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

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

OBJECTIVE: To explore the better freezing protocol to preserve peripheral blood monuclear cells (PBMCs), islets antigen-specific T cells responses compared with freshly isolated samples in type 1 diabetic (T1D) patients. METHODS: The T cell Workshop Committee of the Immunology of Diabetes Society (IDS-TCW) organized the Freezing Study I and we were one of the 9 centers in the world to participate in the study. According to the two standardized T cell freezing protocols (warm and cold) to freshly isolated PBMCs in terms of recovery, viability, cell subset composition (FACS) and performance in Enzyme-linked immunospot (ELISPOT) assays, we chose 5 newly onset T1D patients and 5 age and sex matched healthy controls. Besides the protocols, all the freezing reagents and antigens were also centralized. The antigens used in ELISPOT were labeled blindedly. RESULTS: 1) Although warm frozen-thawed (W) samples had a slightly higher recovery rate (61.2% vs 60.1%, P>0.05) and viability (77.5% vs 74.9%, P>0.05) as compared with the cold frozen ones (C), the difference was not significant. 2) Both protocols led to a relative loss in monocytes as compared with the fresh samples (F) [3.2±1.1% (C) and 3.0±0.9% (W) vs 7.0±1.1% (F), both P<0.05], while other subsets including CD4+T, CD8+T, B cells, NK cells and NKT cells didn't. 3) Freezing and fresh samples showed similar IFN-γ secretion responses to polystimuli in ELISPOT. Irrespective of the freezing protocol, recall antigen Pediacel and islet antigen-reactive responses were both lower in the frozen cells compared with fresh PBMCs. The stimuli index (SI) of GADspecific T cell response in the fresh samples from T1D patients was 5.1, higher than that of frozen samples with either cold protocol (1.3) or warm one (1.4) (both P<0.05). Only fresh samples from T1D showed significantly higher GAD-specific T cell responses than the healthy controls no matter in SI (5.1 vs 0.9, P<0.05) or spot forming cells (8.1 vs 0.1, P<0.05), whereas the frozen samples did not show such difference. CONCLUSION More studies are needed to verify a freezing method to bring comparable islets antigen specific T cell responses in T1D patients to fresh PBMCs.
Adult ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cryopreservation ; Diabetes Mellitus, Type 1 ; blood ; immunology ; Female ; Humans ; Islets of Langerhans ; immunology ; Leukocytes, Mononuclear ; cytology ; Male ; T-Cell Antigen Receptor Specificity ; immunology ; T-Lymphocytes ; cytology ; immunology ; Young Adult

