AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymph...AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.展开更多
AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）. METHODS： Lymphotactin...AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）. METHODS： Lymphotactin gene modified DCs （DCLptn） were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor （GM- CSF）, interleukin-4 （IL-4） and tumor necrosis factor alpha （TNF-α）. DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTl_s by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay. RESULTS： Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-α, and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn＋H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn ＋ H22, DC/H22 groups. CONCLUSION： Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.展开更多
Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the ty...Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNα-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells (CTLs) in patients with hepatitis B. METHODS:Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNα-2a treatment. RESULTS:Before IFNα-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNα-2a treatment,Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNα-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS:Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNα-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.展开更多
Objective： To investigate whether human dendritic cells （DC） derived from peripheral blood mononuclear cells （PBMC）, which were pulsed by heat shock protein 70 （HSP70） isolated from human bladder tumor cell lin...Objective： To investigate whether human dendritic cells （DC） derived from peripheral blood mononuclear cells （PBMC）, which were pulsed by heat shock protein 70 （HSP70） isolated from human bladder tumor cell lines of E J, were able to induce peptide specific cytotoxic T-lymphocytes （CTL） response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods： The E J-derived HSP70 co-cultured with DC from the healthy volunteers＇ PBMC, along with the crude lysate （the supematant before HSP70 purification） from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes （SI） and interferon-y （IFN-γ） were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51^Cr releasing test. Results： Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate （the supernatant before HSP70 purification） （P 〈 0.05）. DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells （P 〈 0.05）. Conclusion： The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.展开更多
Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measureme...Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.展开更多
Recently, a new type of CD8^+ T cell subset, namely, the CXCR5^+ CD8^+ T cell subset(also called the follicular cytotoxic T cell(TFC) subgroup), has been discovered around B cell follicles. The discovery has aroused w...Recently, a new type of CD8^+ T cell subset, namely, the CXCR5^+ CD8^+ T cell subset(also called the follicular cytotoxic T cell(TFC) subgroup), has been discovered around B cell follicles. The discovery has aroused widespread interest.However, the processes and mechanisms of TFCs taking part in the immune response of the germinal center and their specific roles must still be clearly identified. This paper reviews domestic and foreign studies on factors regulating the phenotype,physiological functions, maturity, and differentiation of TFCs and roles and clinical significance these cells in human immunodeficiency virus infection. Our review has shown good application prospects for TFCs. We believe that further studies on TFCs can provide another tool for cytotherapy of controlling or curing chronic viral infections or tumors.展开更多
Enhancement of the Human Immunodeficiency Virus (HIV) specific cytotoxic T-cells mechanisms in an HIV-1 and Mycobacterium tuberculosis (Mtb) co-infected individual seems to improve the clinical picture of an individua...Enhancement of the Human Immunodeficiency Virus (HIV) specific cytotoxic T-cells mechanisms in an HIV-1 and Mycobacterium tuberculosis (Mtb) co-infected individual seems to improve the clinical picture of an individual by reducing Acquired Immuno Deficiency Syndrome (AIDS) state progression rate. In this paper, we develop a system of deterministic differential equations representing the immune cells involved in an HIV-1 and Mtb co-infected individual. Results show that although the non-lytic arm of the HIV-1 cytotoxic T-cells affects the co-infection dynamics more than the lytic factors, a combination of both factors results in a more positive reduced progression to the AIDS state. This is due to the increased protection of the CD4<sup>+</sup> T-cells by the CTL mechanisms by further reducing infections and replications by the HIV. Thus, HIV-1 specific CTLs mechanisms’ involvement is here recommended to be part of a solution to the HIV and Mtb co-infection problems.展开更多
Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte an...Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte antigen(CTLA)-4/B7 interaction(using CTLA-4Ig) and the CD40/CD40 L interaction(using anti-CD40 L antibodies) prevents T cell mediated autoimmune diseases, transplant rejection and graft vs host disease in experimental models. Moreover, CTLA-4Ig is in clinical use to treat rheumatoid arthritis(abatacept) and to prevent rejection of renal transplants(belatacept). Under certain experimental conditions, this treatment can even result in tolerance. Surprisingly, the underlying mechanisms of immune modulation are still not completely understood. We here discuss the evidence that costimulation blockade differentially affects effector T cells(Teff) and regulatory T cells(Treg). The latter are required to control inappropriate and unwanted immune responses, and their activity often contributes to tolerance induction and maintenance. Unfortunately, our knowledge on the costimulatory requirements of Treg cells is very limited. We therefore summarize the current understanding ofthe costimulatory requirements of Treg cells, and elaborate on the effect of anti-CD40 L antibody and CTLA-4Ig treatment on Treg cell activity. In this context, we point out that the outcome of a treatment aiming at blocking the CD28/CTLA-4/B7 costimulatory interaction can vary with dosing, timing and underlying immunopathology.展开更多
Objective:To investigate the cytotoxic effect of ethanol extract of the stem bark of asam kandis[Garcinia cowa Roxb.(G.cowa)]on T47 D breast cancer cell line.Methods:The cytotoxicity of ethanol extract was carried out...Objective:To investigate the cytotoxic effect of ethanol extract of the stem bark of asam kandis[Garcinia cowa Roxb.(G.cowa)]on T47 D breast cancer cell line.Methods:The cytotoxicity of ethanol extract was carried out against human breast cancer cell line(T47D) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay.The extract was added at various concentrations(0.1.1,10 and 100 μg/mL).The level of cytotoxicity was determined by calculating the level of IC_(50),that was based on the percentage of the cell death after 24 h treatment with the extract.Cell morphological changes were observed by using inverted microscope.Results:The 3-(4.5-dimelhylthiazol-2-yl)-2.5-diphenyltelrazolium bromide assay showed that ethanol extract of G.cowa exhibited significant cytotoxic effect on T47 D with IC_(50) value of(5.10+1.68) μg/mL.Morphological alteration of the cell lines after exposure to ethanol extract of G.cowa was observed under phase contrast microscope in a dosc-dependent manner.ConclusionsThe results suggest the possible use of ethanol extract of asam kandis for preparing herbal medicine for cancer-related ailments.展开更多
AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral ...AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining. RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBV specific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n - 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells. CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.展开更多
AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated wit...AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. Bone marrowderived DCs （BM-DCs） were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1：3 DC：tumor cell ratio. Finally, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNγ） secretion was evaluated ex vivo. RESULTS： c-kit^＋ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs.Morphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of la, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these cells gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed （TP） DCs were able to induce effective CTL activity against SGC-7901 tumor cells （E：T = 100：1, 69.55% ± 6.05% specific lysis）, but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells （1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11± 18.52 pg/mL in B16 group, P 〈 0.01）. CONCLUSION： BM-derived DCs pulsed with tumor lysates Can induce anti-tumor immunity specific to gastric cancer ex vivo.展开更多
