The Mechanism Of The Adjuvanticity Of CpG ODN Based On The Relationship Between The Level And Migration Of B Cell Surface TLR9 And The Activation Of B Cells

CpG oligodeoxynucleotide(CpG ODN)is an unmethylated synthetic ODN containing CpG motifs,and the agonist of the pattern recognition receptor TLR9(Toll-like receptor 9).After recognizing CpG ODNs,the downstream signal cascades of TLR9 are activated,which lead to the activation of B cells,plasmacytoid dendritic cell,T cells,therefore,CpG ODNs are considered to be potential adjuvant candidates.TLR9 is constitutively expressed in human and mouse B cells and plasmacytoid dendritic cells(p DCs),as well as in mouse dendritic cells(DC)and monocytes/macrophages.During resting state,TLR9 locates in endoplasmic reticulum(ER),and has to translocate to endosomes for the recognition with CpG ODNs.Interestingly,it was reported that TLR9 could be also expressed on the cell membrane of B cells and neutrophils as surface TLR9(sTLR9).However,it remains unclear that whether the sTLR9 could be regulated by CpG ODN and whether the regulation could contribute to the adjuvanticity of CpG ODN.Thus,we detected the effect of a series of self-designed CpG ODNs on the B cell sTLR9,and tried to enrich the mechanisms of the adjuvanticities of CpG ODN from an unrecognized viewpoint based on the relationship between the levels and migration of B cellsTLR9 and the B cell activation.Firsty,we used bioinformatic analysis and predicted that CpG 5805 and CpG5801 might hve the strongest mmunostimulatory effects among all the self-designed CpG ODNs.Then,we investigated the immunological activity of the CpG ODNs and classify them by anti-VSV assay and splenocyte proliferation assay.Finally,we indentified one C-type CpG ODN(CpG 5805),three B-type CpG ODNs and no A-type CpG ODN.To investigate whether CpG ODN could regulate the expression of B cell sTLR9,we cultured the mouse splenocytes with each CpG ODN,and detected the levels of B cell sTLR9 and total TLR9(t TLR9)by cell surface staining and intracellular staining,respectively.The results showed that B-type and C-type CpG ODN could reduce the level of B cell sTLR9,the regulation of CpG ODN on sTLR9and t TLR9 was opposite.Although the normally used CpG ODNs as adjuvant were B-type CpG ODN,C-type CpG ODN contained not only the characteristics of B-type CpG ODN,but the ability to induce the type I interferon(IFN-I)production which might assist the humoral immunity.We selected the B-type CpG 5805 to co-cultured with mouse splenocytes,and then examine the proliferation and activation of antibody response related immune cells,such like B cells,dendritic cells(DCs)and T cells.The results showed that CpG 5805 could strongly promote the proliferation and activation of B cells,have no effect on the proliferation of the CD4~+T,CD8~+T cells and DCs,but significantly strengthen the activation these cells.Thus,CpG 5805 had stronger immunostimulatory ability,showing great potential as a vaccine adjuvant.To examine the adjuvanticity of CpG 5805,we immunized the mice with CpG5805 and?CP recombinant protein vaccine,inactivated H9N2 virus vaccine and hepatitis B virus vaccine(Vac),respectively,and detected the levels of antigen specific antibody in the sera of the immunized mice by ELISA at different timepoints.The results showed CpG 5805 could increase the antibody levels of?CP recombinant protein vaccine and Vac,but could not increase the antibody levels of inactivated H9N2 virus vaccine.Then,we cultured the mouse splenocytes with CpG 5805 and hepatitis B virus vaccine in vitro,and detected the expression of MHC II,CD80,CD86,CD40 on B cells to detect the effect of CpG 5805 as an adjuvant on B cells.These results suggested that CpG 5805 as an adjuvant could up-regulate the expression of MHC II,CD80,CD86,CD40 on the B cells to promote the B cell activation.To detect the effect of CpG 5805 as an adjuvant on the expression levels of B cell sTLR9 and the activation of B cells,we isolated the draining lymphoid(DLN)cells and splenocytes,and detected the levels of sTLR9,CD80,CD86,CD40 and MHC II on the B cells.CpG 5805 as an adjuvant increase the expression level of CD80,CD86,CD40 and MHC II on the B cells,and