| Natural Killer cells (NK cells) are a very important type of cytotoxic lymphocytes ininnate immunity which is the first barrier of the immune system. NK cells are a unique,different from acquired immune system which needs antibody and MHC to act. They makevery fast responses when mammality was infected by virus or invaded by tumor cells.NK cell activation is determined by the balance of activating and inhibitory receptorstimulation. If the activating signal is more prominent then NK cell will be activated,similarly if the inhibitory receptor signaling dominants then NK cell activity will be inhibited.NKG2D and NKp44, the two activating NK receptors, are involved in tumor cell lysis byactivated NK cells. NK cells have a lot of cytolytic granules in their cytoplasm. When NKcells are activated, they release these granules to the site of cell–cell contact. These granulescontain cytolytic proteins, such as perforin and granzyme, that are involved in inducingtarget cell death. Lining the membrane of the granules is the lysosomal-associatedmembrane protein, LAMP-1or CD107a. CD107a is a glycoprotein representingapproximately50%of the proteins in the lysosomal membrane. During degranulation,CD107a on the luminal membrane is exposed in the immunological synapse and thenantibody could bind. Alter et al. showed that CD107a can also be detected on the surface ofNK cells following stimulation with target cells, so CD107a is considered as a marker ofdegranulation.Cytotoxic T-lymphocyte-associated antigen4(CTLA4) is expressed on the surface of Tcells hours or days after activation and functions as a negative regulator of T cell activation.CTLA4Ig is constructed by genetically fusing the external domain of human CTLA4to theheavy-chain constant region of human IgG1. CTLA4Ig has been shown to induce T celltolerance by binding to both CD80and CD86on antigen-presenting cells (APC), whichprevents the binding of CD28to CD80and CD86to deliver costimulatory signals to T cells.Many in vitro and in vivo studies demonstrated that CTLA4Ig could be used for controlling ofautoimmune diseases and allograft rejection. Both commercial products of CTLA4Ig, Abatacept and Belatecept (Bristol-Myers Squibb), have been approved by the FDA as atherapy for treating autoimmune diseases such as arthritis, and for controlling graft rejection.One interesting aspect of CTLA4Ig is that patients treated with this fusion proteinexperienced not much higher incidence of tumors and infectious episodes than controls. Weare interested in why immunocompetence against tumors remains while T cell activation issuppressed by CTLA4Ig. It is believed that surveillance for tumorigenesis and anti-infectionare mediated by both adaptive and innate immune cells. Whether the innate immunity is intactwhen the adaptive immunity is suppressed by CTLA4Ig has not been examined. Grohmannet.al. found that CTLA4Ig could influence APC function via the interaction with B7molecules on APC. Moreover, recent studies demonstrated that resting NK cells couldexpress CD86and that activated NK cells express both CD80and CD86receptors that mightbe bound by CTLA4Ig. These results prompted us to study the possibility that CTLA4Ig canregulate tumor surveillance and anti-infection immune by modulating NK cell function.In the present study, we reveal the novel function of CTLA4Ig via enhancement of NKcell cytotoxicity to target cells in vivo and in vitro and also show the underlying moleculemechanism was involve in this activity. Our main findings and conclusions of this study aresummarized as follows:1. CTLA4Ig reduces melanoma metastasis in vivo and prolonged host survival.To define the role of CTLA4Ig in tumor immunity, sex-and age-matched B6mice wereinjected with B16F0cells on Day0, followed by intravenous injection of either CTLA4Ig(n=10) or isotype control IgG (n=10) on Days0,3and6, respectively. The survival time andthe melanoma lung metastasis of each animal were monitored. The results showed that micetreated with CTLA4Ig had significantly longer survival time and significantly lower numbersof lung metastatic tumor nodules than those treated with control IgG. These results suggestthat CTLA4Ig contribute