OBJECTIVETo explore the specific anti-leukemia immune response of CD8+ cytotoxic T lymphocyte (CTL) derived from cord blood (CB) ex vivo and evaluate the feasibilities and values of the CTL for specific immunotherapy.

METHODSDendritic cells (DC) were induced from mononuclear cells (MNC) by combination cytokines in 10 CB samples. Loading U937 cell lysate antigen on the mature DC, they could stimulate the lymphocytes of the same origin to generate CTL. MidiMACS was used to isolate CD8+ CTL. Analysis of DC was performed by inverted microscopy, scanning electron microscopy and flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of the CTL.

RESULTSCocultured with GM-CSF, IL-4, TNF-alpha and PGE2, CB-MNC could be induced into functional DC with typical morphology. The mean cytotoxicity of CD8+ CTL to U937 cells was significant stronger than that of CD8- CTL and TL at the same E: T ratios. The mean cytotoxicity rate of CD8+ CTL to U937 cells was higher than that to K562 cells [(66.36 +/- 12.43)% vs (41.97 +/- 14.24)%] at E: T ratio of 40: 1 (P < 0.05). The cytotoxicity of CD8- CTL to K562 cells showed no difference from that to U937 cells (P > 0.05).

CONCLUSIONMature CB-DC loading U937 cell antigens could induce CB-T lymphocytes to generate leukemia-specific CD8+ CTL. The cytotoxicity of the CD8+ CTL is specific against U937 cells and is more potent than that of CD8- CTL.

CD8-Positive T-Lymphocytes ; cytology ; immunology ; Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; In Vitro Techniques ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology ; U937 Cells

This study was aimed to investigate the specific anti-leukemia cell effect of cytotoxic T lymphocytes (CTLs) induced by HL-60 or K562 cell-sensitized dendritic cells (DCs) from umbilical cord blood. 12 units of human umbilical cord blood (UCB) were collected and the mononuclear cells (MNCs) were isolated from UCB, then cultured with granulocyte monocyte colony- stimulating factor (GM-CSF), interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and EPO for 3 - 4 weeks. Flow cytometry was used to determine the number of DCs and cell surface antigens before and after culture with monoclonal antibodies including CD83, CD1a, CD11c and CDw123. HL-60 and K562 were frozen-thawed, and released their tumor antigen peptides (TAP). The CTLs were produced by sensitizing T lymphocytes with DC-loaded HL-60 and K562 cell antigens. The test of (3)H-TdR incorporation was used to detect the immunostimulation activity of DCs. MTT assay was applied to evaluate specific cytotoxicity of CTL on leukaemia cells. The results indicated that the MNCs of UCBs cultured with GM-CSF, IL-3, EPO and SCF were shown to differentiate into CD1a(+) CD11c(+) CD83(+) CDw123(+) DCs. Numbers of DCs from UCBs remarkably increased in 2 - 4 weeks and then decreased. After culture with cytokines DCs increased (10.6 - 28.2) x 10(5)/ml in actual numbers. The CTL induced by DC pulsed with HL-60, K562 frozen-thawed lysates were effective to kill HL-60 and K562. Cytotoxicity of CTL to HL60 and K562 were (42.04 +/- 8.46)% and (31.25 +/- 11.07)% respectively. It is concluded that the MNCs of UCBs cultured with cytokines of GM-CSF, SCF, EPO and IL-3 can differentiate into CD1a(+), CD83(+), CD11c(+) and CDw123(+) DCs. The CTL induced by DCs pulsed with HL-60, K562 frozen-thawed lysates can effectively kill HL-60 and K562. These DCs as antigen presenting cells play an important role in cancer immunotherapy.
Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; immunology ; HL-60 Cells ; Humans ; K562 Cells ; T-Lymphocytes, Cytotoxic ; immunology

