Proliferation And Anergy Of T Cells Induced By Superantigen SEA In Vitro And In Vivo

Superantigen is a powerful immuno-stimulant, which can stimulate T cells bearing certain TCR vβelements when bound as an unprocessed protein outside the antigenic groove of MHC-Ⅱmolecules. Superantigen-activation of lymphocytes results in cytokines production, proliferation and cytotoxicity, which can be used in the tumor therapy. Tumor vaccines have been studied for several years in our lab. We have constraucted the prokaryotic expression vector of SEA and gained SEA protein with biological activity. We have mixed the MAGE-1-HSP70 fusion protein with superantigen SEA as a new complex protein vaccine (MHS), which was encapsulated in nanoemulsion, and evaluated the vaccine's immune responses and therapeutic effects on tumor. We have also constructed hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene driven by AFP gene transcription- regulatory elements, which could inhibit the growth of Hepa1-6 tumor and increase the survival rates of mouse.Although exposure of mature T cells to superantigen generally induces a marked proliferative response in vivo, this often followed by unresponsiveness of T cells, also called anergy. Anergy is one of mechanisms of tolerance. Such property of superantigen strongly limits its antitumor therapy effect. The mechanisms of superantigen in tolerance have been highly studied. However, the tumor vaccine could not be used at clinic only by the studies of mechanisms, it is also important to know the changes and regulations of the responses of T cells in vitro and in vivo affected by the doses and operation method of superantigen, which have not been reported comprehensively. In this study, we designed the models of anergic T cells in vitro and in vivo, and evaluated the responses and regulations of the induction of T cells induced by SEA stimulations by detecting comprehensively the proliferation and surface molecule expressions and the levels of cytokines.1. Activation and proliferation of T cells stimulated by SEA in vitro and in vivoObjective: to evaluate the regulations and characteristics of proliferation of activated T cells after SEA stimulation in vitro and in vivo. Methods:①Proliferation of spleen cells was detected by MTT assay after SEA stimulation.②surface molecule expressions were analyzed by FACS.③The IL-2 levels were measured with CTLL-2 cells and intracellular IL-2 was analyzed after permeabilized using 0.5% saponin before stained. Results: In vitro, the results showed a strong proliferation after a single SEA stimulation. A dose-dependent relationship was clearly observed and 100ng/ml was the optimal dose of SEA, which could accelerate the proliferation of T cells and could not lead to the early death of the cells. In vivo, we selected i.p. as the optimal method of injection and the optimal dose of SEA was 10μg in our study. The data showed both in vitro and in vivo that the expressions of CD69 and CD25 were markedly upregulated and expressions of CD4 and CD8 increased similarly. At the same time, large amounts of IL-2 were released. Conclusion: a significant activation and proliferation of T cells could be stimulated by SEA both in vitro and in vivo, which was similar to CD4+T cells and CD8+T cells. 2. Anergy of T cells induced by SEA in vitro and in vivo.Objective: to construct the model of anergy of T cells in vitro and in vivo, and evaluate the characteristics of anergic T cells induced by repeated SEA stimulations. Methods:①In vitro, Proliferation of spleen cells was detected by MTT assay after repeated SEA stimulations at 48h interval. In vivo, the expressions of CD3 were analyzed by FACS after repeated SEA injections at different time-intervals.②The expressions of CD4,CD8,CD69,CD25 were analyzed by FACS.③IL-2 and TNF-αlevels were measured with CTLL-2 cells and L929 cells, and IL-4,IL-10 and IFN-γproductions were measured with sandwich ELISA kits. Intracellular IL-2 and IL-10 were analyzed after permeabilized using 0.5% saponin before stained.④The responsiveness of anergic T cells induced in vitro and in vivo was measured by MTT. Results:①There was a maintained expansion in response to SEA twice stimulations. In vitro, 100ng/ml was the optimal dose of SEA. The twice stimulations induced a different increase between the two T subsets and the up-regulation of CD8+ T cells appeared a dominant position. The expressions of CD25 were going up, while CD69 expressions didn't appear upregulation. Productions of IL-4 and IL-10 increased markedly. In vivo, the effects on expression of CD4+ and CD8+ T cells were different at different time-intervals between SEA injections. The 3-days intervals led to an obvious increase of CD4, not to the increase of CD8; while the increase of CD4 and CD8 was both seen markedly at 28 days intervals. The expressions of CD25 were seen markedly regulated in both time-intervals. The maintained level of IL-2 was seen and productions of IL-10 increased markedly.②the unresponsiveness could be induced after three SEA stimulations in vitro and in vivo. In vitro, there was no proliferation of T cells after third SEA stimulation. No up-regulations of CD4 and CD8 expressions were seen and the expression of CD69 decreased significantly. The CD25 expression only kept high levels during 48h, and then gradually decreased. Only IL-10 productions still increased. In vivo, It was CD4+T cells that were reduced markedly after three SEA injections at 3 days interval. The expression of CD25 was not increased, which kept high levels during 3 days after last injections. At 28 days interval, the expression of CD4 was not seen markedly up or down, and the CD25 expression was low. Only IL-10 production increased markedly after injections.③The responsiveness of anergic T cells induced in vitro could be seen after 21 days in culture. In vivo, faint responsiveness of anergic T cells induced by SEA injections at 3 days interval and at 28 days interval could be seen respectively after 21 days and 28 days. Conclusion:①the model of T cells anergy were constructed successfully in vitro and in vivo. The responsiveness of anergic T cells could be restored gradually after some times.②there were still expansion of SEA-reactive T cells after SEA re-stimulation. In vitro, the second stimulation mainly resulted in the proliferation of CD8+T cells. In vivo, it was firstly proved that only the proliferation of CD4+T cells was induced by twice SEA injections at a short interval, while the proliferation of both subsets was induced by twice SEA injections at a long interval.③there was no relationship between the induction of anergy and the time-interval. However, the time of interval affected the clonal deletion. the time of interval was shorter, the more amount of CD4+T cells were deleted..④Although the high expression of CD25 was related with the induction of anergy, the unresponsivnession of T cells could be induced in the state of low expression of CD25.⑤IL-10 was related to the induction and maintenance of anergy.3. CD28/CTLA-4 expressions during induction of T-cell anergy in vitro and in vivo.Objective: to evaluate characteristics of CD28 and CTLA-4 expressions in induction of T cells anergy in vitro and in vivo. Methods: the spleen cells were stimulated with SEA single or repeatedly in vitro and in vivo. Cells were collected and analyzed for the expressions of CD28 and CTLA-4. Results: Expression of CD28 was significantly upregulated after single SEA treatment, with low expression of CTLA-4. Twice SEA stimulations led to increases of CTLA-4, while upregulation of CD28 was also seen. The expression of CTLA-4 and CD28 were gradually down after three SEA stimulations. Conclusion: The downregulation of CD28 and upregulation of CTLA-4 accelerated the induction of anergy, and CTLA-4 might have no relation with the maintenance of anergy.