AIM: To investigate the regulatory effect of Vδ1 T cells and the antitumor activity of Vδ2 T cells in rectal cancer.METHODS: Peripheral blood, tumor tissues and para-carcinoma tissues from 20 r...AIM: To investigate the regulatory effect of Vδ1 T cells and the antitumor activity of Vδ2 T cells in rectal cancer.METHODS: Peripheral blood, tumor tissues and para-carcinoma tissues from 20 rectal cancer patients were collected. Naïve CD4 T cells from the peripheral blood of rectal cancer patients were purified by negative selection using a Naive CD4<sup>+</sup> T Cell Isolation Kit II (Miltenyi Biotec). Tumor tissues and para-carcinoma tissues were minced into small pieces and digested in a triple enzyme mixture containing collagenase type IV, hyaluronidase, and deoxyribonuclease for 2 h at room temperature. After digestion, the cells were washed twice in RPMI1640 and cultured in RPMI1640 containing 10% human serum supplemented with L-glutamine and 2-mercaptoethanol and 1000 U/mL of IL-2 for the generation of T cells. Vδ1 T cells and Vδ2 T cells from tumor tissues and para-carcinoma tissues were expanded by anti-TCR γδ antibodies. The inhibitory effects of Vδ1 T cells on naïve CD4 T cells were analyzed using the CFSE method. The cytotoxicity of Vδ2 T cells on rectal cancer lines was determined by the LDH method.RESULTS: The percentage of Vδ1 T cells in rectal tumor tissues from rectal cancer patients was significantly increased, and positively correlated with the T stage. The percentage of Vδ2 T cells in rectal tumor tissues from rectal cancer patients was significantly decreased, and negatively correlated with the T stage. After culture for 14 d with 1 μg/mL anti-TCR γδ antibodies, the percentage of Vδ1 T cells from para-carcinoma tissues was 21.45% ± 4.64%, and the percentage of Vδ2 T cells was 38.64% ± 8.05%. After culture for 14 d, the percentage of Vδ1 T cells from rectal cancer tissues was 67.45% ± 11.75% and the percentage of Vδ2 T cells was 8.94% ± 2.85%. Tumor-infiltrating Vδ1 T cells had strong inhibitory effects, and tumor-infiltrating Vδ2 T cells showed strong cytolytic activity. The inhibitory effects of Vδ1 T cells from para-carcinoma tissues and from rectal cancer tissue were not significantly different. In addition, the cytolytic activities of Vδ2 T cells from para-carcinoma tissues and from rectal cancer tissues were not significantly different.CONCLUSION: A percentage imbalance in Vδ1 and Vδ2 T cells in rectal cancer patients may contribute to the development of rectal cancer.展开更多
基金Supported by National Natural Science Foundation of China,No.81071982
文摘AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.
基金Supported by the Science & Technology Foundation for Academicians of Zhejiang Province, China, No. 203201513
文摘AIM： To investigate the in-vitro activation of cytotoxic T lymphocytes （CTLs） by fusion of mouse hepatocellular carcinoma （HCC） ceils and lymphotactin gene-modified dendritic cells （DCs）. METHODS： Lymphotactin gene modified DCs （DCLptn） were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor （GM- CSF）, interleukin-4 （IL-4） and tumor necrosis factor alpha （TNF-α）. DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTl_s by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay. RESULTS： Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-α, and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn＋H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn ＋ H22, DC/H22 groups. CONCLUSION： Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.
文摘Interferon(IFN) with antiviral and im-munomodulatory activities is one of the most important therapeutic agents for the treatment of chronic hepatitis. The apoptotic effect of IFN is influenced by cell type and the types of IFN, which suppresses proliferation and induces apoptosis in some cell types while inhibiting apoptosis in others. The aim of this study was to explore the effect of IFNα-2a on Fas expression and the apoptosis rate of peripheral blood cytotoxic T cells (CTLs) in patients with hepatitis B. METHODS:Peripheral blood mononuclear cells were isolated from 26 patients with hepatitis B including 16 patients with chronic hepatitis B and 10 patients with chronic severe hepatitis B. Fas expression and apoptosis rate of CTLs were analyzed with flow cytometry before and after IFNα-2a treatment. RESULTS:Before IFNα-2a treatment, Fas expression and apoptosis rate of CTLs from patients with chronic hepatitis B were significantly higher than those from patients with chronic severe hepatitis B and healthy controls respectively. No significant difference was observed between Fas expression and apoptosis rate of CTLs from patients with chronic severe hepatitis B and healthy controls. After IFNα-2a treatment,Fas expression and apoptosis rate of CTLs from different groups were compared with those before IFNα-2a treatment, showing no significant difference despite alternation of different degree. CONCLUSIONS:Activation induced cell death (AICD) exists in peripheral blood CTLs from patients with hepatitis B. No effect of IFNα-2a exerts on Fas expression and apoptosis rate of Fas in patients with hepatitis B.
基金Supported by a grant from the National Natural Science Foundation of China (No. 3000754).
