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

Bone Marrow Cells ; Cell Line ; Cell Proliferation ; Humans ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes ; cytology ; T-Lymphocytes, Regulatory ; cytology

There has been a history of 30 years in the study of CD4(+)CD25(+) T regulatory cells (Treg) which primarily play a role of immune suppression in vivo. Many autoimmune diseases are related to the decrease and the disorder of these cells, such as multiple sclerosis, non-obese diabet (NOD) and lupus erythematosus. In the field of transplantation tolerance, the role played by Treg is also very important. All of these features have drawn the attention to the prevention of autoimmune diseases and the rejection of transplantation. However, the low frequency of Treg in vivo affected their use and study. Currently, many techniques about expansion of Treg in vitro have been established so as to overcome the problem of their limited cell numbers in vivo. Recent studies suggest that antigen-specific T regulator cells (sTreg) expanded by dentritic cells (DC) showed a superior immunosuppression in comparison with polyclonal Treg expanded by anti-CD3/CD28Ab, which is the focus of current studies. This article mainly reviews and compares the expansion techniques and the mechanism of regulatory T cells.
Cell Culture Techniques ; Humans ; Immunosuppression ; T-Lymphocytes, Regulatory ; cytology ; immunology

OBJECTIVETo investigate the frequency distribution features of innate-like lymphocytes (iNKT cells, γΔT cells and B1 cells) in peripheral blood of normal adults.

METHODSThe flow cytometry with 6 fluorescence staining was used to detect the percentages of iNKT lymphocytes, γΔT lymphocytes, B1 lymphocytes and adaptive T lymphocyte, B2 lymphocytes in peripheral blood lymphocytes of 50 normal adults. The difference and correlation between these lymphocyte subsets were analyzed by statistical software.

RESULTSThe percentage of iNKT cells in peripheral blood of 50 normal adults was 0.18% (0.01%-2.01%), the percentage of γΔT cells was 4.90% (1.45%-20.14%), the percentage of B1 lymphocytes was 1.62% (0.20%-3.77%), the percentage of adaptive T cells was 63.52% (33.20%-83.22%), the percentage of B2 cells was 6.64% (3.07%-13.80%). B1 and B2 were two subsets of B lymphocyte, the percentage of B2 in B lymphocyte was 81.43% (57.90%-94.12%) and more than that of B1 lymphocyte; the percentage of B1 lymphocytes was 17.28% (5.28%-41.13%). In T lymphocyte group the percentage of iNKT cell was 0.32% (0.01%-3.6%), the percentages of γΔT cells and adaptive T cells were 7.55% (3.04%-27.66%) and 91.98% (72.22%-96.86%) respectively. Spearman correlation analysis was used to analyze the correlation between the percentages of several lymphocyte subsets. There was a positive correlation between iNK T cells and γΔT cells, γΔT cells and adaptive T cells, B1 cells and B2 cells (r=0.39, P=0.0056; r=0.6028, P<0.0001; r=0.4791, P=0.0004). It was also found that the percentage of iNKT cells in female peripheral blood lymphocytes was 0.29% (0.06%-2.01%), and significantly higher than that in male peripheral blood lymphocytes 0.12% (0.01%-1.37%) (P<0.05).

CONCLUSIONThe percentages of γΔT cells, B1 cells and iNKT cells in peripheral blood lymphocytes of normal adults are significantly lower than that of adaptive lymphocytes, and their contents in peripheral blood decrease in turn. There are no sex differences in the percentages of these lymphocyte subsets except iNKT cells.

Adult ; B-Lymphocytes ; cytology ; Female ; Flow Cytometry ; Humans ; Male ; Natural Killer T-Cells ; cytology ; T-Lymphocyte Subsets ; cytology

Multiple myeloma (MM) is a hematologic malignancy resulted from genetic mutations in the process of B lymphocyte differentiating into plasma cells, the chemotherapy is the main treatment method, especially with the development of proteasome inhibitors and other drugs, the overall survival rate of MM patients has improved greatly, but the chemoresistance is still an important reason for treatment failure. Chimeric antigen receptor (CAR)-modified T lymphocyte therapy is a new method for tumor adoptive immunotherapy. By means of genetic modification, T cells are able to identify the target antigen specifically, and to kill target cells without major histocompatibility complex (MHC) restriction, therefore the specific killing activity is conspicuous, which has got considerable attention by the public, and has made remarkable achievements particularly in the treatment of B-lineage leukemia and lymphoma, but no systematic literatures were reported in the field of multiple myeloma using CAR therapy. Therefore, this review summarizes the research results of different CAR target in vivo and in vitro experiments for multiple myeloma.
Genetic Therapy ; Humans ; Immunotherapy, Adoptive ; methods ; Multiple Myeloma ; therapy ; Receptors, Antigen, T-Cell ; T-Lymphocytes ; cytology

