Activation of Human T Lymphocytes by Crosslinking of Anti-CD3 Monoclonal Antibodies
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Abstract The proliferation of human T lymphocytes induced by anti-CD3 monoclonal antibodies (mAb) is used as a model for antigen-induced activation via the T cell receptor-CD3 complex. Since both systems are accessory cell (AC)-dependent, an understanding of the role of AC in anti-CD3-induced proliferation may provide an understanding of physiological activation via the T cell receptor. Previous work has implicated receptor crosslinking as an important AC function. To determine its necessity in anti-CD3-induced lymphocyte proliferation, we prepared highly purified T lymphocytes and found that these cells did not respond to the anti-CD3 mAb UCHT1, either alone or with interleukin 1 (IL1), interleukin 2 (IL2), or tetradecanoyl phorbol acetate (TPA). However, the response, as measured by appearance of IL2 receptors and proliferation, was restored by crosslinking with immobilized goat anti-mouse antibodies (GAM) and did not require the addition of IL1, IL2, or TPA. Thus, crosslinking of CD3 receptors was a sufficient signal for proliferation of these cells. Cyclosporine A (CsA) inhibited the activation induced by immobilized UCHT1. Since macrophages are the principal targets of CsA-mediated suppression of mitogen-induced proliferation, but macrophages do not participate in the response to immobilized anti-CD3, this may indicate that CsA was inhibiting crosslinking or a signal generated by it.Abstract Nonfractionated peripheral blood lymphocytes from patients with systemic lupus erythematosus (SLE) showed enhanced proliferative responses when stimulated via the CD3 pathway. In contrast, proliferative responses induced by phytohemagglutinin were diminished in SLE patients. Levels of CD3‐induced interleukin‐2 production and interleukin‐2 receptor expression were comparable with normal levels. Highly purified T cells also showed augmented CD3 responses, but only in the presence of phorbol myristate acetate or a combination of phorbol myristate acetate plus calcium ionophore A23187, and not with calcium ionophore alone. The data suggest integrity of the T cell receptor/CD3 pathway for T cell activation in patients with SLE, as examined in cultures stimulated with specific anti‐CD3 monoclonal antibodies rather than with multivalent lectins. An increased response via the CD3 complex could contribute to the autoimmune activity in human SLE.
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Background : TotalT lymphocytes can be measured by CD3-fluorescein isothiocyanate (FITC)/CD4-phycoerythrin (PE) and CD3-FITC/CD8-PE. The difference in the CD3 percentages between these two determinations was evaluated. And, we characterized the T lymphocytes subset using the monoclonal antibody that detectsT lymphocytes receptors. Methods : TheT lymphocyte subset assay was performed on 221 samples. A two-color direct immunofluorescence flow cytometric assay was done using a Simultest IMK-Lymphocyte kit (Becton- Dickinson, San Jose, CA, USA). If the difference between the CD3 determinations were greater than 3%, the entire procedure was reviewed and the flow cytograms were reanalyzed. In 71 among 221 samples the proportion ofT lymphocytes was determined. Results : The difference between the CD3-FITC/CD4-PE tube and CD3-FITC/CD8-PE tube was 3.0%, 3.6%, 3.0%, 3.4%, and 2.4% in normal subjects, patients with chronic liver disease, patients with cancer, patients with other diseases, and children, respectively. The between-tube differences for CD3 exceeding 3% were found in 69 samples (31.2%). The proportion ofT lymphocytes was 0.81%, 2.46%, 2.50%, and 0.85% in normal controls, patients with chronic liver disease, patients with cancer and patients with other diseases, respectively. No correlation betweenT lymphocytes and T lymphocytes was observed. Conclusions : The reproducibility of the totalT lymphocytes should be improved because of the between-tube difference exceeding 3% in about one third of the cases. Additionally, T lymphocytes were composed of heterogeneous subsets includingT lymphocytes and their proportion might be considered to be related to individual variation.
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Mixed lymphocyte reaction
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TCR/CD3 receptor complex plays a central role in antigen recognition and T cell activation. Therefore, the present study investigates TCR alpha/beta (TCR-1) and CD3 receptor density (RD, number of receptors per cell) on uremic CD4 T lymphocytes in relation to T cell proliferative response induced by anti-CD3 monoclonal antibodies (mAb). The influence of uremic serum on TCR/CD3 receptor expression of normal and uremic CD4 T lymphocytes was evaluated as well. We found, that: a) percentage of TCR-1 and CD3 positive cells of freshly isolated CD4 T lymphocytes is the same in controls and ESRD-patients, but the TCR/CD3 RD is lower on uremic CD4 T lymphocytes, b) Incubation for 24 h with uremic serum lowers TCR/CD3 RD on normal and uremic CD4 T cells, c) There is positive correlation between TCR/CD3 RD and anti-CD3 induced lymphocyte proliferation. These data might also support the hypothesis that blunted T-cell response to antigen in uremia is due to down-regulation of the TCR/CD3 receptor complex by uremic milieu.
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Specific lysis of allogeneic cells after activation of CD3- lymphocytes in mixed lymphocyte culture.
