| Background: Immune response is in the focus of the control of virus infection, tumor growth, autoimmune disease, organ transplantation and so on. Recently, interest has been shown in understanding the immunoregulatory of the immune net. While considerable study has been focused on the innate shaping of the adaptive immune response, only modest attention has been paid to the adaptive influencing the innate immune system beyond well-characterized antibody–Fc receptor interactions. To reveal this mechanism could be very helpful for understanding the immune balance, providing a new link between innate immunity and adaptive immunity, and increasing outcome of immuno therapy against tumor and virus infection.In our previous study on thymus transplantation to establish immune tolerance model, we have noticed a very interesting phenomenon. Briefly, the athymic nude mice that lack mature T cells are able to reject allo- and concordant xeno-skin grafts, and NK cells have been believed to be involved in, although the rejection time was significantly delayed. However, if the neonatal mouse thymus was grafted into nude mouse, the corresponding skin graft could be accepted for lifetime. It has been well known that there is no mature T cell in nude mouse before thymus grafting, and there are mature auto-tissue antigens tolerized T cells after thymus grafting. That implies if there is no mature T cell, NK cells will destroy allo-skin grafts. While after neonatal allo-thymus grafted, NK cells do not attack allo-skin grafts. The quite possible interpretation for this phenomenon we propose is that mature auto-tissue antigens tolerized T cells might be able to inhibit NK cell activity and let the recipient accept skin graft.The inhibition of T cell activated by antigen or mitogen, partial-activated or full-activated to NK cell functions had been demonstrated in our group. Moreover, the suppressive effects of T cell on NK cell is APC-independent. We know that T cell population is consisted of different kinds of subsets. What kind of subset of T cells is responsible for this inhibition of NK cell cytotoxicity? We proved that CD4~+T cell is the main subunit cell that affects the function of NK cell. Naturally occurring CD4~+CD25~+ T cells are considered as the main regular cell that possess wide range of regulatory activities, Then, is the inhibition of NK cells by CD4~+T cells triggered by na?ve Treg cells? We therefore investigate the role of CD4~+ T cells and their subgroup in NK cell activity in vitro and in vivo. This work could be very helpful for understanding the immune balance, providing a new link between innate immunity and adaptive immunity, and increasing outcome of immuno therapy against tumor and virus infection. MethodsPreparation of different kinds of cellsAll kind of cells in our experiments was obtained from Balb/c mouse spleen. CD4~+ T cells, CD4~+CD25~+T cells, CD4~+CD25-T cells, and NK cells were acquired by means of magnetic cell separation as described in the Instruction of Kit. Flow cytometric analysisFor determination of the purity of the isolated cells, either PE-labeled or FITC-labeled antibodies against CD3, CD4, CD25, CD49b was used for CD4~+ T cells, CD4~+CD25~+ T cells, CD4~+CD25- T cells and NK cells, respectively. Then, the expression of above markers on labeled T cells and NK cells was used for the determination of the purity as measured by FACS.Intracelluar Foxp3 protein levels were detected by using Mouse Regulatory T cell Staining Kit according to the manufacturer's instructions by FACS. The effect of activated CD4~+ T cells, CD4~+CD25~+T and CD4~+CD25-T cells on the NK cell cytotoxicityIsolated CD4~+ T cells, CD4~+CD25~+T cells and CD4~+CD25-T cells were activated by ConA in vitro for 24h, followed by adding NK cells into the culture. T cell proliferation was determined by measuring 3H thymidine incorporation. The cytolytic activity of NK against YAC-1 was determined in a 4h 51Cr release assay. The blockage of TGF-β1 and CD25 moleculaes in vitroTo test whether TGF-β1 and CD25 molecules involved in the interaction between CD4~+T cells and their subgroups and NK cells, antibody against TGF-β1 or CD25 was added into the growth culture medium 24 hours or 48 hours after stimulation of CD4~+ T cells and their subgroups at a final concentration of 10μg/ml. After 4 hours of adding antibody, NK cells were added to the wells to co-culture with activated CD4~+ T cells and their subgroups.Animal experimentThe isolated Balb/c