The myelodysplastic syndromes (MDS) comprises a heterogeneous group of clonal hematopoietic stem cell disorders, while immunological abnormalities are frequently observed in such patients. T-cell mediated suppression of hematopoietic precursors contributes to the cytopenia and is related to the initiation and development of myelodysplastic syndromes (MDS). In this review, current knowledge concerning the feature of T-cell clonal expansion in MDS based on the analysis of T-cell receptor repertoire is summarized, including following issues: autoimmunity in MDS patients associated with T-cell mediated suppression of hematopoiesis, characteristics of T-cell clonal expansion in MDS patients, change of T-cell repertoire and recent thymus output function in MDS patients, efficiency of immunosuppressive treatment associated with T-cell clonal expansion in MDS patients and significance of T-cell clonal analysis in clinical application.
Clone Cells ; Humans ; Myelodysplastic Syndromes ; immunology ; Receptors, Antigen, T-Cell ; immunology ; T-Lymphocytes ; cytology ; immunology

Maturation of T cells in thymus plays key roles in antigen-specific immune response. In maturation, thymocytes undergo positive and negative selection. The MHC restriction of T cell is determined in positive selection. The tolerance of T cells to autoantigen is acquired during negative selection. Quantitation (avidity) theory points out that difference of avidity between thymocytes and stroma cells leads to difference in the signals integrated inside cells. When the signal reaches a particular threshold, positive or negative selection takes place. In this article, the selection mechanism and the relationship between positive and negative selection are mini-reviewed.
Animals ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Differentiation ; Humans ; Receptors, Antigen, T-Cell ; immunology ; Signal Transduction ; T-Lymphocytes ; immunology ; Thymus Gland ; cytology

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

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

BACKGROUND: We investigated the characteristics of the mononuclear cells remaining in the leukoreduction system (LRS) chambers of Trima Accel(R) in comparison with those of standard buffy coat cells, and evaluated their potential for differentiation into dendritic cells. METHODS: Twenty-six LRS chambers of Trima Accel(R) were collected after platelet pheresis from healthy adults. Flow cytometric analysis for T, B, NK, and CD14+ cells was performed and the number of CD34+ cells was counted. Differentiation and maturation into dendritic cells were induced using CD14+ cells seperated via Magnetic cell sorting (MACS(R)) Seperation (Miltenyi Biotec Inc., USA). RESULTS: Total white blood cell (WBC) count in LRS chambers was 10.8x108 (range 7.7-18.0x108). The median values (range) of proportions of each cells were CD4+ T cell 29.6% (18.7-37.6), CD8+ T cell 27.7% (19.2-40.0), B cell 5.5% (2.2-12.1), NK cell 15.7% (13.7-19.9), and CD14+ cells 12.4% (8.6-32.3) respectively. Although total WBC count was significantly higher in the buffy coat (whole blood of 400 mL) than the LRS chambers, the numbers of lymphocytes and monocytes were not statistically different. The numbers of B cells and CD4+ cells were significantly higher in the buffy coat than the LRS chambers (P<0.05). The median value (range) of CD34+ cells obtained from the LRS chambers was 0.9x10(6) (0.2-2.6x10(6)). After 7 days of cytokine-supplemented culture, the CD14+ cells were successfully differentiated into dendritic cells. CONCLUSIONS: The mononuclear cells in LRS chambers of Trima Accel(R) are an excellent alternative source of viable and functional human blood cells, which can be used for research purposes.
Adult ; B-Lymphocytes/cytology/immunology ; CD4-Positive T-Lymphocytes/cytology/immunology ; CD8-Positive T-Lymphocytes/cytology/immunology ; Cell Differentiation ; Dendritic Cells/*cytology/immunology ; Flow Cytometry ; Humans ; Killer Cells, Natural/cytology/immunology ; Plateletpheresis/*instrumentation