The Role Of CTLA4Ig In The Regulation Of NK Cell Function And The Management Of Burn Injury

Patients who suffer from massive burn injury are hyporesponsive and susceptible to infection (1, 2), while the rejection towards skin graft is severe. Therefore, maintenance of long-term skin graft survival without increasing incidence of disseminated infection is the key problem to be solved in treatment of massive burn patients.NK cells, as the most important cells in innate immune system, play a considerable role in tumor surveillance and anti-infection. Whether NK cells are activated or not is mostly dependent on the balance between the inhibitory (i.e.NKG2A) and activated (i.e.NKG2D) receptors expressed on the cell surface. However, NK cell function is proved to be severely suppressed after massive burn, which leads to susceptibility to infection. It is believed that nosocomially-acquired resistant Pseudomonas aeruginosa infection is the major cause of infection and sepsis after burn injury(3). Pseudomonas aeruginosa exotoxin A is found to be able to inhibit NK cell-mediated cytotoxicity and IFN-gamma synthesis (4). Furthermore, it has been 10 years since Pseudomonas aeruginosa was identified, while unfortunately, it became multidrug resistance (5). However, it is demonstrated that NKG2D is critical for NK cell activation in host defense against Pseudomonas aeruginosa respiratory infection (6, 7). Accordingly, we are very interested in the question that whether the enhancement of NK cell-mediated cytotoxicity can decrease the incidence of disseminated infection.Recombinant protein CTLA4Ig has been shown to be able to induce immune tolerance. The necessary co-stimulatory signal for T cell full stimulation is provided by ligation of CD28 with either B7-1 or B7-2 molecules expressed on antigen-presenting cells and can be blocked by a soluble form of CTLA4Ig which binds with high affinity to both B7-1 and B7-2(8). Recently, selective immunomodulatory agents such as Belatacept and Abatacept have shown great promise in treatment of rheumatoid arthritis and promoting allograft survival (9-12). Additionally, not only our previous research (13), but also other work(14) have proved that systemic or local administration of recombinant adenovirus CTLA4Ig can promote skin allograft survival. Nonetheless, CTLA4Ig utilization in promoting skin allograft survival in massive burn patients is rarely because of the consideration of the side-effects, especially disseminated infection.Abatacept is a fusion protein composed of a mutant immunoglobulin fused to the extracellular domain of CTLA-4, a molecule capable of binding B7. It is licensed by FDA for the treatment of rheumatoid arthritis. One interesting aspect of CTLA4Ig is that patients treated with this fusion protein experience much lower incidence of tumors and infectious episodes than the prediction (15-17). Meanwhile, Abatacept does not impair the ability of mice to control a chronic M tuberculosis infection(18). However, the corresponding mechanism of these phenomenons has not yet been determined. Grohmann et al. found that CTLA4Ig could influence on the antigen presenting cell function via the interaction with B7 molecules on APC (19), which means CTLA4Ig is possibly able to regulate all the cells that expressed CD80/CD86 molecules. Moreover, recent studies demonstrated that resting NK cells can express CD86, and activated NK cells express both CD80 and CD86 receptors(20, 21), which prompted us to study the possibility role of CTLA4Ig in NK cell function. Beissert S et al. found that Chronic UV exposure of CTLA4Ig transgenic mice resulted in significantly reduced numbers of skin tumors, when compared to control mice (22). Taken together, previous data showed that CTLA4Ig could inhibit immune rejection of skin grafts without increasing the infection morbidity via blocking co-stimulatory pathway, meanwhile,CTLA4Ig might regulate the functions of CD80/CD86 positive cells such as NK cells that play an important role in control of infections and in tumor cell clearance (23). Thus it prompts us to determine the questions as to: 1,whether the ligation of CTLA4Ig and B7 molecules expressed on NK cell surface leads to the enhancement of NK cell-mediated cytotoxicity and clearance of tumor cells and bacteria.2, what the underlying mechanisms are. 3, whether the regulation of CTLA4Ig on NK cell-mediated cytotoxicity can explain CTLA4Ig systemic administration does not increase the morbidity of infections and tumorogenesis.In the present study, we detected the possible roles of CTLA4Ig in the regulation of NK cell function both in vitro and in vivo, and in the models of Pseudomonas aruginosa infection, scald injury and B16 melanoma metastasis. The corresponding molecular mechanisms were also been examined. Our main findings and conclusions of this study are summarized as follows:1. CTLA4Ig and Abatacept are strong activator of NK cells:1) CTLA4Ig could enhance PBMC or purified human NK cell function in vitro:In order to determine that whether CTLA4Ig can regulate PBMC or purified human NK cell function in vitro, we used FACS to detect the cytotoxicity after administration of different concentrations of CTLA4Ig. In our study, we found that:Ⅰ.CTLA4Ig significantly enhanced PBMC or purified human NK cell cytotoxicity to NK cell-sensitive target cells (K562 cell line) in vitro when its concentration was more than 1μg/ml.Ⅱ.The enhanced cytotoxicity was comparable to IL-2 that has long been identified as the T cell and NK cell stimulator.