mouse CD4~+CD25- T cells were incubated either with ConA (at a final concentration of 10μg/ml for 24-hour or 48-hour) or without ConA. Then, total 1.5×106 either of 24-hour-Con-A activated Balb/c mouse CD4~+CD25- T cells or of non-activated Balb/c mouse CD4~+CD25- T cells and the same volume normal sodium were injected via tail vein into Balb/c nude mouse. Twenty hours later, total 1.5×106 either of 48-hour-Con-A activated Balb/c mouse CD4~+CD25- T cells or of non-activated Balb/c mouse CD4~+CD25- T cells and the same volume normal sodium were injected via tail vein into Balb/c nude mouse again. Another 24-hour, spleen cells from injected Balb/c nude mouse were harvested, and the cytotoxicity of splenocytes to YAC-1 cells were tested as described above.Results:1. ConA-activated CD4~+T cell is the main contributor to the inhibit effect of activated T cells towards to NK cells in vitro We showed that under the condition of our experimental system, ConA-activated CD4~+ T cells acquired the inhibitory capacity after 24h, 48h, 72h, and 96h NK cell-activated T cell co-culture system. The inhibition rates of NK cell cytotoxicity were 25%,31.6%,34.7% and 46.5% respectively.2. CD4~+CD25~+T cells can inhibit NK cells founction Both na?ve and ConA-activated CD4~+CD25~+T cells can significantly inhibit the cytotoxicity of NK cells and the inhibition rates were 50.8% and 49.1% at 24h co-culture system, 19% and 20.9% at 48h. 3. ConA-activated CD4~+CD25~-T cells can inhibit NK cells cytotoxicity both in vitro and in vivoWe here for the first time to demonstrated that ConA-activated CD4~+CD25~-T cells ,not naive CD4~+CD25~-T cells, could significantly suppress NK cell cytotoxicity in vitro. The corresponding inhibition rate of NK cells by ConA-activated CD4~+CD25~-T cells were 31.7% and 25.5% after 24h and 48h NK cell-activated T cell co-culture system. Cytotoxicity of splenocytes harvested from spleen of injected Balb/c nude mice was significantly suppressed against the mouse NK target cell line YAC-1, as compared with controls. The corresponding inhibition rate of splenocytes cytotoxicity by injected ConA-activated CD4~+CD25~- T cells was 60%.4. TGF-βand CD25 play different role in the inhibitory action of different subsets An antibody against TGF-β1 was used for neutralizing TGF-β1 in our experiment. Surprisingly, administration of antibody against TGF-β1 to the co-culture system of both NK cells-activated CD4~+T cell rather than attenuates the inhibitory effect of ConA-activated CD4~+ T cell on NK cell activity, but enhance the inhibitory function. After co-cultured 24h, 48h, 72h and 96h, the inhibition rate were from 25%, 31.6%, 34.7% and 46.5% up to 63.8%, 59.43%, 60.7% and 90.9% respectively. The same result was required in the NK cells-activated CD4~+CD25ˉT cell co-cultural system. In detail, the inhibition rates were from 31 .65% and 44.56% up to 47.26% and 68.24% after co-cultured 24h and 48h. in the NK cells-naive CD4~+CD25~+T cell co-cultural system, the inhibition was reversed by anti- TGF-β1 antibody, but in the NK cells-activated CD4~+CD25~+T cell co-cultural system, the inhibition rate were changed from 19% (24h), 49.1%(48h) to 31.9%(24h) and 35.9%(48h).Antibody against CD25 was used for testing whether CD25 molecule on CD4~+CD25~+ T cell and CD4~+CD25~- T cell membrane was involved in the suppression of NK cell activity. The result showed that the inhibition of NK cells by CD4~+CD25~+ T cells both na?ve and activated is completely reversed and the function of NK cells by CD4~+CD25~- T cells stimulated by ConA is partly recovered, the inhibition rate were changed from 31 .65%(24h),20.64%(48) to 25.2(24h) and 16.3%(48). 5. The expressionof Foxp3 on ConA-activated CD4~+CD25~-T cells.CD4~+CD25– T cells isolated from mouse (Balb/c) splenocytes were cultured in presence or absence of ConA. T cells were collected at 48h. CD4~+CD25– T cells activated by ConA, do not express Foxp3 despite the expression of CD25 on the cell surface.Conclusion:We show that ConA- activated CD4~+ T cells, CD4~+CD25~+T and CD4~+CD25ˉT cells can significantly inhibit NK cells cytotoxicity in vitro and ConA- activated CD4~+CD25~- T cells can also significantly inhibit NK cells cytotoxicity in vivo. TGF-βand CD25 plays different role in different co-cultural systems. We first stated that ConA-activated CD4~+CD25~- T cells acquired regulatory actions without expression of Foxp3 and high expression of CD25.
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