Effects Of NKG2DL On The Rejection Of Allogeneic Tumors In Mice And Its Possible Mechanism

NKG2D ligands(NKG2DL)are induced self-proteins and expressed poorly by most normal cells but up-regulated on tumor cells. Their receptor, called NKG2 D, is expressed on the surface of many immune cells, such as NK cells and CD8+ T cells.As reported, the immune cells could kill tumor cells through NKG2D-NKG2 DL interaction both in vivo and in vitro. In syngeneic mice, NKG2 DL high expressing tumor cells could be rapidly rejected and NKG2 D deficient mice exhibited a higher incidence or greater severity of tumors. In addition, NKG2 DL high expressing cancer cells were more susceptible to NKG2D+cell killing in vitro.Similarly, in allogeneic mice, the graft rejection could be also triggered by the NKG2D-NKG2 DL interaction. Benign NKG2 DL high expressing cells could be eliminated by NKG2D+ cells in allogeneic mice. Besides, the NKG2D+ cells could recognize and kill NKG2 DL high expressing allogeneic tumor cells in vitro. Little was known about how allogeneic tumors are eradicated by innate immune responses in vivo, although the allogeneic tumor cells could be eradicated by adaptive cellular immune responses, which were launched by CD8+ T cells or CD4+ T cells after recognizing with MHC-?molecules or MHC-?molecules on the cells. It remains unclear whether the innate immune cells, such as NKG2D+ cells, get involved in the rejection of allogeneic tumor cells in vivo by the NKG2D-NKG2 DL interaction.In order to investigate the role of NKG2 DL in the allogeneic tumor cell eradication, we first established NKG2 DL low expressing and NKG2 DL high expressing tumor cell derived allogeneic tumor bearing mouse model. Then we observed the growth of the allogeneic tumors derived from NKG2 DL high expressing and NKG2 DL low expressing tumor cells, and the percentage of NKG2D+ cells of allogeneic tumor bearing mice. Furthermore, we up-regulated the NKG2 DL expression on the tumor cells and knocked out the NKG2 DL gene in the tumor cells,and then inoculated the cells into their allogeneic mice, respectively to observe thetumor development. The results were shown as follows:1. The tumor formation of tumor cells with different level of NKG2 DL in allogeneic mice.To study weather different level of NKG2 DL could determine the allogeneic tumor growth, we detected the expression of NKG2DL( RAE-1, H60 and MULT-1)on EMT-6 cells, B16 cells and GL261 cells, respectively. Then we subcutaneously inoculated the cells into their allogeneic mice, respectively, to observe the tumor growth. The results showed that 1) EMT-6 cells and B16 cells were expressed low level of NKG2 DL, defined as NKG2 DL low expressing cells, while GL261 cells were expressed high level of RAE-1, defined as NKG2 DL low expressing cells; 2)NKG2DL low expressing tumor cells(EMT-6 cells and B16 cells) could be developed into palpable tumors in allogeneic mice at the early stage, whereas, NKG2 DL high expressing tumor cells(GL261 cells) could not. The data indicated that high expression of NKG2 DL on tumor cells may determine the early tumor rejection in the allogeneic mice and NKG2D+ cells may be involved in the process.2. The percentage of NKG2D+ cells in spleens and lymph nodes of allogeneic mice inoculated with tumor cells with different level of NKG2 DL.To explore whether NKG2D+cells participated in the rejection of allogeneic tumors in mice, we subcutaneously inoculated NKG2 DL high expressing tumor cells,GL261 cells, and NKG2 DL low expressing tumor cells, EMT-6 cells and B16 cells,into allogeneic mice, respectively, and then analyzed the percentage of NKG2D+ cells and the adaptive immune cells in the spleens and lymph nodes of the allogeneic mice inoculated with the tumor cells for 3 days(early stage) and 50 days(late stage),respectively. The results showed that 1) at the early stage, the NKG2D+NK cells and NKG2D+CD8+ T cells were significantly increased in the spleens and lymph nodes of allogeneic mice inoculated with NKG2 DL high expressing tumor cells; 2) at the late stage, the NKG2D+ NK cells and NKG2D+CD8+ T cells were also increased in the allogeneic mice injected with NKG2 DL high expressing tumor cells. Additionally, the adaptive immune cells, such as cytotoxicity T lymphocytes(CTL), dentritic cells(DC)and B cells, were largely proliferated, while