The study was purposed to explore the quantity, morphology and immunophenotype of dendritic cells (DC) acquired by co-cultivated system with 3 types of cytokines and sodium selenite (Se) from peripheral blood mononuclear cells (PBMNCs), and to investigate the effects of Se on inducing the cytotoxic T lymphocyte (CTL) to get specific anti-leukemic activity in vitro by DC pulsed with K562 cell frozen-thawed antigen (antigen cell loading). PBMNCs isolated from healthy donors were cultured in RPMI 1640 medium contained 10% FBS supplied with 3 cytokines (rhGM-CSF, rhIL-4, TNF-alpha) for 4 days, DCs harvested were divided into 4 groups, DCI: DC alone; DCII: DC + Se (adding 0.5 micromol/L of Se); DCIII: DC + K562 (pulsed with lysed K562 cells); DCIV: DC + Se + K562. Morphology of DCs was observed under microscope at day 7. The CD1a, CD40, CD83, and CD86 were detected by FCM. Cytotoxicity of T cells induced by DC were measured with LDH release test at day 12. The level of IL-12 in supernatant of cultured DCs were determined with ELISA. The results indicated that at 7th day DC in 4 groups showed characteristic morphology, the colony numbers of 4 groups were all higher than those before cultivation. There were no obvious differences of morphology and colony counts between DCI group and DCII group. The colony numbers of DCIII group and DCIV group increased, as well as the ratio of suspended cells enhanced. The expressions of CD1a, CD40, CD83 and CD86 in 4 groups of DC were significantly higher than those in PBMNC group (p < 0.01), the expressions of CD1a and CD40 in 4 groups of DC did not display significant difference (p > 0.05), the expressions of CD83 and CD86 in both DCIII group and DCIV group were all higher than those in DCI group and DCII group (p < 0.01), but their expressions of CD83 and CD86 in DCI and DCII were not significantly different (p > 0.05), as well as those in DCIII group and DCIV group. With the ratio of 25:1 between E:T, killing rate of CTL on K562 cells in 4 DC groups were 15.3 +/- 2.3%, 26.3 +/- 3.7%, 28.2 +/- 4.5% and 36.2 +/- 3.7% respectively, all obviously higher than those of T cell group without being sensitized by DCs (5.9 +/- 2.4%) (p < 0.01), The CTL effect in DCIV group was the highest, which was higher than those in other 3 DC groups (p < 0.01); the effects in both DCII and DCIII group were also higher than that in DCI group (p < 0.01), but their difference between DCII and DCIII groups did not show significance (p > 0.05). The levels of IL-12 in supernatant of DCI, DCII, DCIII and DCIV groups were 257.0 +/- 64.2, 328.1 +/- 43.9, 323.0 +/- 53.5 and 353.9 +/- 46.2 pg/ml respectively, all significantly higher than that in supernatant of T cell alone group without being sensitized by DCs (35.27 +/- 27.1 pg/ml) (p < 0.01), The levels in DCII, DCIII and DCIV groups were all higher than that in DCI group (p < 0.01), but their levels between DCII, DCIII and DCIV groups were not of significant difference (p > 0.05). It is concluded that matured DCs can be successfully obtained from PBMNCs by a culture system contained rhGM-CSF, rhIL-4 and TNF-alpha with or without low-dose of Se (0.5 micromol/L) in vitro. Using K562 cell frozen-thawed antigen, DC express more adhesive molecules and co-stimulating molecules (CD83, CD86), and increase the secretion of IL-12, as well as the killing effects of CTL on special target cells. Low dose of Se did not showed effects on quantity and morphology of matured DC harvested, as well as their expression of mature phenotypes, it raised levels of IL-12 secreted by DCs, reaching the same level as using K562 cell frozen-thawed antigen, and it showed synergistic effect on induction of CTL with K562 cell frozen-thawed antigen.
Cells, Cultured ; Cytokines ; pharmacology ; Dendritic Cells ; cytology ; immunology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; cytology ; Sodium Selenite ; pharmacology ; T-Lymphocytes ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology

OBJECTIVETo construct vitiligo-specific HLA-A*0201-peptide tetramers and to apply the constructed tetramers in detection of vitiligo-specific cytotoxic T lymphocytes (CTL).

METHODSProteins HLA-A0201*-BSP and β2M were obtained by effective prokaryotic expression. The purified proteins were refolded with vitiligo antigen peptides MelanA 26-35, gp100 209-217, and tyrosinase 1-9, respectively to form HLA-A*0201-peptide complex. The complex was biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with phycoerythrin (PE)-streptavidin at a ratio of 4∶1 and identified by Dot-blot assay. The capacity of tetramer to detect vitiligo-specific CTL was analyzed by flow cytometry.

RESULTSThe biotinylation of vitiligo-specific HLA-A*0201-peptide tetramers were successfully performed by Dot-blot. Flow cytometry analysis indicated that the tetramer effectively bound to specific CTL from peripheral blood of patients with vitiligo.

CONCLUSIONThree kinds of biotinylated vitiligo-specific HLA-A*0201-peptide tetramers have been constructed successfully. The tetramer can detect antigen specific CTL from patients with vitiligo.