to the anti-tumor protection.2. CTLA4Ig enhances NK cell cytotoxicity in vivo.1). CTLA4Ig reduced the melanoma metastasis in SCID mice and B6mice with NKcells depletion.It has been demonstrated that CTLA4Ig inhibits the function of the adaptive immunesystem by blocking B7/CD28interactions. We hypothesized that innate immunity, but notadaptive immunity, plays a critical role in the CTLA4Ig-mediated anti-tumor activity. To test our hypothesis, sex-and age-matched SCID mice were used, and the results showed thatCTLA4Ig could also significantly inhibit the lung metastasis of melanoma tumors in SCIDmice. Because NK cells play an important role in tumor surveillance in the body, we furtherexamined whether NK cells were involved in the CTLA4Ig-mediated anti-tumor activity.Therefore, we used the PK136depleting antibody to deplete NK cells in mice. The resultsshow that depletion of NK cells results in the abolishment of the CTLA4Ig killing target celleffect and indicate that NK cells might play some role in the CTLA4Ig-mediated killingactivity.2). CTLA4Ig enhances the tumor infiltrating NK cell cytotoxicity in vivo.The ability of NK cells to kill tumor cells are critical immune surveillance mechanismsfor eradicating developing tumors. To assess the NK cells cytotoxicity in NK-dependent-CTLA4Ig-anti-tumor activity in vivo, the B16melanoma mice treated with either CTLA4Igor control IgG were sacrificed10days after tumor inoculation; magnetic-activated cellsorting was used to isolate the infiltrating NK cells from lung tissue for the analysis ofcytolytic activity and cytokine production. The results showe that tumor-infiltrating NK cellsfrom mice treated with CTLA4Ig possess significantly higher cytolytic activity than thosetreated with control IgG. These results suggest that CTLA4Ig retards tumor metastasis byenhancing the NK cell cytotoxicity to tumor cells in vivo.3). CTLA4Ig induces higher number of perforin-producing and CD107a-positive NKcells in vivo.Because the degranulation marker CD107a and effector molecules perforin are alsoclosely associated with NK cell cytotoxicity against tumor cells, we examined the expressionof these molecules in tumor infiltrating NK cells of mice treated with either CTLA4Ig orcontrol IgG. The results showed that there were significantly higher numbers ofperforin-producing and CD107a-positive NK cells in CTLA4Ig-treated mice than controlIgG-treated mice. These results clearly show that the cytotoxicity of the infiltrating NK cellsis markedly enhanced in the process of CTLA4Ig-mediated killing activity.3. CTLA4Ig stimulats NK cell cytolytic activity in vitro.1). CTLA4Ig enhances mouse spleen NK cell cytotoxicity in vitro.To assess the direct effect of CTLA4Ig on NK cells, we first examined the role ofCTLA4Ig in NK cell cytotoxicity against tumor cells in vitro. Mouse splenic NK cells were purified by MACS and were co-cultured with either CTLA4Ig or control IgG to analyze thecytolytic activity to YAC-1cells. As compared to control IgG, CTLA4Ig could significantlyenhance NK cell cytotoxicity to YAC-1cell in vitro.2). CTLA4Ig enhances tumor-infiltrating NK cells cytotoxicity ex vivo.Then, we checked the effect of CTLA4Ig on NK cell cytotoxicity to tumor cells ex vivo.The tumor-infiltrating NK cells were purified from lungs of mice bearing B6melanomatumor and co-cultured with either CTLA4Ig or control IgG to analyze the cytolytic activity toYAC-1cells. As compared to control IgG, CTLA4Ig significantly enhanced the cytotoxicityof the infiltrating NK cells ex vivo.3). CTLA4Ig enhances human NK cells cytotoxicity in vitro.Then, we also checked the effect of CTLA4Ig on human NK cell cytotoxicity againsttumor cells in vitro. The human NK cells were purified from PBMC and co-cultured witheither CTLA4Ig or control IgG to analyze the cytolytic activity to K562cells. As compared tocontrol IgG, CTLA4Ig significantly enhanced the cytotoxicity of human NK cells in vitro.4). CTLA4Ig enhances the NK92MI cell cytotoxicity in vitro.Because the NK cells isolated by using MACS were not highly purified (the purity wasless than90%), it was possible that the CTLA4Ig was acting on other cell types in the culture.To address