文摘Objective： To investigate whether human dendritic cells （DC） derived from peripheral blood mononuclear cells （PBMC）, which were pulsed by heat shock protein 70 （HSP70） isolated from human bladder tumor cell lines of E J, were able to induce peptide specific cytotoxic T-lymphocytes （CTL） response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods： The E J-derived HSP70 co-cultured with DC from the healthy volunteers＇ PBMC, along with the crude lysate （the supematant before HSP70 purification） from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes （SI） and interferon-y （IFN-γ） were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51^Cr releasing test. Results： Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate （the supernatant before HSP70 purification） （P 〈 0.05）. DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells （P 〈 0.05）. Conclusion： The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.
文摘Theileria parva is a protozoan parasite that causes the disease East Coast fever in cattle which results in major economic losses in Eastern, Central and Southern Africa. Efforts to generate vaccines involve measurements of cytotoxic activity since CD8 cells are believed to be responsible for protection of the animals. CTL assays are time consuming, and may require use of radioactive material and they also impose a considerable amount of in vitro work, which may skew the response compared to ex vivo assays. Hence it would be beneficial to identify other markers that correlate with the cytotoxicity. In this in vitro study we examined if the number of tetramer positive CD8 cells and the staining intensity of these correlated with lysis of the target cells. Furthermore, we investigated if the expression of the activation marker perforin correlated with the cytotoxicity. Perforin is involved in permeabilization of the cell membrane of the target cell. Bulk CTL lines and purified CD8 cell lines generated from cattle of the A18 BoLA (MHC) type were analysed for the Theileria parva specific immune responses using a peptide-MHC tetramer and antibodies for perforinin FACS analysis. Thelysis of target cells was determined by a chromium release assay. Results demonstrate that the percentage of tetramer positive cells, in six cell lines generated against the whole parasite, correlate with killing of PBMC pulsed with the peptide. The product of the percentages of perforin and tetramer double positive cells and the net MFI of perforin showed a perfect correlation with the cytotoxicity of the peptide pulsed PBMC. Likewise, the product of percentage perforin positive cells and the staining intensity had the best significant correlation with killing of the pulsed PBMC. The present results suggest that perforin could be a possible biomarker for the cytotoxicity to Theileria parva infections/immunizations.
文摘Recently, a new type of CD8^+ T cell subset, namely, the CXCR5^+ CD8^+ T cell subset(also called the follicular cytotoxic T cell(TFC) subgroup), has been discovered around B cell follicles. The discovery has aroused widespread interest.However, the processes and mechanisms of TFCs taking part in the immune response of the germinal center and their specific roles must still be clearly identified. This paper reviews domestic and foreign studies on factors regulating the phenotype,physiological functions, maturity, and differentiation of TFCs and roles and clinical significance these cells in human immunodeficiency virus infection. Our review has shown good application prospects for TFCs. We believe that further studies on TFCs can provide another tool for cytotherapy of controlling or curing chronic viral infections or tumors.
文摘Enhancement of the Human Immunodeficiency Virus (HIV) specific cytotoxic T-cells mechanisms in an HIV-1 and Mycobacterium tuberculosis (Mtb) co-infected individual seems to improve the clinical picture of an individual by reducing Acquired Immuno Deficiency Syndrome (AIDS) state progression rate. In this paper, we develop a system of deterministic differential equations representing the immune cells involved in an HIV-1 and Mtb co-infected individual. Results show that although the non-lytic arm of the HIV-1 cytotoxic T-cells affects the co-infection dynamics more than the lytic factors, a combination of both factors results in a more positive reduced progression to the AIDS state. This is due to the increased protection of the CD4<sup>+</sup> T-cells by the CTL mechanisms by further reducing infections and replications by the HIV. Thus, HIV-1 specific CTLs mechanisms’ involvement is here recommended to be part of a solution to the HIV and Mtb co-infection problems.
