ConA-activated CD4~+CD25~-T Cells Inhibited NK Cell Functions

Innate and adaptive immunity has been considered as a story that begins with cooperation and ends with codependence. The innate immune system is considered as the first line of defense to combat the pathogen. When the innate immune system is unable to contain an infection, the cells of the adaptive immune system step in as a second line of defense. There is ample evidence about the concerted integration between innate and adaptive immune functions in an effort to minimize immunopathology and maximize host defense. Although sufficient immune responses are required to protect hosts from invading pathogens, excessive responses of immune cells could lead to immunopathology. Several of immune system components maintain the balance between immune response and immunopathology. Recently, Kim et al. uncovered that when the innate immune cells attacked the pathogens, the incompletely activated adaptive immune system had already began to monitor and regulate the innate immune response to prevent an overzealous early innate response and severe immunopathology. This finding spurs us to understand whether the completely activated adaptive immunity could influence the innate immunity. Furthermore, some phenomena suggest the potential interaction of adaptive immunity to innate immunity.In the heart-xenotransplant models, NK cells were notably contributed to grafts rejection in the nude rats (without T cells). However, in the wild type rats (containing T cells), although NK cells could also be detected in the graft, it had no influence on the grafts rejection. The phenomenon suggest us that as the the innate immune cells (NK cells) own the impair function as adaptive immune cells (T cells) completely activated. What is the relationship between the immune cells?NK cells are one of the most important innate immune cells for protection against virus invasion and tumor growth and are best characterized by their ability of cytotoxicity towards target cells without preactivated and immunoregulatory cytokines production. An intimate relationship could be observed between NK cells and T cells at the aspect of the development, homing and function. It is now widely accepted that naturally occurring CD4+CD25+ T cells could inhibit NK cell function through TGF-β1, most likely in its membrane-bound form, down-regulating NKG2D receptors on the NK cell surface. In addition to the naturally occurring CD4+CD25+T cell population, peripheral mature CD4~+CD25~- T cells can also be induced to acquire inhibitory function in vitro as well as in vivo. Luis et al. stated that both CD4+CD25+ and CD4~+CD25~- T cells from tolerant mice could mediate suppression, although CD4+CD25+ T cells were more efficient than CD4~+CD25~- T cells. CD4~+CD25~- T cells activated by anti-CD3 monoclonal antibody (MoAb) were able to inhibit the activation and proliferation of bystander CD4+ T cells in a cell contact way, reversible by exogenous IL-2 and anti-Glucocorticoid Induced Tumor Necrosis Factor Receptor (GITR). Peripheral T cells capable to prevent autoimmune diabetes in rats are found in both CD25+ and CD25- subpopulations. These data suggest that T cells with regulatory activities are likely to be extremely diverse, but it is not clear how such diverse regulatory T cells assume common responsibility in regulating productive immune responses, which often involve cells in both adaptive and innate immune systems.In our previous study, using tumor model and mixed lymphocyte reaction, we demonstrated that activated T cells could inhibit NK cells function. Activated CD4+ T cell is the main contributor to the inhibit effect of activated T cells towards NK cells in vitro. Importantly, after depleting nTreg in CD4+ T cell subset, the conventional T still efficiently inhibited NK cell cytotoxicity. Therefore, we hypothesized that CD4~+CD25~- T cell could obtained a certain regulatory quality after activated by ConA, which could also regulate NK cell function.In the present study, we detected the regulatory characters of ConA-activated CD4~+CD25~- T cells, and then we co-cultured preactivated CD4~+CD25~- T cells with NK cells and established the tumor model to demonstrate the influence of activated CD4~+CD25~- T cells on NK cell-mediated cytotoxicity both in vitro and in vivo. The underlying molecule mechanism was also been examined. Our main findings and conclusions of this study are summarized as follows:1. Determination of ConA-activated murine CD4~+CD25~- T cells regulatory characters: Similar as other regulatory T cells, ConA-activated CD4~+CD25~- T cells could significantly inhibit CD4+ T cell proliferation. However, the expression of typical regulator cytokine in ConA-activated CD4~+CD25~- T cells are not as same as previously reported iTreg.1) ConA-activated CD4~+CD25~- T cells could inhibit CD4+ T cell proliferation: CD4~+CD25~- T cells, freshly purified from splenocytes of BALB/c mice using MACS beads were activated with ConA (10μg/ml) for 24h. The preactivated CD4~+CD25~- T cells were fixed by 0.5% glutaraldehyde. Fixed CD4~+CD25~- T cells were then cocultured with freshly isolated CD4+ T cells in the presence of ConA for 72h. Cells