The Immunological Mechanism Of TLR7 Agonist Loxoribine On The Inhibition Of Tumor Growth

Objection: There are various types of immunosuppressive cells existing in the region of tumor. Among these cells, tregs(regulatory T cell, Tregs) play an important role in the development of tumor cells. Tregs express in different tumors,including lung cancer,breast cancer, hepatocellualr carcinoma, ovarian cancer, gastric cancer, lymphoma,increase in different levels. Moreover, the local Tregs quantity in tumors are closely related to the prognosis of cancer. Tregs-induced immunosuppression is now recognized as a key element in enabling tumors escape immune-mediated destruction. The suppression of Tregs immunosuppressive effect is a key step of immunotherapy. This paper intends to explore the mechanism of tumor growth inhibition of TLR7 agonist Loxoribine by establishing CT26 murine colon tumor and LLC lewis murine lung tumor models.Methods: 1) After the successfully construction of bearing mice with CT26 murine colon tumor cells and LLC lewis murine lung cells, Loxoribine was injected intraperitoneally twice a week.The total volume was 1 umol per mice and the control group was replaced with PBS(9.8%DMSO). 2) The effect of Loxribine in vivo was closely observed after injection. Cell assay proliferation methods(CCK8) are taken to observe the direct effect of Loxoribine on CT26 murine colon tumor cells and LLC lewis murine lung cells. 3) The methods of RT-PCR were carried out to detect the expression of TLR7 on CT26 murine colon tumor cells and LLC lewis murine lung cells. 4) SCID mice which lack of T lymphocytes and B lymphocytes bring about the defect of acquired immunity,but the innate immunity is intact. With the construction of SCID bearing mice contains two kinds of cells, Loxoribine was injected intraperitoneally to detect the activation of innate immune or acquired immune pathways. 5) MACS magnetic sorting methods were conducted to obtain CD4+T cells, DCs and Tregs from both experimental group and control group. Loxoribine was administered in variety ways to observe the effect on the proliferation of the cells. 6) Gene knockout mice TLR7(-/-) was handled in the same way with MACS magnetic sorting methods to obtained cells. The administration of Loxoribine and Cell proliferation methods were also handled to observe the effect on the proliferation of the cells. 7) Methods were conducted to detect the role of Loxoribine and the maturation of DCs after Loxoribine treatment. 8) In transwell chamber, the underlying part joins with the Loxribine and DCs, the upper part joins with Tregs, observe the effect of Loxoribine on the secretion of DCs. 9) The Methods of ELISA were used to detect the cytokines and the antibody was used to verify the role of the cytokine. Loxoribine treated DCs were injected to test the inhibition of tumor growth in vivo.Results: 1) After the injection of Loxoribine, tumor growth are significantly inhibited in experimental bearing mice with contrast of control group. 2) The methods of RT-PCR show that there is no expression of TLR7 on the two kinds of tumor cells. Likely,Loxoribine has no direct killing effect on the CT26 murine colon tumor cells and LLC lewis murine lung cells. 3) From the SCID mice models, we can draw the conclusion that Loxoribine does not activate innate immunity. 4) The co-culture of different immune cells show that Ligation of TLR7 onto DCs reverses Tregs mediated immune suppression function. 5)The different immune cells divides into diverse groups, single culture of CD4+T cells, Tregs, DCs, co-culture of CD4+T/DCs, CD4+T/Tregs, DCs/Tregs. With the treatment of Loxoribine, we can draw the conclusion that Loxoribine promotes the proliferation of CD4+T cells and Tregs. 6) After the addition of Loxoribine, the result in CD4+T/DCs/Tregs group show that Loxoribine reverses Tregs mediated suppression in the presence of DCs. 7) From the gene knockout mice models, we find that Ligation of TLR7 onto DCs are essential to promote CD4+T cells proliferation and reverse Tregs mediated suppression. 8) The Methods of ELISA and transwell chamber and the addition of IL-6 antibody indicate that IL-6 secreted by DCs is critical for abrogating suppression.9) Loxoribine-treated DCs inhibit tumor growth in vivo.Conclusion:TLR7 ligand Loxoribine inhibits tumor growth in vivo and the inhibition are not by the means of the direct killing effect or the activation of innate immunity.The anti-tumor effect of Loxoribine is elicited by rendering CD4+T cells refractory to the suppressive effect of Tregs. Ligation of TLR7 onto DCs promotes CD4+T cells proliferation and Ligation of TLR7 onto DCs reverses Treg cell mediated suppression.IL-6secreted by DCs is critical for abrogating suppression.