, P<0.01］. Dexamethasone-treated DCs exhibited decreased expression of CD83 (P<0.01) and increased expression of CD14 (P<0.01) in both asthmatic patients and healthy controls. CONCLUSION: DCs of asthmatic patients induce a Th2-skewed cytokine production from autologous T cells. Dexamethasone-treated DCs inhibit the Th2 reactions, and this effect is probably mediated through the pathway that dexamethasone inhibits DCs maturation and skews the macrophage/DC balance towards the macrophage side and thus directs the development more towards the macrophage lineage.     

Phenotype and immune activity of dendritic cells under interleukin-18 intervention

AIM: To investigate the phenotype and immune activity of dendritic cells using interleukin-18 as intervent.METHODS: Monocytes were isolated from human peripheral blood and induced into DCs with GM-CSF and IL-4. The cellular morphous was observed under inverted microscope. On the 5th day, 3 groups including IL-18 group, TNF-α group and IL-18+TNF-α group were set. IL-18, TNF-α or IL-18+TNF-α was used as intervents respectively to facilitate cell maturity. Supernatants were collected at 24 h, 48 h and 72 h. IL-12 in the supernatant, CD1a, HLA-DR, CD83 and CD86 were analyzed using flow cytometry. DCs of the 3 groups were co-cultured with T cells respectively on the ratio of 1∶〖KG-*2〗100, 1∶〖KG-*2〗50 and 1∶〖KG-*2〗10. T cell proliferation stimulated by DC was determined using MTT method. DCs were co-cultured with T cells on the ratio of 1∶〖KG-*2〗10, and the supernatant were collected at 24 h, 48 h and 72 h. IFN-γ in the supernatant was detected with ELISA method.RESULTS: Induced by GM-CSF and IL-4, then stimulated by IL-18, TNF-α or IL-18+TNF-α, monocytes showed typical morphous of DC. No morphological difference was observed among DCs of the 3 groups. No statistical difference showed in expression level of CD1a, HLA-DR, CD83 and CD86 between IL-18 group and TNF-α group (P>0.05). The positive rates of CD1a and CD83 in IL-18+TNF-α group were higher than those in other 2 groups. The positive rate of HLA-DR in IL-18+TNF-α group was higher than that in IL-18 group. No difference between IL-18 group and TNF-α group in the potency of stimulating T cell proliferation was found, whereas the stimulating potency in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group. IL-12 in IL-18+TNF-α group at 48 h and 72 h was higher than that in IL-18 group and TNF-α group (P<0.05). However, there was no difference between the latter 2 groups. There was also no difference between IL-18 group and TNF-α group in IFN-γ secretion. IFN-γ in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group (P<0.05).CONCLUSION: Using IL-18 as intervent, DC expresses high level of surface molecules, secretes high level of IL-12, stimulates T cell proliferating effectively and produces IFN-γ potently. The actions are stronger when used in combination with TNF-α. It suggests that IL-18 may serve as a promoting agent of DC maturity, or combination with TNF-α in DC induction will strengthen the immune activity of DC.     

Dendritic cells transfected with tumor total RNA induce specific immune responses against hepatocellular carcinoma in vitro