were harvested and cell proliferation was assays. The result showed that ConA-activated CD4~+CD25~- T cells could notably inhibit CD4+ T cell proliferation.2) To further characterize ConA-activated CD4~+CD25~- T cell, we examined the mRNA expression of CD103, TGF-β, IL-10, IL-4, perforin, CTLA-4 (cytotoxic T-lymphocyte antigen 4), GITR (glucocorticoid-induced TNF receptor family related protein) and CD39 in naturally CD4+CD25+T cells, na?ve CD4~+CD25~-T and ConA activated CD4~+CD25~- T cells by Real-Time PCR.Real-Time PCR analysis confirmed that all of the targeted molecules could be detected and up-regulated in concanavalin A-activated CD4~+CD25~- T cells despite in different degrees.These appears to be concanavalin A-activated CD4~+CD25~- T cells possessing a unique array of molecules that differs from other regulatory cells.2. ConA-activated CD4~+CD25~- T cells prevent the IL-2–induced proliferation of resting NK cells: To assess whether ConA-activated CD4~+CD25~- T cells could exert a inhibitory effect on NK cells, we cultured the fixed activated- CD4~+CD25~- T cells with resting NK cells in the presence of exogenous IL-2 (100 U/mL). As illustrated in result, ConA-activated CD4~+CD25~- T cells significantly inhibited the proliferation of NK cells under IL-2 stimulation.3. ConA-activated CD4~+CD25~- T cells inhibited NK cell tumor rejection in mouse tumor model: B16 melanoma-bearing SCID mouse model was set up to detect the function of NK cells. By transferring cells and antibody depletion experiments, we confirmed that ConA-activated CD4~+CD25~- T cells could increase the number of metastatic melanoma nodules by inhibiting NK cell function.1) Transferring of ConA-activated CD4~+CD25~-T cells facilitated melanoma metastasis in SCID mice: ConA-activated CD4~+CD25~- T cells were transferred into B16 melanoma-bearing SCID mouse. Ten days later, the number of metastatic melanoma nodules was significantly higher in ConA-activated CD4~+CD25~- T cell treated group than that in control groups. Moreover, the survival rate of the experimental group was lower than that of control groups.2) Facilitation of melanoma metastasis by ConA-activated CD4~+CD25~- T cell was through the suppression of NK cell function: Under the condition of depletion of NK cells, adoptively transferred ConA-activated CD4~+CD25~- T cells as well as na?ve CD+CD25- T cells did not influence on the growth and metastasis of tumor cells.4. Studying the underlying mechanism: ConA-activated CD4~+CD25~- T cell could inhibit NK cell function by Qa-1 molecule.1) As nTregs can regulate T cell immune suppression by the downstream production of adenosine acting via the A2A receptor, and both nTreg and previously reported iTreg produce copious amounts of adenosine to inhibit T cell proliferation, we thus tested whether ConA-activated CD4~+CD25~- T cells did produce adenosine. The result showed that ConA-activated CD4~+CD25~- T cells did not generate meaningful level of adenosine.2) ConA-activated CD4~+CD25~- T cell could not kill NK cell directly: We detected the survival state of NK cells in the coculture system. The results demonstrated that the death rate of NK cells was not increased in the experimental group.3) ConA-activated CD4~+CD25~- T cell inhibited NK cell cytotoxicity in cell-cell dependent way: glutaraldehyde fixed ConA-activated CD4~+CD25~- T cells not only kept the inhibitory capacity on NK cell function, but also possessed stronger suppressive potent on NK cell cytotoxicity than un-fixed ConA-activated CD4+Foxp3- T cells, which suggested that the suppression was cell-cell contact dependent.4) Suppression of NK cell cytotoxicity by ConA-activated CD4~+CD25~- T cells was Qa-1 dependent.We used the anti-Qa-1 antibody to block the molecule on ConA-activated CD4~+CD25~- T cells, and the outcome was that the blocking antibody could counteract the inhibitory effect of ConA-activated CD4+CD25 - T cells on NK cell lytic activity against target cells in vitro. We then administered anti-Qa-1 antibody together with ConA-activated CD4~+CD25~- T cells into SCID mice. The result showed that the impaired NK cell function could be recovered from the suppression caused by adoptive transferring of ConA-activated CD4~+CD25~- T cells after infusion of anti-Qa-1 antibody. This result was further confirmed in the tumor-model, in which the administration of Qa-1 blocking antibody in B16 melanoma-bearing SCID mice could significantly lower the number of metastatic nodules in lung than that in control group.Conclusion: We demonstrate that ConA-activated CD4~+CD25~- T cells can attenuate tumor surveillance by inhibition of NK cell cytotoxicity in cell-cell contact and membrane-bound Qa-1-dependent manner. This ConA-activated CD4+CD25 - T cell population is distinct from previously known natural and induced regulatory T cells according to the different regulatory molecular mechanisms. These findings, i.e., activation-dependent inhibition, have proved a functional regulatory feedback between these two kinds of cells and provided a new bridge between T and NK cells. Of particular concern are the implications for the clinical use of anti-tumor and anti-virus strategies that stimulate T cell responses.