A Preliminary Research On The Inhibition Mechanism Of Tolerant T Cell To NK Cell In Vitro With Co-stimulating-blocking Models

T lymphocyte plays a key role as executive and regulative cells in cell immunity. In the study of cellular immune rejection of allo-transplantation, T cell is the most important target. But study showed that the deletion of T cell did not induce lifetime tolerance. On the otherhand, various evidences indicated that NK cell played a very important role in the graft rejection. When anti-NK1.1 Mab is applied to remove NK cell before organ transplantation, the xeno-graft can survive longer remarkably. Obviously, the induction of tolerance on T cell alone is not enough to completely overcome graft rejection. In order to induce long-term or even lifetime immune tolerance, we should take into account of other factors which participate in the rejective reaction , such as MF,NK cells, etc. As an important constituent of innate immunity, NK cell is one of the cells firstly appearing in graft, and is also an important effect cell of specific cell immunity. The functional condition of NK cell has a close relationship with the result of organ transplantation. As one of the main cells in immune response, NK cell participates in the regulation of immune response, but its functions are affected by many factors. This project was supported by the National Natural Science Fundation (30200261). We mixed thymus of fetal Balb/c mice with thymus from fetal rats (F344), and then transplanted it to Balb/c nude mice, through which we successfully established a heterogenous tolerance model. When we carried out skin xenotransplantation on Balb/c nude mice (F344-nude mice), the grafts were rejected in about 20 days. Meanwhile, the same experiments on the model mice, which previously received transplantation of the mixed thymus, the survival time of xenografts was prolonged remarkably. Obviously, there should exist a thymus-independent way in thyms-defected nude mice. For example, NK cell and other cells may reject the graft and be inhibited by donor-tolerant T cell. Some documents mentioned that, in model of thymus transplantation tolerance and animal model of transplantation tolerance induced by blocking CD28/B7 costimulatory signal, the cytotoxic effects of NK cell would be inhibited in a long time. What is the connection of the immune inhibition of NK cell with the tolerance of T cell? Immune rejective reaction is a very complicated event. In this event, in addition to T lymphocyte that occupies the principle position, NK cell also has very important function. Previous studies indicated that tolerant T cell could inhibit or interfere the function of NK cell. So we choose NK cell as a target cell in our study and use anti-CD80 antibody to blockade CD28/B7 signal pathway to establish a T cell tolerance model in vitro . Base on this model, we try to explore the possible regulatory effects and mechanism of that how the tolerant T cell effect on NK cell function, including the effect of various T cell subunit, the influence of APC cell and possible its molecular mechanisms. This not only facilitates us to further elucidate the tolerant mechanisms of T cell and NK cell, but also provides new ideas to control transplantation immunological rejective reaction, which significantly help us to further understand the relationship between acquired immunity and innate immunity in organ transplantation. Our main findings and conclusions of this experiment as following: 1. Harvest of experimental cells: we use nylon wool column to separate T lymphocyte. CD4+T cell and NK cell is isolated by MACS magnetic beads. The respective pick-up rates of these cells are: 10%, 48.9%, 3.42%; the purity coefficients respectively get to 90.04%, 96.62%, 86.3%. 2. The establishment of costimulatory blockage tolerant model in vitro: APC: T=1:3,total volume is 200μl with antigen is added until the terminal concentration reaching 10mg/ml and terminal concentration of anti-CD80 antibody being 10mg/ml, we can successfully induce T cell's low response to specific antigen. 3. It was found first time in vitro that both antigen specific tolerant and activated T cells can inhibit NK cell's functions efficiently by cells contact.. To investigate the inhibition mechanism of NK cell functions under tolerant condition. CD28/B7 passage of co-stimulating-blocking model of T cell under tolerant condition was blocked by anti-CD80 antibody in vitro. YAC-1 cells were labeled with 51Cr as the target of NK cells. 