Biotinylation ; Flow Cytometry ; HLA-A2 Antigen ; Humans ; Peptides ; T-Lymphocytes, Cytotoxic ; cytology ; Vitiligo ; diagnosis ; immunology

The aim of this study was to investigate whether exosomes derived from K562 cells and human monocyte-derived dendritic cells (DCs) transfected with total RNA of K562 cells are capable of inducing antigen-specific cytotoxic T lymphocytes (CTL) responses in vitro. DCs were generated from peripheral blood mononuclear cells (PBMNC) of healthy volunteers in the presence of GM-CSF and IL-4, and then were transfected with K562 RNA by using DOTAP lipofection. Exosomes was extracted from the supernatant of DCs and K562 cells. The T cell were activated to be tumor specific CTL after DCs and exosomes were co-cultured with autologous T cells derived from healthy volunteers' PBMNC. The effect of CTL on K562 cells was detected by MTT assay. The results showed that treatment of T cells with exosomes derived from K562 cells or DCs transfected with total RNA of K562 cells could significantly promote their killing ability on K562 cells as compared with untreated T cells (P < 0.05). The killing ability of T cells treated with exosomes on K562 cells was stronger than on HL-60 cells (P < 0.05). It is concluded that the specific CTL immune response to leukemia cells can be induced by exosomes derived from K562 cells.
Dendritic Cells ; cytology ; immunology ; Endosomes ; immunology ; Exocytosis ; immunology ; Humans ; K562 Cells ; Monocytes ; cytology ; RNA, Neoplasm ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

This study was aimed to investigate the effects of tumor antigen-loaded dendritic cells (DC) stimulating the specific cytotoxic T lymphocytes (CTL) on Jurkat cells in vitro. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood, the adherent monocytes were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), alpha tumor necrosis factor (TNF-alpha) and sCD40L, DCs were co-cultured with frozen-thawed antigen of Jurkat cells or WT1 peptides, and then T cells were triggered into specific CTL. The results showed that most suspended cells exhibited distinctive morphological features of DC which expressed CD40 (96%), CD86 (97%), CD80 (77%), CD1a (69%), and gained the powerful capacity to stimulate proliferation of allogeneic lymphocytes. Under the effector: target ratio of 20:1, CTLs derived from cultures with DC and frozen-thawed antigen of Jurkat cells showed 91.1% cytotoxicity against Jurkat cells, CTL derived from cultures with DC and WT1 peptides showed 87.5% cytotoxicity against Jurkat cells, cytotoxicity of CTL derived from cultures with unloaded DC against Jurkat cells was 42.1% and cytotoxicity of monocytes was 22.7%. Cytotoxicity of CTL derived from culture with frozen-thawed antigen or WT1 peptides loaded DC was stronger than that in control groups (P < 0.01). It is concluded that the tumor antigen-pulsed DC can induce efficient and specific anti-tumor immunity, may play a great role in clinical therapy for leukemia.
Antigens, Neoplasm ; immunology ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; immunology ; Humans ; Jurkat Cells ; Leukemia, T-Cell ; immunology ; pathology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured

This study was aimed to explore the method for induction and expansion of EB virus specific cytotoxic T lymphocytes (EBV-CTL) in vitro, and to detect their killing effect. Peripheral blood mononuclear cells (PBMNC) were collected from 6 EBV seropositive healthy donors, and EBV-transformed B lymphoblastoid cells (BLCL)were used as the antigen-presenting cells and antigen stimulant which was irradiated by 40 Gy (60)Co irradiator. The autologous PBMNC and irradiated BLCL were cultured to induce and expand the EBV-CTL, and the immunophenotype was identified by the flow cytometry. The killing effect of the EBV-CTL against the autologous BLCL (autoBLCL), the autologous PHA cultured B lymphoblastoid cells( PHA-BLCL), the allogeneic BLCL (alloBLCL) and the K562 cells were measured with LDH release assay under different effector-to-target ratio. The results showed that the 6 cell lines of EBV-CTL were induced and expanded from the EBV seropositive healthy donors, the overall increase in cell numbers varied from 18.6 to 55.0 times. After 10 stimulations, the specific killing efficiency of the EBV-CTL for the autoBLCL were 59.4%, 43.2% and 29.0% under the effector-to-target ratio of 20: 1, 10: 1 and 5: 1. The nonspecific killing efficiency for the PHA-blast, alloBLCL and K562 cells were 7.1%, 9.4% and 10.3% (P < 0.05) under the 20: 1 ratio; 6.6%, 8.3% and 8.1% (P < 0.05) under 10: 1; 5.4%, 7.3% and 6.3% (P < 0.05) under 5: 1, respectively. It is concluded that the EBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLA matched BLCL and may become a potential treatment for EBV related post-transplant lymphoproliferative disorders.
B-Lymphocytes ; immunology ; Cell Line, Transformed ; Herpesvirus 4, Human ; immunology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; immunology ; virology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology

This study was purposed to analyze the changes of T-cell clonality after induction of peripheral T lymphocytes by autogenous DC and cytokines in the preparation of adoptive immunotherapy for leukemias. The bone marrow and peripheral blood from 21 leukemia patients at remission stage after treatment and subjected to adoptive immunotherapy were collected. Their DCs and T-cells were stimulated with cytokines and then were mixed to activate T-cells. T-cell receptor beta variable region (TCRBV) families were amplified by RT-PCR, and genescan method and sequencing of the PCR products were used to observe the clonality changes of T-cells before and after the induction and cultivation of T-cells. The flow cytometry was used to identify CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOXP3(+) cells to disclose the ratio change of cytotoxic T lymphocytes (CTL), helper T-cells, regulatory T-cells and NK T-cells before and after induction and cultivation of T-cells. The results showed that in the 21 patients, most of the 24 TCRBV families presented as oligoclonal distribution on genescan, several families were not expressed, and only a few families remained polyclonal. TCRBV24 was found to be oligoclonal in all of the 21 patients. DNA sequence analysis of TCRBV24 revealed a common motif of VAG in CDR3 in 3 cases and a common motif of GGG in CDR3 in 2 cases. In patient 5, both TCRBV 24 and TCRBV8 contained the same motif of GGG in CDR3. The identical motif in these patients may suggest that these T-cells recognize the same antigen. The peripheral lymphocytes demonstrated recovery of clonal profile on genescan from oligoclonal profile and absence of several families before the induction and cultivation to typical polyclonal profile in all TCRBV families after the induction by DC and cytokines for 13 days. After the induction and cultivation, the number of lymphocytes increased to 3.38 +/- 1.20 times. CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOX P3(+) cells were 71.1 +/- 11.8%, 26.7 +/- 11.4%, 35.7 +/- 12.9%, 3.1 +/- 1.6% and 0.12 +/- 0.1% respectively before the induction and cultivation, and changed to 95.4 +/- 3.2% (p < 0.01), 27.0 +/- 13.1% (p > 0.01), 55.5 +/- 13.8% (p < 0.01), 9.8 +/- 6.1% (p < 0.01) and 0.22 +/- 0.18% (p < 0.01) respectively after the induction and cultivation. It is concluded that the major action of this induction and cultivation method on T-lymphocytes in vitro is the promotion of CTL and NK T-cell proliferation. In leukemic patients at the remission stage, the TCRBV profile is characterized by the oligoclonal proliferation of T-lymphocytes. Several proliferated clones may have the same motif in CDR3, suggesting the recognition of the same antigen by these lymphocyte clones. Cytokine induction and co-culture with autogenous DCs can stimulate the T-lymphocytes to recover their immunocompetence as manifested by the polyclonal profile and the proliferation of CTL and NK-T cells.
Adolescent ; Adult ; Aged ; Child ; Female ; Genes, T-Cell Receptor beta ; Humans ; Immunotherapy, Adoptive ; Leukemia ; genetics ; immunology ; therapy ; Lymphocyte Activation ; Male ; Middle Aged ; T-Lymphocytes ; chemistry ; cytology ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; T-Lymphocytes, Regulatory ; chemistry ; immunology ; Young Adult

OBJECTIVETo establish a method for efficient induction and expansion of Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) in vitro and evaluate the possibility of using this strategy for treatment of nasopharyngeal carcinoma (NPC).

METHODSEBV-transformed B lymphoblastoid cells (BLCLs) were used as the antigen stimuli and antigen-presenting cells. EBV-specific CTL was induced by co-culture of the autologous peripheral blood mononuclear cells (PBMCs) and the irradiated BLCLs, and expanded with a cocktail method consisting of OKT-3, irradiated homologous PBMC, and IL-2. The specific activity of the CTL against the NPC cells was measured with MTT assay.

RESULTSEBV-specific CTL was successfully induced and expanded by 600 folds. The killing efficiency of the CTL was 76% for autologous BLCLs, 13% for homologous BLCLs, 51% for autologous NPC cells, and 27% for homologous CNE cell line, and after expansion, the corresponding killing efficiencies were 63%, 25%, 49%, and 33%, respectively. The non-specific killing only slightly increased after the expansion.

CONCLUSIONEBV-specific CTL can be successfully induced and expanded in vitro for specific killing of autologous NPC cells, suggesting the potential of this strategy in the treatment of NPC.

Antigen-Presenting Cells ; cytology ; immunology ; Antigens, Viral ; immunology ; B-Lymphocytes ; cytology ; immunology ; virology ; Cells, Cultured ; Coculture Techniques ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology ; Tumor Cells, Cultured

Animals ; Cancer Vaccines ; immunology ; Cell Fusion ; methods ; Cell Line ; Dendritic Cells ; cytology ; immunology ; Fibrosarcoma ; immunology ; pathology ; Hybrid Cells ; immunology ; Mastocytoma ; immunology ; pathology ; Mice ; Mice, Inbred BALB C ; T-Lymphocytes ; immunology ; pathology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Cells, Cultured