regulatory T cells(Treg) were decreased in the allogeneic mice inoculated with EMT-6 cells. The results suggested that NKG2D+ cells were not only involved in the eradication of NKG2 DL high expressing allogeneic tumor cells at the early stage, but at the late stage participated in theelimination of the allogeneic tumor cells with the adaptive immune cells.3. The correlation between the regulation of NKG2 DL and the allogeneic tumor growth.To further investigate the relationship between the high expression of NKG2 DL on the tumor cells and the rejection of the allogeneic tumors in mice, we first up-regulated the NKG2 DL expression on NKG2 DL low expressing tumor cells in syngeneic mice and observe the tumor growth. Then we increased the NKG2 DL expression on NKG2 DL low expressing tumor cells(EMT-6 cells and B16 cells) in vitro or knocked out NKG2 DL gene in NKG2 DL high expressing cells(GL261 cells),and subcutaneously inoculated the cells into the allogeneic mice, respectively, to observe their tumor growth. The results were shown as following:3.1 The correlation between NKG2 DL up-regulation and tumor inhibition in syngeneic mice.To study the correlation between the NKG2 DL up-regulation and syngeneic tumor rejection, we subcutaneously inoculated NKG2 DL low expressing tumor cells(EMT-6 cells and B16 cells) into their syngenic mice, respectively, followed by six-time administration with a type of oligodeoxynucleotide(Cp G ODN) designed by us after the tumors were palpable in one-day interval at tumor draining lymph node(TDLN) area to observe tumor growth, and after 48 hours of the last injection, the tumors were removed for analyzing the expression of NKG2 DL on the tumor cells.The results showed that 1) the syngeneic tumors were growing slower in the Cp G ODN-treated mice; 2) the expression of NKG2 DL on tumor cells was up-regulated in the Cp G ODN-treated mice. The results indicated that Cp G ODN could up-regulate NKG2 DL expression on NKG2 DL low expressing tumor cells and the up-regulation could inhibit the tumor growth in syngeneic mice.3.2 The up-regulation of NKG2 DL on NKG2 DL low expressing tumor cells in vitro.To clarify how Cp G ODN up-regulated the NKG2 DL expression on the tumor cells, we incubated NKG2 DL low expressing tumor cells(EMT-6 cells or B16 cells)with Cp G ODN, Cp G ODN-conditioned supernatant or the typical cytokine in Cp G ODN-conditioned supernatant, IFN-? in vitro, and then analyzed the expression of NKG2 DL on the cells. The results showed that 1) Cp G ODN could not up-regulate the NKG2 DL expression on the tumor cells; 2) Cp G ODN-conditioned supernatant couldincrease the NKG2 DL expression on the tumor cells; 3) IFN-? could up-regulate the NKG2 DL expression on the tumor cells. The results revealed that some cytokines in the Cp G ODN-conditioned supernatant, such as IFN-?, could up-regulate the NKG2 DL expression on the NKG2 DL low expressing tumor cells.3.3 The growth of NKG2 DL up-regulated tumor cells in allogeneic mice.Based on the above results, we subcutaneously inoculated Cp G ODN-conditioned supernatant cultured NKG2 DL low expressing tumor cells(EMT-6cells or B16 cells) or NKG2 DL gene transfected B16 cells into their allogeneic mice to observe the tumor growth. The results showed that the growth of NKG2 DL up-regulated tumor cells was significantly inhibited in the allogeneic mice.3.4 The growth of NKG2 DL gene knockout tumor cells in allogeneic mice.To further consolidate the contribution of high level of NKG2 DL to the allogeneic tumor rejection, we used the CRISPR/Cas9 system to knock out NKG2 DL genes in NKG2 DL high expressing tumor cells(GL261 cells), and then subcutaneously inoculated the NKG2 DL deficient cells into their allogeneic mice to observe the tumor growth and survivals. The results showed that the NKG2 DL deficient cancer cells could be developed into allogeneic tumors at the early stage.In conclusion, our study demonstrated that NKG2 DL triggered innate immune responses may contribute to the allogeneic tumor inhibition in mice at the early stage after cancer cell inoculation. The inhibition was mainly mediated by NKG2D+cells,especially NK cells. Practically, the findings presented here might provide insights for combining the allogeneic NK cell adoptive transfer with the approaches of up-regulating NKG2 DL to treat cancer patients.