To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.
CD4-Positive T-Lymphocytes ; cytology ; CD8-Positive T-Lymphocytes ; cytology ; Flow Cytometry ; Humans ; Immunomagnetic Separation ; methods ; Leukocytes, Mononuclear ; cytology ; immunology

Colony-Forming Units Assay ; Cryopreservation ; methods ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunotoxins ; immunology ; Leukocytes, Mononuclear ; cytology ; Ricin ; immunology ; T-Lymphocytes ; cytology ; immunology ; Time Factors

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

This study sought to shed light on the killing effect of peripheral blood mononuclear cells (PBMCs) irradiated by gamma ray at a dose of 1 Gy on cultured human gastric tumor cell line MKN-28. The radiation dose rate of 17 Gy/min was used. The groups in the experiment were MKN-28 cell control group, PBMCs control group, MKN-28 tumor cells with irradiated or non-irradiated PBMCs co-cultured groups. Radiation dosage was one Gray, acridine orange/ethidium bromide (AO/EB) staining was used for observation of the killing effects of PBMCs on tumor cells in different period. Cells were harvested 240 h later and observed by transmission electron microscopy. The result showed the living period of irradiated PBMCs was shorter than that of non-irradiated PBMCs. In the irradiated and non-irradiated groups,a few PBMCs were still alive after being cultured for 240 h, but the cell volume was larger than that of lymphocytes. These cells were identified as monocytes (95%) or DCs (5%) by transmission electron microscopy. The co-culture of irradiated PBMCs and MKN-28 cells showed that tumor cells were eliminated after 96 h. As compared with the non-irradiated goup, the irradiated PBMCs had more potent ability for killing tumor. The results demonstrate that 1 Gy gamma irridiation can improve the killing effect of PBMCs on the tumor cells, and that 1 Gy gamma irritation can also induce shorter living period of lymphocytes in PBMCs cultured in vitro, but such irritation has little effect on the living period of monocytes and DCs in PBMCs.
Cell Survival ; Coculture Techniques ; Gamma Rays ; Humans ; Leukocytes, Mononuclear ; cytology ; immunology ; radiation effects ; Stomach Neoplasms ; immunology ; pathology ; Tumor Cells, Cultured

OBJECTIVETo prepare tumor antigen peptides from HL-60 cells and to induce specific immune response.

METHODSHL-60 antigen peptides were obtained using techniques including freezing and thawing, heat precipitation and acid precipitation. The stimulating effect of the in vitro Hsp70 binding HL-60 peptides on PBMC and the proliferation of stimulated PBMC were observed by T cell activation test. The cytotoxicity of proliferated PBMC is detected by incubating HL-60 cells or K562 cells with PBMC respectively.

RESULTSThe obtained tumor antigen peptides were a peptides mixture. The mixed peptides could activate PBMC and cause PBMC proliferation in vitro after presented by Hsp70. The proliferated PBMC showed specific cytotoxicity to HL-60 cells but not to K562 cells.

CONCLUSIONThe method for preparing of human leukemia tumor antigen peptides used in this paper is simple and easy; the obtained antigen peptides can induce specific immune response in vitro.

Cell Division ; HL-60 Cells ; HSP70 Heat-Shock Proteins ; immunology ; Humans ; K562 Cells ; Killer Cells, Natural ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Neoplasm Proteins ; immunology ; Peptides ; immunology

To investigate the characteristics of interstitial inflammatory cells and possible involvement of nudelta T cells, 16 renal allograft biopsies showing chronic rejection were stained by immunohistochemical method and correlated with the data of peripheral blood evaluated by flow cytometry. For immunophenotyping, fresh frozen sections were stained with monoclonal antibodies against CD3, CD4, CD8, CD68, CD56, TCRdelta1 and HLA DR. Paraffin embedded tissue was stained with CD45RO, CD20-Cy and CD68. Nine cases of nonspecific tubulointerstitial change and 4 cases of nonallograft tubulointerstitial nephritis were used as a control. Inflammatory infiltration was present in all cases studied. T cells predominated in the interstitium of chronic rejection and were followed by macrophages and B cells. The degree of interstitial infiltration of frozen section was not accordant with that of paraffin sections. Allografts with nonspecific tubulointerstitial changes or tubulointerstitial nephritis of native kidneys showed similar distribution pattern in terms of type and degree. However, the degree of infiltrate did not give any statistical significance among groups. The CD4/CD8 ratios in interstitial infiltrates were less than 1.0 in 6 cases and was not accordant with those of peripheral blood. Proportion of nudelta T cells increased over 10% in 2 cases in tissue and in 3 cases in peripheral blood. In 3 cases of chronic rejection in which both tissue and blood results were available, there was no concordance of CD4/CD8 or nudeltaT/CD3 between them. Tubular expression of HLA DR was, however, present only in 4 cases of chronic rejection. In conclusion, T lymphocytes were predominant regardless of diagnosis or disease activity. T lymphocyte subset did not give any suggestion as to the diagnosis or disease activity in chronic rejection. Furthermore nudelta T cells had only limited value. Lymphocytic subsets in peripheral blood would not be predictors of tissue destruction in chronic rejection.
Flow Cytometry ; Graft Rejection/*immunology ; Human ; Kidney/cytology/*immunology ; Kidney Transplantation/*immunology ; Leukocytes, Mononuclear/*immunology ; Phenotype ; Receptors, Antigen, T-Cell, gamma-delta/immunology ; Support, Non-U.S. Gov't