Ⅲ.PBMC or purified human NK cell cytotoxicity did not change significantly with the increase of CTLA4Ig concentration in our experiment. These data indicated that CTLA4Ig can enhance NK cell-mediated cytotoxicity in vitro, which is comparable to IL-2 and the enhancement did not change significantly with the increase of CTLA4Ig concentration in our experiment.2) CTLA4Ig (Abatacept) could augment NK cell-mediated cytotoxicity of mice in vivoTo determine that whether the augmentation of Abatacept on NK cell-mediated cytotoxicity was related to the phenomenon that systemic administration of Abatacept did not significantly increase the incidence of tumor and infection, we injected Abatacept into mice via tail vein at a dose higher than clinical utilization and then detected the splenetic NK cell-mediated cytotoxicity. It was demonstrated that administration of Abatacept via mouse tail vein could significantly increase NK cell cytotoxicity as compared with PBS at 24 and 48 hours after injection. These data indicated that CTLA4 molecule could significantly enhance NK cell-mediated cytotoxicity in vivo. It might be the major reason that administration of Abatacept did not significantly increase the incidence of tumor and infection.3) The augmentation of CTLA4Ig on NK cell-mediated cytotoxicity is mainly CTLA4 dependent:NK cell cytotoxicity could be activated via FcR signaling as referred as antibody-dependent cellular cytotoxicity (ADCC)(24). To test whether the enhanced cytotoxicity of NK cells is ADCC-dependent in the presence of the Fc portion of CTLA4Ig, IgG1 containing the same Fc portion was added into the co-culture system in the form of either soluble human IgG1 or plate-bound human IgG1. Either soluble human IgG1 or plate-bound human IgG1 could significantly affect NK cell cytotoxicity, which, however, was much lower than that in the presence of CTLA4Ig. These data suggested that the CTLA4 portion rather than the Fc portion was the main part of CTLA4Ig that was responsible for enhancement of NK cell cytotoxicity. To further determine whether CTLA4Ig mediated NK cell cytotoxicity was mainly CTLA4 dependent, we chose human NK cell-line (NK92) as the effective cells that only express minimal low-affinity FcR CD16 and are lack of CD32/CD64(25). We found that CTLA4Ig could also significantly increased NK92 cell cytotoxicity. These data strongly argued that CTLA4Ig-mediated NK cell activation is mainly CTLA4 dependent.2. CTLA4Ig(Abatacept)could strengthen host defense against tumors:As is known that NK cells are very important in tumor surveillance in the body, we then tested whether this enhanced NK cell cytotoxicity could favor the control of tumor formation. In the B16 melanoma bearing-mice, injection of Abatacept significantly decreased the melanoma formation in the number of tumor nodules on the lung surface and finally prolonged the survival of B16 melanoma bearing-mice as compared with PBS control. Experiment in vivo model of B16 melanoma showed that Abatacept could significantly retard B16 melanoma metastasis. These date indicated that Abatacept could strengthen host defense against tumors (B16 melanoma).3. CTLA4Ig(Abatacept)application in massive burn:It was reported that systemic or local administration of CTLA4Ig both in our(13) and other work(14) could promote allogenic skin graft survival. Take account of disseminated infection, we valued the safety of CTLA4Ig utilization in massive burn patients via Pseudomonas aeruginosa infectious model and scald mice model. We for the first time found that systemic administration of Abatacept could not only strengthen host defense against Pseudomonas aeruginosa but also enhance splenetic NK cell-mediated cytotoxicity of scald mice in vivo, which was coincidence with in vitro experiments. These data prompted that CLTA4Ig might be safety when utilized in massive burn patients in order to induce immune tolerance and promote skin graft survival. 4. CTLA4Ig enhanced NK cell-mediated cytotoxicity by up-regulation of activated receptors (NKG2D and NKp44) via ligation of CD80/CD86 molecules:1) CTLA4Ig enhanced NK cell-mediated cytotoxicity via ligation with CD80/CD86 molecules expressed on NK cell surface: To test whether CD80/CD86 contributed to the activation of NK cell cytotoxicity induced by CTLA4Ig, anti-CD80 and anti-CD86 monoclonal antibodies were used in vitro. The results showed that both antibodies could apparently stimulate NK cytotoxicity function, and the level of the increased cytotoxicity was similar to that of CTLA4Ig. It indicated that CTLA4Ig enhanced NK cell-mediated cytotoxicity via ligation with CD80/CD86 molecules expressed on NK cell surface.2) CTLA4Ig could up-regulate NKG2D and NKp44 expression on NK cell surface: As previous reports, NK cell function is controlled by NK cell activated receptors and inhibitory receptors. We thus examined the NK cell activated receptors, such as NKG2D, NKp44 by means of FACS. After stimulation of NK cells by CTLA4Ig, the expressions of NKG2D and NKp44 on NK cell membrane were increased in the percentage of positive cells and the level was comparable to that after stimulation by IL-2. However, hIgG1 showed no effect in receptors expression. These data indicated a possibility that NKG2D and NKp44 might be critically involved in the CTLA4Ig-mediated NK cytotoxicity enhancement.Conclusion:Our findings have demonstrated that CTLA4Ig could enhance NK cell function by up-regulation of activated receptors (NKG2D and NKp44) via ligation of CD80/CD86 molecules. Systemic administration of CTLA4Ig (Abatacept) could not only enhance NK cell function in scald mice but also strengthen host defense against Pseudomonas aeruginosa and tumors. This is extremely important in the understanding of CTLA4Ig in regulation of immune responses, the expansion of the indications of CTLA4Ig and in the management of massive burn injuries, some types of tumors, viral and bacteria infections.