文摘Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte antigen(CTLA)-4/B7 interaction(using CTLA-4Ig) and the CD40/CD40 L interaction(using anti-CD40 L antibodies) prevents T cell mediated autoimmune diseases, transplant rejection and graft vs host disease in experimental models. Moreover, CTLA-4Ig is in clinical use to treat rheumatoid arthritis(abatacept) and to prevent rejection of renal transplants(belatacept). Under certain experimental conditions, this treatment can even result in tolerance. Surprisingly, the underlying mechanisms of immune modulation are still not completely understood. We here discuss the evidence that costimulation blockade differentially affects effector T cells(Teff) and regulatory T cells(Treg). The latter are required to control inappropriate and unwanted immune responses, and their activity often contributes to tolerance induction and maintenance. Unfortunately, our knowledge on the costimulatory requirements of Treg cells is very limited. We therefore summarize the current understanding ofthe costimulatory requirements of Treg cells, and elaborate on the effect of anti-CD40 L antibody and CTLA-4Ig treatment on Treg cell activity. In this context, we point out that the outcome of a treatment aiming at blocking the CD28/CTLA-4/B7 costimulatory interaction can vary with dosing, timing and underlying immunopathology.
基金Supported by Hibah Doktor scheme of Directorate General of Higher Education,Ministry of Education and Culture,Republic of Indonesia(Grant No.DIPA 09/UN.16/D-DD/2014)
文摘Objective:To investigate the cytotoxic effect of ethanol extract of the stem bark of asam kandis[Garcinia cowa Roxb.(G.cowa)]on T47 D breast cancer cell line.Methods:The cytotoxicity of ethanol extract was carried out against human breast cancer cell line(T47D) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay.The extract was added at various concentrations(0.1.1,10 and 100 μg/mL).The level of cytotoxicity was determined by calculating the level of IC_(50),that was based on the percentage of the cell death after 24 h treatment with the extract.Cell morphological changes were observed by using inverted microscope.Results:The 3-(4.5-dimelhylthiazol-2-yl)-2.5-diphenyltelrazolium bromide assay showed that ethanol extract of G.cowa exhibited significant cytotoxic effect on T47 D with IC_(50) value of(5.10+1.68) μg/mL.Morphological alteration of the cell lines after exposure to ethanol extract of G.cowa was observed under phase contrast microscope in a dosc-dependent manner.ConclusionsThe results suggest the possible use of ethanol extract of asam kandis for preparing herbal medicine for cancer-related ailments.
基金Supported by in part Grant-in-Aid for Scientific Research (C) (toK.K)
文摘AIM: Persistent hepatitis B virus (HBV) infection is characterized by a weak CD8+ T cell response to HBV. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may facilitate viral clearance in chronically infected individuals. Therefore, we examined whether CD25+CD4+ regulatory T (Treg) cells might be involved in a inhibition of CD8+T cell priming or in the modulation of the magnitude of the 'peak' antiviral CD8+ T cell response primed by DNA immunization. METHODS: B10.D2 mice were immunized once with plasmid pCMV-S. Mice received 500 μg of anti-CD25 mAb injected intraperitoneally 3 d before DNA immunization to deplete CD25+ cells. Induction of HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) was measured by S28-39 peptide loaded DimerX staining and their function was analyzed by intracellular IFN-γ staining. RESULTS: DNA immunization induced HBV-specific CD8+ T cells. At the peak T cell response (d 10), 7.1±2.0% of CD8+ T cells were HBV-specific after DNA immunization, whereas 12.7±3.2% of CD8+ T cells were HBV-specific in Treg-depleted mice, suggesting that DNA immunization induced more antigen-specific CD8+ T cells in the absence of CD25+ Treg cells (n = 6, P<0.05). Similarly, fewer HBV specific memory T cells were detected in the presence of these cells (1.3±0.4%) in comparison to Treg-depleted mice (2.6±0.9%) on d 30 after DNA immunization (n - 6, P<0.01). Both IFN-γ production and the avidity of the HBV-specific CD8+ T cell response to antigen were higher in HBV-specific CD8+ T cells induced in the absence of Treg cells. CONCLUSION: CD25+ Treg cells suppress priming and/or expansion of antigen-specific CD8+ T cells during DNA immunization and the peak CD8+ T cell response is enhanced by depleting this cell population. Furthermore, Treg cells appear to be involved in the contraction phase of the CD8+ T cell response and may affect the quality of memory T cell pools. The elimination of Treg cells or their inhibition may be important in immunotherapeutic strategies to control HBV infection by inducing virus-specific cytotoxic T lymphocyte responses in chronically infected subjects.