AIM: To observe the ability of dendritic cells (DC) vaccine transfected with human hepatocellular carcinoma (HCC) total RNA induce specific cytotoxic T lymphocyte(CTL) response in vitro. METHODS: DCs generated from HCC patient’s peripheral blood mononuclear cells (PBMC) were incubated with recombinant human granulocyte macrophage colony-stimulationg factor (GM-CSF) and human interleukin (IL-4). Tumor total RNA was isolated from Hep G-2 cells and HCC cells. DCs transfected with tumor total RNA were used to induce specific CTL proliferation. Specific cytotoxicity was measured using MTT method. RESULTS: DC transfected with HepG-2 cell RNA and HCC RNA exhibited increased expression of CD83, CD86 and HLA-DR. The CTL from DCs transfected with HepG-2 cell RNA killed 5.84％， 14.26％， 25.19％, or 35.78％ of HepG-2 cells, and 5.26％， 11.67％， 14.68％, or 23.24％ of HCC cells, respectively, at an E/T ratio of 2.5, 5, 10, or 20. The CTL from DCs transfected with HCC cells RNA killed 4.65％， 12.23％， 15.61％, or 19.15％ of HepG-2 cells, and 7.20％， 12.83％， 27.21％, or 31.15％ of HCC cells,respectively, at an E/T ratio of 2.5, 5, 10, and 20. These CTL did not kill allogeneic malignant cells as human gastric carcinoma cells SGC-7901. CONCLUSION: DC transfected with tumor-derived total RNA could induce specific antitumor immune CTL response. These results suggest that CTL generation is applicable to adoptive immunotherapy of HCC.     

Immune function of dendritic cells in patients with hepatocellular carcinoma

AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.     

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.     

Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2. METHODS: RT-PCR and Western blot was used to determine the expression of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29. HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay. ELISPOT assay was used to investigate the levels of IFN-γ. The cytotoxicity assay in vitro was also used to determine the ability of inducing T cell response by the peptides. RESULTS: The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P485 and P965 induced CTLs of IFN-γ release form CTLs. The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION: The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.     

Human dendritic cells transfected with MUC1 mRNA induce lethal effect of cytotoxic T-lymphocytes on non-small cell lung cancer in vitro

AIM：To investigate the specific anti-tumor effects of mature dendritic cells (DCs) transfected with amplified mucin 1 (MUC1) mRNA in vitro. METHODS：DCs separated and purified from the peripheral blood mononuclear cells were induced in vitro and then identified by flow cytometry. pcDNA3.1（+）-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro. The MUC1 mRNA was transfected into DCs by electroporation. MUC1-transfected DCs were used to induce T cells to be cytotoxic T-lymphocytes. Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs. The proliferation of T cells was examined by MTT assay. The proportion of CD8+ cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay. The secretion of IFN-γ was detected by ELISA. RESULTS：The marker gene expression in the DCs transfected with MUC1 mRNA was significantly increased compared with control group, peaking at 24 h. The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10. The proportion of CD8+ cells in transfection group was higher than that in control group. The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group. The level of IFN-γ in the cell supernatant of transfection group was higher than that in control group. CONCLUSION：DCs plus MUC1 mRNA by electrical transfection induces specific anti-tumor effects, which provides an experiment evidence of using MUC1 as a target for immunotherapeutic strategy against non-small cell lung cancer.     

Effects of dendritic cell stimulation on cytotoxic activity of cord blood derived cytokine-induced killer cells,natural killer cells and CD45RO expression in CIK cells

AIM: To investigate the efficacy of dendritic cells (DCs) that augments the cytotoxic activity of cytokine-induced killer (CIK) cells, natural killer (NK) cells from a same donor and the CD45RO expression on CIK cells. METHODS: The expanded killer cells were divided into two groups: group A was pre-cocultured with DCs for 6 days, group B was the control that without any stimulation. Cytotoxicity of CIK and NK cells was measured at different effect-target ratio against K562 and HL-60. CD45RO and CD45RA expression on CIK cells in different groups were detected by flow cytometry. RESULTS: Cytotoxicity of CB derived killer cells was positive correlation with effect-target ratio. The cytotoxicity of group A against HL-60 was higher than that of group B significantly. At 20∶1 effector-target ratio, the lytic activity of group A CIK, NK cells against K562 was higher than that of group B significantly, but no significant difference between them at 10∶1 effector-target ratio. The CD45RO expression on CIK cells in groups A was significantly higher than that in groups B. CONCLUSION: CIK and NK cells cocultured with DCs can augment the killer's cytotoxicity against tumor cells and promote the CD45RO expression on CIK cells.