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

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

To study the effects of supernatant derived from acute myeloid leukemia (AML) cell lines on proliferation and apoptosis of CD4(+) and CD8(+) T cell subsets and to investigate the mechanism by which AML escapes from immune recognition, lymphocytes were labeled with CFSE and were stimulated with anti-CD3 and anti-CD28 in presence or absence of supernatants from three AML cell lines (HL-60, NB4, U937). After culture, cell suspensions were labeled with 7AAD and CD4 PE (or CD8 PE). Cells were then detected by flow cytometry and their proliferation and apoptosis were analyzed. The results showed that supernatants from two of three cell lines (HL-60 and NB4) inhibited the proliferation of CD4(+) and CD8(+) T cells, and the degree of inhibition showed a dose-dependent way. Similarly, the apoptosis of stimulated CD4(+) T cells was inhibited, but stimulated CD8(+) T cells remained unaffected by supernatant from HL-60 and NB4. In contrary, the apoptosis of proliferative CD8(+) T cells were increased significantly by HL-60 and NB4 supernatant. It is concluded that soluble factors derived from AML cell lines inhibit the proliferation of CD4(+) and CD8(+) T cells and induce the apoptosis of proliferative CD8(+) T cells, that may be one of the mechanisms by which the immunity was suppressed.
Apoptosis ; physiology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Cells, Cultured ; Culture Media ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; T-Lymphocytes ; cytology ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVE: To study the effect of electro- acupuncture at Zusanli acupoint in regulating perioperative cell immune functions in rats. METHODS: Forty-two SD rats were divided into blank control group (=6), model group (=18), and electroacupuncture group (=18). The rats in the latter two groups underwent thigh incision and femoral dissection under anesthesia; the rats in electro-acupuncture group received electro-acupuncture at bilateral Zusanli acupoint for 15 min before anesthesia and 1 h after the surgery. The rats in the model group and electro-acupuncture group were sacrificed at 6 h, 24 h, and 72 h after the operation and blood samples were taken from the ventricle for analyzing CD3, CD4, and CD8 T cell subpopulations and calculation of CD4/CD8 using flow cytometry. ELISA was used to detect the levels of interleukin-1 (IL-1) and IL-6. RESULTS: The CD3 T cell subpopulation was significantly lower in the model group and electro-acupuncture group than in the blank group at 6 h and 24 h after the operation. At 72 h after the operation, CD3 subpopulation levels still remained low in the model group, but recovered the control level in electro-acupuncture group. At each time point of measurement, CD3 level was significantly lower in the model group than in the electro-acupuncture group. CD4 level in the model group was significantly lowered at 6 h and 24 h after the operation, and recovered the control level at 72 h. In the electro-acupuncture group, CD4 level was significantly lowered at 6 h after the operation, but recovered the control level at 24 h. At 24 h and 72 h, the levels of CD4 were significantly lower in the model group than in the electro-acupuncture group. CD8 level underwent no significant changes after the operation in either the model group or electro-acupuncture group. CD4/CD8 was significantly lowered at 24 h and 72 h after the operation in the model group but showed no significant variation in the electro-acupuncture group. Compared with that in the control group, IL-1 level was significantly lowered in both the model group and electroacupuncture group at 6 h, 24 h, and 72 h after the operation, and was significantly lower in the model group than in the electroacupuncture group at these time points. IL-6 level increased significantly in the model group and the electro- acupuncture group at 6 h and 24 h. at 72 h, IL-6 level was obviously lowered in the electro-acupuncture group but remained elevated in the model group. CONCLUSIONS Electro-acupuncture alleviates postoperative immune suppression and promotes recovery of the immune function in rats, suggesting a protective effect of electro-acupuncture at Zusanli acupoint on cellular immune function after surgery.
Acupuncture Points ; Animals ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Electroacupuncture ; methods ; Femur ; surgery ; Flow Cytometry ; Humans ; Immunity, Cellular ; Perioperative Period ; Rats ; Rats, Sprague-Dawley ; T-Lymphocyte Subsets ; cytology ; immunology

OBJECTIVETo investigate the relation between CD4(+) and CD8(+) T lymphocyte infiltration and apoptosis of the neurons in the local traumatic brain tissue after brain trauma in rats.

METHODSIn rat models of brain trauma, the changes in the number of CD4(+) and CD8(+) T lymphocytes and the apoptosis of neurons in the local traumatic brain tissue were observed by immunohistochemistry at different time points after brain trauma.

RESULTSTwenty-four hours after brain trauma, a significant increase in the number of CD4(+) and CD8(+) T lymphocytes occurred in the injured brain tissue, both reaching the highest levels on day 10, at the point of which the number of CD4(+) cells increased by about 15 folds and that of CD8(+) cells by about 20 folds compared with the control groups. The CD4(+) and CD8(+) T lymphocytes both began to decrease 30 days after the injury. A similar pattern of alterations was found in the apoptosis of neurons in the local brain tissue. Correlation analysis demonstrated a close positive correlation between the changes in CD4(+) and CD8(+) lymphocyte numbers and the number apoptotic neurons in the injured brain tissue.

CONCLUSIONSBrain trauma induces obvious increases in CD4(+) and CD8(+) T lymphocytes and enhanced cellular immune response in the injured brain tissue to mediate neuronal apoptosis and further exacerbate the brain tissue injuries.

