Effect Of CTLA4Ig On T Cells From Bone Marrow In Aplastic Anemia (AA) Mice In Vitro

Part ⅠExpressions of CD28 and CTLA4 on T cells from bone morrow in aplastic anemia mice.Objective: To investigate the expressions and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia (AA) mice,Methods: In vitro bone marrow mononuclear cells (BMMNCs) were activated through being incubated with PHA (15μg/ml). The CD28 and CTLA4 expressions on T cells incubated with or without PHA were analyzed by two color flow cytometry. The expressions of CD28 and CTLA4 significantly increased after PHA stimulation. In the AA mice, the expressions of CD28 with or without PHA stimulation were both higher than that in the normal mice (P<0.01 and P<0.01), but the expressions of CTLA4 with or without PHA stimulation had both no significant difference compared with that in the normal mice (P>0.05 and P>0.05). In the AA mice, there were more activation and activated potential of T cell than the normal, and the abnormal expressions of CD28 and CTLA4 maybe participate in immunological disorder mediated by T cell. Part ⅡT cell specific transcription factors T-bet and GATA-3 contribute to shifted development of T help cell toward type I from bone marrow in aplastic anemia (AA) miceObjective: To investigate the T helper cell predominant differentiation in aplastic anemia (AA) mice and to explore the modulation of T cell specific transcription factors T-bet and GATA-3.Methods: Established model of AA mice. Lymphocytes were isolated from bone marrow incubated with phytohemagglutinin (PHA)(15μg/ml) in vitro. Cytokines IL-2, IFN-γ, IL-4 and IL-10 concentration in bone marrow were detected by ELISA. The gene expression levels of transcription factor T-bet/GATA-3 were assayed by RT-PCR.Results: In AA mice, the levels of IL-2 and IFN-γ were markedly increased but IL-4 and IL-10 were markedly decreased than normal mice. And the gene and protein expressions of T-bet in AA mice were much higher than normal, but GATA-3 were lower than normal.Conclusion: In AA mice, there is a shifted development of Th toward to Th1, which maybe involved in induction of T-cells cytotoxic to hematopoietic stem cell. Furthermore, type I shift of Th cause by the level of T-bet was significantly over expressed and GATA-3 decreased. Part ⅢEffect of CTLA4Ig on T cells which from bone marrow in aplastic anemia (AA) mice in vitroObjective: To study the effect of CTLA4Ig on T cells which from bone marrow in aplastic anemia mice in vitro.Methods: T cells from bone marrow of normal and AA mice (as reaction cells) with same amount of mitocin-C treated DC (as stimulation cells) were co-cultured for 5 days in the presence CTLA4Ig. MTT colorimetry was used to detect allogeneic T cells proliferation. Using ELISA assay to detect the level of allogeneic response T cells cytokine: IFN- γ and IL-4. Co-cultured the above allogeneic response T cells (as stimulation cells) with DC (as reaction cells), using LDH Cytotoxicity Assay Kit detect the cytotoxic activity of CTL.Result: In aplastic anemia the proliferation response of T cells induced by DC and in presence CTLA4Ig was significantly lower than which without CTLA4Ig, the level of INF- γ was markedly decreased and the level of IL-4 markedly increased by CTLA4Ig, the cytotoxic activity was markedly decreased by CTLA4Ig in vitro.Conclusion: In aplastic anemia, CTLA4Ig could inhibit T cells proliferation, down-regulate the Th1 cytokines and up-regulate the Th2 cytokines, and inhibit the cytotoxic activity in vitro, so that induced T cell anergy by blocking B7/CD28pathway.