51Cr standard 4h release experiment was conducted. The inhibiting effecti on NK cells were observed after the co-incubation of tolerant T cell andNK cell. The release rates of 51Cr were 21%, 24.4%, 34% and 31.6% in tolerance-induced group (TI, APC+ T+Ag+AntiCD80+NK), antigen-activated group (AA, APC+T+Ag +NK), cell control group (CC), and cell control group without T cells (NT) respectively. In TI, AA and CC groups, the 51Cr release rates of target cells were 34%, 31.6%, and 33.1% respectively, after the incubation of supernates with NK cells. the 51Cr release rates of TI and AA groups were lower than those of the other groups (P<0.01). No significant difference was found between the 51Cr release rates of TI and AA groups or among group NT and supernates of other groups. These results suggested that not only tolerant T cell but also the antigen activated T cell can inhibit cytotoxic activity of NK cell by cells contact, and the two kinds of T cells might share the same mechanism. 4. The suppressive effects of antigen-induced T cell on NK cell is APC-independent After T cell had been induced by antigen. APC was removed with L-leucine-methylester( terminal concentration: 10mmol/L) and standard 4h 51Cr release experiment was applied to detect the functional of NK cell. The results showed: the induced T cell with or without APC did not have remarkable suppressive effects on NK cell. APC, which induced with antigen, alone didn't have effects on NK cell and also cannot affect NK cell through T cell. No reports are found on this finding. 5. CD4+T cell is the main subunit cell that affects the function of NK cell, and there exist CD4+CD25+ Treg-independent way. In our experimental system in vitro, T cell is a mixture of different subunit. We studied the effects of CD4+CD25+ T, CD4+CD25-T and CD4-T on the functions of NK cell respectively. It was found that: CD4+CD25+ T cell had remarkable inhibitory effects on NK cell. CD4+CD25-T cell induced by antigen also had remarkable inhibitory effects on NK cell, but CD4-T cell had no effect on NK cell. No similar study is found. 6. Although the main mechanism that NK cell be inhibited by activated T cell is cell contact, there may exist some different mechanisms accord with the different activators. We mentioned the inhibitory effects of antigen-activated T cell on the cytotoxicity of NK cell. We found that whether using Anti-CD80 or not to carry out constimulatory blockage didn't affect inhibition ability of T cell on NK cell. Is antigen the prerequisite on inhibition of NK cells?Anti-CD3, and then the inhibitory capability of T cell on NK were studied dynamicly. Results are: twenty-four hours later after T cell stimulation, T cell from ConA or anit-CD3-stimulated group could suppress the activity of NK cell. Forty-eight hours later, T cell in both tolerance-induced group and antigen-activated group showed remarkable inhibitory effects to NK cell functions. However, supernatants from 24hr, 48hr and 72hr group had not remarkable effects on function of NK cells. It is clear that activated T cell can inhibit NK cell's functions through cell contact. The results also tell us that polyclonal activators had quick effects on T cell, and the effects lasted a long time, at least 96hr.Mean while, T cell of tolerance-induced group and antigen-activated group showed remarkable inhibitory effects to the functions of NK cell about 48h after induction. We also found that after 96h, supernatant of both groups could promote the kill ability of NK cell. Obviously under the effects of different activators, T cell can use different mechanisms to regulate the behavior of NK cell. 7. The expression of bc-1 and bc-2 of mice can be produced by the induction of antigen, which is the important factor that antigen-induced T cell inhibits the functions of NK cell. The expression of bc-1 and bc-2 of mice is the possible molecular basis that T cell inhibits the activity of NK cell. Bc-1 and bc-2 are atypical MHC-I molecules that are expressed by embryo-sac or placental trophoblastic cell of mice. Like human HLA-G, people generally think these molecules can inhibit the functions of NK cell. Usually mature lymphocytes don't express these molecules. RT-PCR was used to detect the bc-1 and bc-2 mRNA of T cell stimulated by ConA, Anti-CD3 and allogeneic lymphocyte antigen. F9 cell strain was used as the positive controler. We can detect bc-1 and bc-2 in T cell on 48h after the stimulation of antigen but not on other phases and under effects of other polyclonal cell activators. These results suggested that bc-1 and bc-2 might be one of the main factors that antigen-induced T cell inhibits the functions of NK cell. 8. Because T cell activated by ConA and Anti-CD3 doesn't express bc-1 and bc-2,there should exist bc-1 and bc-2 independent ways that T cell down regulates the NK cell cytotoxicity.