4-1BB,ICOS Regulate Survivin Expression And Sustain T Cell Survival

The ability of T cells to persist in a functional state for extended periods dictates the strength of immunity and protection. Optimal activation of T lymphocytes at least requires two signals. Signals provided by the interaction of the peptide-antigen-MHC complex with the T-cell receptor (TCR) are called 'signal 1', which control commitment to interleukin 2 receptor (IL-2R) expression and proliferation. Signals delivered to T cells by co-stimulatory cell surface molecules expressed on APCs are called 'signal 2', which are essential for enhancing cytokines production and long-term survival of T cells. The CD28/CTLA-4-B7-1/B7-2 pathway is the best characterized T-cell co-stimulatory pathway and is crucial in T-cell activation and tolerance. In conjunction with signaling through the T cell receptor, CD28 ligation increases antigen specific proliferation of T cells, enhances production of cytokines, stimulates differentiation and effector function, and promotes survival of T cells. However, although the importance of both CD28 and CD154 in primary T cell activation is well established, these molecules appear to be far less important in the generation and maintenance of memory and effector T cell functions. Suggests that during the late phase of immune response, costimulation of CD28 is not essenstail and even not important, and there should exist additional active key costimulatory pathways. ICOS, OX40, 4-1BB are induced many hours or days after antigen exposure, during the late phase of IR, signals delivered by these molecules posses main effect to sustain T-cell survival and promote T-cell function. Here we are interested in the T-cell's long-live response. Under the stimulation of TCR and with the help of CD28 and CD40L, T-cell start proliferation, and then, T-cell still need division to enhance clone expansion. At the same time, T-cell still need inhibit apoptosis to ensure effectiveness. ICOS, OX40, 4-1BB are important for long-live immunity, to determine the downstream molecule regulated by these costimulatory molecules is helpful to understand costimulatory molecules signal transduction and clinical utilization. At 16.5kD, Survivin is the smallest member of the mammalian IAP family. The phosphorylation of Survivin on Thr34 by P34cdc2-cyclin B1 is apparently necessary for its anti-apoptotic activaty. Force expression of Survivin Thr34→Ala (T34A) mutant or use Survivin antisense oligonucleotide can prohibit Survivin function. After T cell activation, an upregulation of Survivin was demonstrated. Maximal Survivin expression was observed after 2-4 days in culture. By day 6, expression was again downregulated. This time course correlates with the expression features of OX40, ICOS and 4-1BB. So we suppose that there must be some correlation in function between Survivin and these co-stimulatory molecules. Under the stimulation of TCR and with the assistance of CD28 and CD40L, T-cell start proliferation, and then, T-cell still need division to enhance clone expansion. At the same time, T-cell still need inhibit apoptosis to ensure effective. Survivin is one of the most powerful anti-apoptotic factor, it also has definite function in cell cycle process, The expression of Survivin will activate CDK2-cyclinE, make Rb phosphorylation, then cells will skip the G1/S checkpoint and enter S phase. Because of the feature of Survivin expression and its duplex function, we guess Survivin is the very molecule we are looking for. There is already evidence suggests that OX40 signal up-regulates Survivin expression and thus sustains T lymphocytes survival, like OX40, ICOS and 4-1BB are both inducible costimulatory molecules, and they are all taking action during late phase of immune response, so we suppose that ICOS and 4-1BB are both take Survivin as their downstream signal, and our studies confirmed it. 1.The wildtype and T34A mutant Survivin recombinant adenovirus and control adenovirus AD-GFP were constructed using the pAdEasy system. The favorite gene was detected by fluorescent microscope and reverse transcription polymerase chain reaction. The titer of Survivin adenovirus was 3.6×1013 U/ml. The adenovirus can mediate the expression of wildtype and T34A mutant Survivin in NIH3T3 cells by infection study. 2.To purify T lymphocytes from human peripheral blood mononuclear cells (PBMCs) using nylon wool cotton column, The T-cell surface mark molecule CD3 was determined by flow cytometry and the purity of T lymphocytes was about eighty percent, it was satisfying result. After conA stimulation, T-cells were sufficient activated, and the costimulatory molecules CD28, OX40, ICOS, 4-1BB were all detected. 3.In order to confirm whether Survivin and OX40, ICOS, 4-1BB have same function,T-cell were activated by ConA stimulation and infected with adenovirus carried Survivin gene. As late-acting costimulatory signals deficient, the T-cells were normal compare to that having costimulatory signals counterparts in proliferation and survival, this finding suggested that Survivin and late-acting costimulatory molecules such as ICOS had similar function in sustaining T cell survival and inhibiting activated-induced-cell-death. 4.In order to confirm whether Survivin and OX40, ICOS, 4-1BB have internal correlation in function, T-cells were activated by ConA stimulation and were given ICOS, OX40 or 4-1BB costimulatory signals, then these T-cells were infected with adenovirus carried GFP alone or GFP and dominant-negative Survivin gene. Compare the two groups, T-cells infected by mutant Survivin adenovirus were deficient in proliferation and display a large apoptotic percentage. This findings indicate that Survivin is the downstream molecular of ICOS and 4-1BB, late-acting costimulatory molecules such as ICOS, OX40, 4-1BB are through up-regulates Survivin expression to sustains T lymphocytes survival. These results confirm our hypothesis: first, without ICOS and 4-1BB, T cells are normal in survival if infected by AD-survivin, then we shown that dominant-negative survivin mutant will inhibit long-term T cell survival even if given sufficient ICOS and 4-1BB signal. In summary, 4-1BB and ICOS regulate Survivin expression and sustain T cell survival.