| T cells play an important role in T cell-dependent antibody response.Cytotoxic T-lymphocyte-associated antigen 4(CTLA-4)is recognized as a critical inhibitory regulator expressed on the surface of activated T cells and it can compete with CD28for binding CD80 or CD86 on antigen presenting cells(APCs),generating the inhibitory signals to wean the T-cell activation.Thus,blocking CTLA-4-mediated inhibitory signal can result in enhanced T-cell activation required for further promotion of T cell-dependent antibody response.In this study,we designed two oligodeoxynucleotides(ODNs)targeting the conserve sequences of both the human and mouse CTLA-4 mRNA 3'untranslated region(3'UTR),tested their inhibitory effects on CTLA-4 and their adjuvanticity for vaccines.The results were as follows:1.Design of ODNs targeting CTLA-4 mRNA 3'UTR.To obtain the ODNs targeting CTLA-4 mRNA 3'UTR,we designed a series of ODN candidates complementary to the conserved sequences of both the human and mouse CTLA-4 mRNA 3'UTR.Then we screened for two ODNs,CMD-1 and CMD-2,by analyzing the secondary structure of the ODN candidates and their targets,and the thermodynamic free energy of the ODN candidates.The results showed that CMD-1 and CMD-2 had relatively higher intraoligo?G,higher interoligo?G,lower binding?G between ODN and mRNA,and hardly bound with the mRNA irrelevant to CTLA-4.Thus,theoretically,CMD-1 and CMD-2 with high specificity were unlikely to form dimers or hairpins and easy to bind with the mRNA targets.2.Inhibitory effect of ODNs targeting CTLA-4 mRNA 3'UTR on CTLA-4To study the inhibitory effect of the ODNs targeting CTLA 4 mRNA 3'UTR on CTLA-4,we firstly analyzed the entry of ODNs into target cells kinetically,then detected the influence of ODNs on CTLA-4 and CTLA-4 mRNA by flow cytometry and real-time PCR(RT-PCR),respectively and finally explore the mechanism.2.1 Kinetics analysis of the entry of CMD-1 and CMD-2 into target cellsSince the major physiological role of CTLA4 seems to be through CD4~+T cells,we used fluorescence labeled CMD-1 and CMD-2 to detect their entry into CD4~+T cells in vitro and in vivo.The results showed that:1)CMD-1 and CMD-2 could enter CD4~+T cells with similar efficacy in a time-dependent manner;2)The distribution of CMD-1 and CMD-2 in CD4~+T cells was similar;3)When injected into the right hind limbs of na?ve ICR mice intramuscularly,CMD-1 and CMD-2 were mainly distributed in popliteal lymph node on the same side as the injection site,and 6,12and 24 h after injection,CMD-1 and CMD-2 could be detected in CD4~+T cells in the popliteal lymph node on the same side as the injection site.These result suggested that both CMD-1 and CMD-2 could enter CD4~+T,which provided the possibility for ODNs to work in vivo.2.2 Inhibitory effect of CMD-1 and CMD-2 on CTLA-4 expressionTo test the inhibitory effect of CMD-1 and CMD-2 on CTLA-4 expression in CD4~+T cells,we used Cp to stimulate the splenocytes from the mice immunized with Cp to activate T cells,added CMD-1 or CMD-2 in the meantime,and detected the influence of CMD-1 or CMD-2 on CTLA-4 expression on the CD4~+T cells from mice.The results showed that CMD-1 rather than CMD-2 could inhibit the CTLA-4expression on the CD4~+T cells from mice.To detect whether CMD-1 or CMD-2could also inhibit CTLA-4 expression in human,we stimulated human peripheral blood cells with phorbol esters(PMA)plus ionomycin(IONO)alone or in presence of CMD-1 or CMD-2 and detected the CTLA-4 expression on CD4~+T cells form human.Both CMD-1 and CMD-2 could inhibit the CTLA-4 expression on CD4~+T cells and the inhibitory effect of CMD-1 was insignificantly better than CMD-2.Together,compared with CMD-2,CMD-1 could significantly inhibit CTLA-4expression on CD4~+T cells from both mice and human,suggesting CMD-1 had the potential to be used in human,and its adjuvanticity could be studied in mice.2.3 Effect of CMD-1 and CMD-2 on CTLA-4 mRNATo further detect whether the inhibitory effect of CMD-1 on CTLA-4 expression was resulted from the interaction of CMD-1 with CTLA-4 mRNA,we