the m RNA levels of TLR9 and its downstream signals.Meanwhile,CpG 5805 as an adjuvant could significantly decrease the levels of sTLR9 on B cells.And the results indicated that CpG 5805 as an adjuvant could down-regulated the expression level of B cell sTLR9,and activated B cells via TLR9 signaling pathway simultaneously.Although CpG 5805 could decrease the expression level of B cell sTLR9,but increase the protein and m RNA level of B cell t TLR9,indicating that the decrease of sTLR9 could not be explained by the increased t TLR9 expression.Since TLR9 has the ability of trafficking from ER to endosome or cell membrane surface.Thus,the only explanation of the reduced sTLR9 expression might be due to forcing sTLR9internalization and mostly internalized to endosomes.In order to verify this hypothesis,we did the following research:we developed a method with the reference of an antibody-feeding assay to label the sTLR9,re-called in vitro with 5805+Vac or Vac and observe the location of CD19~+B cells and sTLR9 under confocal microscope.The results showed that the sTLR9 could be internalized into the CD19~+B cells and relocated to endosomes by CpG 5805.During observing the sTLR9 migration,we unexpectedly found that the sTLR9 internalized cells enlarged,indicating that the B cells with internalized sTLR9 underwent blastogenesis.By flowcytometry,we found that with the stimulation of CpG 5805 and Vac,the percentages of larger CD19~+cells were extremely higher.And these CpG 5805 enlarged B cells expressed less levels of sTLR9,but higher levels of CD40.These results proved our hypothesis that the enlargement of B cells induced by CpG 5805 was the sign of B cell activation,and the sTLR9 expression was decreased on these activated B cells.To investigate the adjuvanticity of CpG 5805 is dependent on the B cell sTLR9internalization and TLR9 signal activation,according to our previous work and related references,we selected CCT ODN(an inhibitory ODN)which could specifically inhibit the TLR9 migration out of ER,MS19(another inhibitory ODN)which could inhibit TLR9 downstream signals,chloroquine which destroy the acidic environment of endosome and si RNA target to AP2,to interfere the TLR9 migration,respectively,and observed the sTLR9 and CD40 expression on B cells induced by CpG 5805.The results showed that:1)When the TLR9 was inhibited to move out of ER by CCT ODN,the B cell sTLR9 expression was decreased with or without CpG5805,and CpG 5805 could not increase the CD40 expression,indicating that CpG ODN could not activate B cells when TLR9 migration was inhibited.2)When the sTLR9 internalization was inhibited by AP2 targeted si RNA,CpG 5805 could not decrease the sTLR9 expression and increase the CD40 expression,indicating that the B cell activation induced by CpG ODN was consistent with the sTLR9 reduction.3)When the acidic environment of endosome was inhibited by chloroquine,CpG 5805could not decrease sTLR9 expression or increase CD40 expression,indicating that the B cell activation was dependent on the acidic environment of endosome,and whether sTLR9 could internalize is also dependent on the acidic environment of endosome.4)When the TLR9 downstream signals were inhibited by MS19,CpG 5805 could still decrease the sTLR9 expression,but could not increase the CD40 expression,indicating that the internalized sTLR9 was incorporated into the endosomal TLR9signaling to exert its immunological activity.Thus,CpG 5805 induced the sTLR9reduction and B cell activation by promoting sTLR9 internalization which enhance the endosomal TLR9 signaling.In summary,we obtained a new C-type CpG ODN from the self-designed CpG ODNs,and verified its adjuvanticity of microbial vaccine to promote antibody production.According to the regulation of B cell sTLR9 expression induced by CpG5805,specific on each aspect of TLR9 migration,we investigated that CpG 5805induced sTLR9 reduction might be one of the mechanisms of CpG 5805 enhanced antibody response.The reduced sTLR9 could migrate into endosome and participate in the endosomal TLR9 signaling pathway which promote B cell maturation and activation to increase the antibody production.This might be a novel mechanism of the adjuvanticity of CpG ODN.