Human CD3- lymphocyte populations were obtained by treating peripheral blood lymphocytes with mAbs directed to CD3, CD4, and CD8 surface antigens. The resulting populations were cultured with irradiated allogeneic cells; at day 4, 100 U/ml IL-2 were added and cultures continued for an additional 10 d. The resulting populations were CD3-CD2+CD7+ and displayed cytolytic activity against PHA-induced blast cells bearing the stimulating alloantigens but not against autologous or unrelated allogeneic blast cells. When CD3- populations were cultured with irradiated autologous cells, no cytolytic activity could be detected either against autologous or allogeneic blast cells. On the other hand, K562 target cells were lysed by both MLC-derived CD3- cell populations regardless of the origin (autologous or allogeneic) of the stimulating cells. CD3- clones were further derived from MLC-stimulated CD3- populations. These clones displayed a cytolytic pattern similar to the original MLC populations as only specific PHA blasts could be lysed. These clones did not express detectable surface TCR-alpha/beta or -gamma/delta molecules and lacked productive mRNA for TCR alpha and beta chains, while small amounts of TCR-gamma mRNA were detectable in one of four clones tested. Also mRNA for CD3 gamma and delta chains were undetectable in all clones, however, CD3 epsilon mRNA was consistently present.
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Objective To investigate the change of T lymphocyte subsets in peripheral blood from patients with Sjgren's syndrome(SS) and its clinical significance.MethodsT lymphocyte subsets were determined by two-color flowcytometry with various monoclonal antibodies.Erythrocytesedimentation rate(ESR) and serum IgG were used to classify the disease activity.The relationship between T lymphocyte subsets and disease activity was analyzed.ResultsPercentage of CD3+ T lymphocyte,CD3+ CD4+ T lymphocyte,CD3+ CD8+ T lymphocyte and the ratio of CD3+ CD4+/CD3+ CD8+ did not significantly differ between in SS and the control healthful subjects.The percentage of CD3+ T lymphocyte and CD3+ CD8+ T lymphocyte significantly decreased but the ratio of CD3+ CD4+/CD3+ CD8+ increased in patients with active SS.Conclusion The percentage of T lymphocyte subsets might not be abnormal in patients with SS,but could relate to SS activity,which may play a role in the development of SS.
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We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.
Jurkat cells
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Objective:To study the relationship between the expression of T lymphocyte subtypes and alopecia areata(AA).Methods:CD4+、CD8+ CD3+ T lymphocytes were sorted out by flow cytometry.Results:The expression of CD4+ and CD8+ lymphocytes in patients with AA was significantly increased than that in controls.The expression of CD3 lymphocytes and the ratio of CD4+/CD8+ in AA were slightly increased compared with controls,but the difference was not significant.Conclusion:The study indicates that AA is a T lymphocyte mediated tissue-restricted autoimmune disease.CD4+ and CD8+ T lymphocytes play an important role in the pathogenesis of AA.
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A mAb, 10D1, was obtained by fusing spleen cells from BALB/c mice immunized with a CD3/TCR- human T cell line, P12/ichikawa, to mouse myeloma cells, P3X63-Ag8-653. 10D1 mAb is specific for T cells in that it reacted with all the T cell lines tested, but not with B or myeloid cell lines. A small fraction of normal peripheral blood T cells, preferentially CD4+, was also reactive with 10D1 mAb. Biochemical studies revealed that 10D1 mAb recognizes a disulfide-linked homodimeric molecule composed of 90-kDa polypeptide. 10D1 mAb induced a substantial proliferation of peripheral blood T cells when cross-linked with goat anti-mouse Ig antibody. The elimination of CD4+ cells totally abrogated the proliferative response induced by 10D1 mAb, whereas the elimination of CD8+ cells rather enhanced it. The proliferative response of peripheral blood T cells induced by 10D1 mAb was almost completely inhibited after modulation of the CD3/TCR complex with anti-CD3 mAb. In addition, a prompt increase in intracellular [Ca2+] was observed in a CD3+ T cell line, Jurkat but not in its surface CD3- mutant when 10D1 mAb was added. These results indicate that the 10D1 molecule is involved in a novel pathway of human CD4+ T cell activation, which is associated with the CD3/TCR-mediated pathway.
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To investigate the expression of T-lymphocyte subsets (CD4+, CD8+ and CD45RO+ cells) in human recurrent nasal polyps.Nasal polyps tissue samples and peripheral blood were obtained from 17 patients, normal human inferior turbinate mucosa and peripheral blood were obtained as well. Flow cytometry was adopted to detect the expression of surface markers of the T-lymphocytes. All data were analyzed with t-test.There were significantly large number of CD4+, and CD45RO+ cells in the tissue of recurrent nasal polyps. The expression percentage of CD3+ CD4+ cells was significantly higher than that of CD3+ CD8+ cells. Ratio of CD3+ CD4+/CD3+ CD8+ was 1.956 +/- 0.093.There were generous of T-lymphocytes expression in human recurrent nasal polyps. The ratio of the T-lymphocytes subsets was abnormal high than the usual and this indicated the immunological function were in disorder in the local area. The abnormally higher expression of CD3+ CD4+ cells in the local area may play an important role in the recurrence of human nasal polyps.
Nasal Polyps
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