detected the effect of CMD-1 and CMD-2 on CTLA-4 mRNA in cells.The results showed that compared with CMD-2,CMD-1 could efficiently down-regulate the CTLA-4 mRNA level,while as CMD-1 was consumed,the interference effect could gradually disappear,and the mRNA level was even raised in feedback way.Moreover,to study the mechanism,we incubated CTLA-4 mRNA obtained by in-vitro transcription with CMD-1 or CMD-2 in the presence of RNase H and detected the cleavage efficacy of RNase H on CTLA-4 mRNA.The results showed that compared with CMD-2,CMD-1 could efficiently bind with CTLA-4 mRNA,and induce the RNase H-dependent cleavage of the mRNA.The results above suggested that CMD-1,an ODN targeting CTLA-4 mRNA 3'UTR,was an effective inhibitor of CTLA-4,and it could enter the CD4~+T cells,bound with CTLA-4 mRNA,induced RNase H to cleavage the target mRNA,and inhibited the CTLA-4 expression on CD4~+T cells from mice or human.3.Adjuvanticity of ODNs targeting CTLA-4 mRNA 3'UTR for vaccinesTo verify whether CMD-1 could work as an adjuvant through inhibiting the CTLA-4 inhibitory signal,we detected the effect of CMD-1 on the antibody levels and immune cells in the mice immunized with vaccines,and explore the putative mechanism.3.1 Effect of CMD-1 on antibody levels in the sera of miceTo detect the adjuvanticity of CMD-1 and CMD-2 for vaccines in mice through CTLA-4 inhibition,we immunized mice with inactivated virus and recombinant subunit vaccines and detected the antibody levels in the sera collected from the mice,and studied the effect of strains of mice,emulsion and dosage of ODNs on the adjuvanticity.The results showed that:1)CMD-1 could significantly enhance antibody levels on days 21 and 28 in the sera from the mice immunized with inactivated foot-and-mouth disease virus(FMDV)vaccines;2)CMD-1 could obviously improve antibody levels on days 14,21 and 28 in the sera from the mice immunized with recombinant CPAC VLPs vaccines;3)CMD-1 could work as an adjuvant in both ICR and BALB/c mice;4)Adjuvanticity of CMD-1 could be partly affected by emulsion;5)Adjuvanticity of CMD-1 was in a dosage-dependent manner,and both 5?g/mouse and 10?g/mouse of CMD-1 could significantly up-regulate the antibody levels in mice.3.2 Effect of CMD-1 on immune cells in miceIn order to explore the effect of CMD-1 on the immune cells in vivo,we detected the change of CD4~+T cells,CD11c~+cells and CD19~+B cells in the draining lymph node cells and splenocytes from the mice sacrificed 48 h after the second immunization with CPAC VLPs vaccines alone or in presence of CMD-1 or CMD-2.The results showed that when formulated in vaccines for mice,CMD-1 could significantly down-regulate the CTLA-4 expression on CD4~+T cells,the CD80expressed on CD11c~+cells and the percentage of CD4~+T cells,while the percentage of CD19~+B cells was not obviously changed.3.3 Mechanism of CMD-1 to work as an adjuvantTo further explore the putative mechanism of CMD-1 to work as an adjuvant for vaccines,we used Cp to stimulate the splenocytes from the mice immunized with Cp alone or in the presence of ODNs and detected the effect of ODNs on CD80/CD86expression on CD11c~+cells,activation and proliferation of CD4~+T cells and CD19~+B cells and the expression of IL-4.The results showed that CMD-1 could promote the activation and proliferation of CD4~+T cells,followed by maintaining CD80 and CD86 molecules on CD11c~+cells,up-regulating IL-4 expression and enhancing activation and proliferation of B cells through inhibiting CTLA-4 inhibitory signal in T cells.The results above suggested that CMD-1 could work as an adjuvant in inactivated virus and recombinant subunit vaccines through inhibiting CTLA-4inhibitory signals and promoting T cell-dependent antibody response.In conclusion,CMD-1 could be used as a kind of novel adjuvants to promote antibody response by inhibiting CTLA-4 inhibitory signal in T cells,and the idea of enhancing immune response by inhibiting immunosuppressive signals provides a new strategy for the design of novel adjuvants. |
PDF Full Text Request