文摘AIM： To investigate whether bone marrow-derived denritic cells pulsed with tumor lysates induce immunity against gastric cancer ex vivo. METHODS： c-kit＋ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFα to induce their maturation. They were analysed by morphological observation, phenotype analysis, and mixed lymphocyte reaction （MLR）. Bone marrowderived DCs （BM-DCs） were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1：3 DC：tumor cell ratio. Finally, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNγ） secretion was evaluated ex vivo. RESULTS： c-kit^＋ hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 d showed the character of typical mature DCs.Morphologically, observed by light microscope, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed.high levels of la, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of CD40, and negative for F4/80. Functionally, these cells gained the capacity to stimulate allogeneic T cells in MLR assay. However, immature DCs cultured with cytokines for 5 d did not have typical DCs phenotypic markers and could not stimulate allogeneic T cells. Ex vivo primed T cells with SGC-7901 tumor cell lysate-pulsed （TP） DCs were able to induce effective CTL activity against SGC-7901 tumor cells （E：T = 100：1, 69.55% ± 6.05% specific lysis）, but not B16 tumor cells, and produced higher levels of IFNγ, when stimulated with SGC-7901 tumor cells but not when stimulated with B16 tumor cells （1575.31 ± 60.25 pg/mL in SGC-7901 group vs 164.11± 18.52 pg/mL in B16 group, P 〈 0.01）. CONCLUSION： BM-derived DCs pulsed with tumor lysates Can induce anti-tumor immunity specific to gastric cancer ex vivo.
文摘AIM: To investigate the regulatory effect of Vδ1 T cells and the antitumor activity of Vδ2 T cells in rectal cancer.METHODS: Peripheral blood, tumor tissues and para-carcinoma tissues from 20 rectal cancer patients were collected. Naïve CD4 T cells from the peripheral blood of rectal cancer patients were purified by negative selection using a Naive CD4<sup>+</sup> T Cell Isolation Kit II (Miltenyi Biotec). Tumor tissues and para-carcinoma tissues were minced into small pieces and digested in a triple enzyme mixture containing collagenase type IV, hyaluronidase, and deoxyribonuclease for 2 h at room temperature. After digestion, the cells were washed twice in RPMI1640 and cultured in RPMI1640 containing 10% human serum supplemented with L-glutamine and 2-mercaptoethanol and 1000 U/mL of IL-2 for the generation of T cells. Vδ1 T cells and Vδ2 T cells from tumor tissues and para-carcinoma tissues were expanded by anti-TCR γδ antibodies. The inhibitory effects of Vδ1 T cells on naïve CD4 T cells were analyzed using the CFSE method. The cytotoxicity of Vδ2 T cells on rectal cancer lines was determined by the LDH method.RESULTS: The percentage of Vδ1 T cells in rectal tumor tissues from rectal cancer patients was significantly increased, and positively correlated with the T stage. The percentage of Vδ2 T cells in rectal tumor tissues from rectal cancer patients was significantly decreased, and negatively correlated with the T stage. After culture for 14 d with 1 μg/mL anti-TCR γδ antibodies, the percentage of Vδ1 T cells from para-carcinoma tissues was 21.45% ± 4.64%, and the percentage of Vδ2 T cells was 38.64% ± 8.05%. After culture for 14 d, the percentage of Vδ1 T cells from rectal cancer tissues was 67.45% ± 11.75% and the percentage of Vδ2 T cells was 8.94% ± 2.85%. Tumor-infiltrating Vδ1 T cells had strong inhibitory effects, and tumor-infiltrating Vδ2 T cells showed strong cytolytic activity. The inhibitory effects of Vδ1 T cells from para-carcinoma tissues and from rectal cancer tissue were not significantly different. In addition, the cytolytic activities of Vδ2 T cells from para-carcinoma tissues and from rectal cancer tissues were not significantly different.CONCLUSION: A percentage imbalance in Vδ1 and Vδ2 T cells in rectal cancer patients may contribute to the development of rectal cancer.