Animals ; Apoptosis ; immunology ; Brain Injuries ; immunology ; pathology ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Female ; Immunity, Cellular ; Male ; Neurons ; immunology ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Minimal residual disease (MRD) is the principal root of relapsed leukemia. Application of specific immunotherapy is effective in eradication of MRD and one of the immunotherapeutic strategies is induction and re-transfusion of leukemia-specific cytotoxic T lymphocytes (CTLs). This study was aimed to investigate the possibility of ex vivo induction and generation of CD8 positive CTLs of umbilical cord blood cell origin and to explore their potential of specific anti-leukemia cytotoxicity so as to evaluate the feasibility of application of cord blood cell derived lymphocytes to specific immunotherapy. Dendritic cells (DCs) were induced and generated ex vivo from cord blood mononuclear cells (MNC) with a cytokine cocktail, and loaded with frozen and thawed U937 leukemia antigen. The matured DCs were used to stimulate T lymphocytes derived from the same cord blood cell sample into CTLs. CD8 positive CTLs were then isolated by magnetic activated cell sorting (MACS). Inverted microscopy, scanning electronic microscopy and flow cytometry were used to detect DCs and methyl thiazolyl tetrazolium (MTT) cytotoxicity method was used to assay the killing activity. The results showed that DCs with typical morphology and mature function were cultured from 10 human cord blood cell samples. The cytotoxicities of CD8 positive CTLs, CD8 negative CTLs and T lymphocytes (TLs) to U937 cells were (66.36 +/- 12.43)%, (34.47 +/- 8.19)% and (15.79 +/- 4.64)% respectively under the same effector target ratio (40:1). Among them, the anti-leukemia cytotoxicity of the CD8 positive group was highest. At effctor target ratio of 40 to 1, the cytotoxicity of CD8 positive CTLs to U937 cells (66.36%) was higher than that to K562 cells (41.97%) (P < 0.05), whereas the cytotoxicity of CD8 negative CTLs to U937 cells was not significantly different from that to K562 cells (P > 0.05). It is concluded that specific CD8 positive CTLs can be generated from cord blood lymphocytes by induction of mature cord blood DCs loaded with U937 leukemia antigen. The cytotoxicity of CD8 positive CTLs against U937 cell is more potent than CD8 negative CTLs, and is strain specific.
Antibody Specificity ; Antigens, Neoplasm ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Dendritic Cells ; immunology ; Fetal Blood ; cytology ; immunology ; Humans ; Immunotherapy ; T-Cell Antigen Receptor Specificity ; T-Lymphocytes, Cytotoxic ; immunology ; U937 Cells

OBJECTIVETo investigate the relationship between subsets of lymphocytes and between its activated status and the clinical manifestations in patients with PNH, and to unfold immunological mechanism in the pathogenesis of PNH.

METHODSThe peripheral blood mononuclear cells (PBMNC) from 18 PNH patients and 20 controls were separated into two subpopulations using anti-CD(59) monoclonal antibody combined with goat-anti-mouse IgG immunomagnetic beads. CD(3)(+), CD(4)(+) and CD(8)(+) lymphocyte subsets were detected by flow cytometry. In 6 newly diagnosed patients, phenotypes associated with T cell activation such as CD(28)(+)/CD(4)(+) or CD(8)(+) cells, CD(8)(+) CD(38)(+) cells, and HLA-DR(+)/CD(4)(+) or CD(8)(+), and NK (CD(3)(-) CD(16)(+)) cells were detected in the peripheral blood.

RESULTPatients with PNH showed significantly increased CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) ratio as compared with controls (1.22 +/- 0.51 vs 0.86 +/- 0.27, P < 0.05), and the CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) ratio in CD(59)(-) PBMC was higher than that in CD(59)(+) PBMC (2.31 +/- 1.56 vs 0.62 +/- 0.27, P < 0.05). The ratios of CD(4)(+) CD(28)(+)/CD(4)(+) markedly decreased and CD(8)(+)HLA-DR(+)/CD(8)(+) increased.

CONCLUSIONPatients with PNH appear to have abnormalities in their lymphocytes. Increased ratios of CD(3)(+) CD(8)(+)/CD(3)(+) CD(4)(+) and HLA-DR(+) CD(8)(+)/CD(8)(+) lymphocytes as well as declined ratio of CD(4)(+) CD(28)(+)/CD(4)(+) lymphocytes might be involved in the pathogenesis of PNH.

ADP-ribosyl Cyclase ; analysis ; ADP-ribosyl Cyclase 1 ; Adult ; Aged ; Antigens, CD ; analysis ; CD28 Antigens ; analysis ; CD4 Antigens ; analysis ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8 Antigens ; analysis ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Female ; Hemoglobinuria, Paroxysmal ; blood ; immunology ; Humans ; Killer Cells, Natural ; cytology ; immunology ; Lymphocyte Subsets ; cytology ; immunology ; Male ; Membrane Glycoproteins ; Middle Aged ; Receptors, IgG ; analysis