this issue, we used a human NK cell line, NK-92MI, as the effector cells to checkthe possible direct role of CTLA4Ig in regulating NK function. The results showed thatcytotoxicity of NK-92MI cells to K562tumor cell was significantly greater increased in thepresence of CTLA4Ig than that in the presence of control IgG in vitro. The results suggestthat CTLA4Ig might directly stimulate NK cell cytotoxicity.4. CD86is the target molecule in CTLA4Ig-mediated enhancement of NK cytolyticactivity.1). NK cells could significantly increase the expression of CD86upon tumor cellstimulation.Based on the fact that CTLA4Ig binds with high affinity to CD80/CD86, wehypothesized that CD80or CD86might play in role in the ability of CTLA4Ig to enhance NKcell functions against tumor metastasis. To understand the expression of CD80and CD86onphysiological NK cells of B6mouse, the CD80/CD86expression on NK cells was examinedby flow cytometry. The results showed that prior to activation, mouse NK cells had6% expression of CD86and little CD80expression. The expression of CD86on NK cells wassignificantly increased following activation with both YAC-1tumor cells and cytokine IL-15in vitro. Furthermore, we detected the expressions of CD86and CD80on the tumor-infiltrating NK cells in vivo10days after injection of B16melanoma cells. The data showedthat injection of B16melanoma cells could significantly increase the expression of CD86, butnot CD80, on infiltrating NK cells. These results indicated that NK cells could significantlyincrease the expression of CD86upon stimulation by tumor cells and that CTLA4Ig activatesNK cells possibly via ligation of CD86on the activated NK cells.2). CTLA4Ig induces higher level of expression of both NKG2D and NKp44.To test the role of CD86in regulating NK cell function, we used NK92-MI thatspontaneously expressed high levels of CD86, but not CD80, as effector cells for ligationwith CTLA4Ig in vitro. The results showed that the ligation of NK-92MI cells withCTLA4Ig significantly induced higher expression level of NK effector molecules NKG2Dand NKp44.3). CD86rather than CD80on NK cells might be involved in the enhancement of NKcell cytotoxicity in competition experiments.To exclude that the anti-CD80and anti-CD86antibody may affect the cytotoxicity ofNK cells, we added anti-CD80or anti-CD86antibody into NK-92MI cell culture system, asexpected, no effect of anti-CD80and anti-CD86antibody on NK-92MI cell cytotoxicity wasfound. Then, an anti-CD86antibody was used to compete for CD86molecules on NK-92MIwith CTLA4Ig. The results showed that when anti-CD86antibody, together with CTLA4Ig,was added into the NK-92MI cell culture system, the CTLA4Ig-mediated NK-92MI cellactivation was partly decreased. If anti-CD86antibody was added2hours earlier thanCTLA4Ig, which meant that the anti-CD86antibody had pre-occupied the CD86moleculeson NK cells, CTLA4Ig-mediated NK-92MI cell cytotoxicity was completely abolished.These data from the competition experiments clearly demonstrates that CD86rather thanCD80on NK cells is involved in the enhancement of NK cell cytotoxicity to target cells.Conclusion: we showed that brief administration of CTLA4Ig significantly reducedtumor metastasis and prolonged the survival of host mice bearing B16melanoma. Depletionof NK cells prior to CTLA4Ig administration eliminated the CTLA4Ig-mediated killingactivity. CTLA4Ig can significantly enhance NK cell cytotoxicity to target cells via up-regulation of NK cell effecter molecules CD107a and perforin in vivo. In addition, wedemonstrated that, upon activation, NK cells could significantly increase the expression ofCD86both in vitro and in vivo, and ligation of CD86with CTLA4Ig significantly increasedthe ability of NK cells to kill target cells. Furthermore, a human NK cell line that expressedhigh level of CD86, but not CD80, was directly activated by CTLA4Ig so that killing oftargets was enhanced; this enhanced killing could be inhibited by blocking CD86. Ourfindings uncover a novel function of CTLA4Ig in innate immunity and suggest that CD86onNK cells is an activating receptor and closely involved in the CTLA4Ig-mediatedcytotoxicity activity. |
PDF Full Text Request