The Role Of TLR Agonist In The Regulation Of HPV11E7 CTL Epitope-specific Immune Response In Mouse Model

Objective1.We aimed to analyze the effects of HPVs infection on Toll-Like Receptors (TLRs) expressions in the lesion of Condyloma Acuminatum (CA).2.To investigate the roles of TLR agonists, Imiquimod,PIC and CpG, in the regulation of specific immune response against HPV11E7 CTL epitope peptide in mouse model.Methods1.Frozen samples of CA lesions and normal human skin were detected for TLRs expressions by immunohistochemical staining.2.BM-DCs were isolated from C57BL/6 mice and stimulated with the HPV11E7 CTL epitope-based peptide and three TLR agonists. In vivo test. Mice were vaccinated intraperitoneally (i.p.) with BM-DCs pulsed previously with the peptide and TLR agonists, and were then injected subcutaneously (s.c) with B16 cells. In vitro test, DCs previously pulsed with the peptide and TLR agonists were co-cultured with untreated mouse splenocytes. The expression of DC surface markers, such as CD11c, Classâ…¡, CD40, CD80,CD86 were analysed using flow cytometry (BD).The TNF-a and IFN-ysecretion of the splenic T cells was assessed by ELISA. The antigen-specific CTL activity was assessed using a LDH (lactate dehydrogenase) release assay. And the tumor volume was measured.Results1. In normal skin samples, there were strong expressions of TLR 6 and 9, medium expressions of TLR 1,3,10 and weak expressions of TLR2,4,5,7,8 in the stratum corneum and stratum granulosum, and weak or negative expressions of TLRs were observed in the Stratum spinosum and stratum basale; While in the stratified CA lesions, strong expressions of TLR1,3,6,9, medium expressions of TLR 4,8,10 and weak expressions of TLR 2,5,7 were detected in almost all the layers of keratinocytes except for stratum basale.2.The expression of co-stimulating factor? CD11c,CD86,CD80,MHC II (I-Ak) on BM-DCs was upregulated, when the mice were stimulatedwith the TLR agonists; T lymphocytes secreted highest levels of TNF-aand IFN-ywhen the BM-DCs were stimulated with CpG and the peptide, comparing to the control group (P<0.05) and other groups; there were no significant differences in tumor growth and the antigen-specific CTL activity between each group.conclusions1.These findings revealed that the expressions of TLRs, especially subtype 1,3,6, 9 in the proliferated prickle keratinocytes in CA lesions were markedly enhanced comparing to that of the normal skin. This will give insights into the pathogenesis of HPV infection especially the initiation of innate immunity against viral invasion. 2. TLR agonists especially CpG up-regulated the effects of HPV11E7 CTL epitope on antigen-specific immune response in the mouse model.