To investigate the characteristics of interstitial inflammatory cells and possible involvement of nudelta T cells, 16 renal allograft biopsies showing chronic rejection were stained by immunohistochemical method and correlated with the data of peripheral blood evaluated by flow cytometry. For immunophenotyping, fresh frozen sections were stained with monoclonal antibodies against CD3, CD4, CD8, CD68, CD56, TCRdelta1 and HLA DR. Paraffin embedded tissue was stained with CD45RO, CD20-Cy and CD68. Nine cases of nonspecific tubulointerstitial change and 4 cases of nonallograft tubulointerstitial nephritis were used as a control. Inflammatory infiltration was present in all cases studied. T cells predominated in the interstitium of chronic rejection and were followed by macrophages and B cells. The degree of interstitial infiltration of frozen section was not accordant with that of paraffin sections. Allografts with nonspecific tubulointerstitial changes or tubulointerstitial nephritis of native kidneys showed similar distribution pattern in terms of type and degree. However, the degree of infiltrate did not give any statistical significance among groups. The CD4/CD8 ratios in interstitial infiltrates were less than 1.0 in 6 cases and was not accordant with those of peripheral blood. Proportion of nudelta T cells increased over 10% in 2 cases in tissue and in 3 cases in peripheral blood. In 3 cases of chronic rejection in which both tissue and blood results were available, there was no concordance of CD4/CD8 or nudeltaT/CD3 between them. Tubular expression of HLA DR was, however, present only in 4 cases of chronic rejection. In conclusion, T lymphocytes were predominant regardless of diagnosis or disease activity. T lymphocyte subset did not give any suggestion as to the diagnosis or disease activity in chronic rejection. Furthermore nudelta T cells had only limited value. Lymphocytic subsets in peripheral blood would not be predictors of tissue destruction in chronic rejection.
Flow Cytometry ; Graft Rejection/*immunology ; Human ; Kidney/cytology/*immunology ; Kidney Transplantation/*immunology ; Leukocytes, Mononuclear/*immunology ; Phenotype ; Receptors, Antigen, T-Cell, gamma-delta/immunology ; Support, Non-U.S. Gov't

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

To explore the possibility of in vitro induction of cord blood cell-derived lymphocytes into cytotoxic T lymphocytes (CTL) with anti-leukemia specificity, umbilical cord blood (UCB)-derived mononuclear cells were cultured with multiple cytokines to generate dendritic cells (DC) in vitro. Leukemia cells were irradiated with (137)Cs and activated by premature cytokines. The characteristics of maturation of DC were evaluated through morphology examination and flow cytometry. DC pulsed with leukemic antigens were co-cultured with lymphocytes. Cytotoxicity of the CTL to corresponding leukemic cells was measured with lactate dehydrogenase-release assay. The results showed that UCB-derived monocytes could be induced into typical DC in all of the 12 samples. Expression of immunological markers such as CD1a(+), HLA-DR(+), CD86(+), CD83(+) on DC were significantly up-regulated (P < 0.05). DC presenting leukemic antigens generated leukemia-specific CTL with a killing rate of (44.76 +/- 17.42)% at the E:T ratio of 50:1 against AML cells and a killing rate of (8.50 +/- 4.25)% at the E:T ratio of 50:1 against ALL cells. Whereas, these CTL present almost no killing effect on the mononuclear cells collected from the same patients in complete remission phase. It is concluded that (1) it is possible to induce UCB-derived monocytes into mature DC with typical morphology. (2) Cord blood derived mature DC presenting leukemia antigen can generate leukemia-specific CTL with vigorous cytotoxic activity against the same leukemia blasts and low killing activity against bone marrow cells of the same patients in complete remission phase.
Cells, Cultured ; Coculture Techniques ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; Female ; Fetal Blood ; cytology ; immunology ; Humans ; K562 Cells ; Leukemia ; immunology ; pathology ; Leukocytes, Mononuclear ; cytology ; immunology ; Male ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo explore the role of indirect antigen presentation pathway on the immunogenecity of epidermal cells.

METHODSHuman epidermal cells (HEC), allogeneic human peripheral blood lymphocytes (PBL) and mononuclear cells (PBM, including monocytes) were isolated and cultured in vitro. HECs were transfected by human-originated CTLA4Ig-adenovirus vector. The CTLA4Ig expression was observed. Allogeneic PBLs or PBMs were added to the transfected and non-transfected HECs with simple cultured PBLs and PBMs as the control. The proliferation of PBL and PBM was determined by (3)H-TdR incooperation.

RESULTSHECs could be successfully transfected by CTLA4Ig-adenovirus vector and expressed corresponding proteins. The non-transfected HECs could stimulate slight proliferation of allogeneic PBLs (P < 0.05) and stimulate remarkable proliferation of PBMs (including monocytes) (P < 0.05). The proliferation reaction of PBLs and PBMs decreased significantly (P < 0.05) after being stimulated by HEC which was modulated by CTLA4Ig genes.

CONCLUSIONIndirect antigen presentation pathway might play important roles in the HEC immunogenicity which could be evidently inhibited by CTLA4Ig.

Adenoviridae ; genetics ; Antigen Presentation ; immunology ; physiology ; Antigens, CD ; Antigens, Differentiation ; genetics ; immunology ; CTLA-4 Antigen ; Cell Division ; immunology ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; immunology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocytes ; cytology ; immunology ; Signal Transduction ; Transfection