Identification Of Dendritic Cells Subsets In Peripheral Blood And Development Of Strategies For Targeted Antigen Presentation In Cattle

Dendritic cells(DCs)are professional antigen presenting cells,thus inspection of the phenotype and function of DCs could provide theory basis for development of the methods to improve the acquired immune response.This work aimed to identify the DC cell expressing thechemokine(C-motif)receptor1(XCR1)and C-type lectin domin family 9 member A(Clec9A)and analysis the phenotype features of XCR1~+/Clec9A~+DCs among the conventional DC1(cDC1)or cDC2 in peripheral blood mononuclear cells(PBMCs)of cattle,and further explore the strategy for antigen targeted delivery to DC by receptor ligand or antibody,which would provide new insights into the design of DCs targeted molecular vaccine for animal infectious disease.Pre-enriched lineage negative cells(lin~-)were collected by megnatic separation to depletion of CD3/CD14/CD21/CD335 positive cells.lin~-DCs were further analyzed by multiple-color flow cytomery and qRT-PCR to detect the surface receptor expression.The results showed that cattle XCR1/Clec9A m RNA were mainly expressed by CD26~+CADM1~+CD205~+MHC?~+CD11b~-lin~-DC in blood,which represents a subset of c DCs,not plasmacytoid DCs(pDCs).This DCs subset partially expresses CD11a,CD44,CD80 and CD86,but they do not express CD4,CD8,CD163or CD172a.By comparison,XCR1/Clec9A mRNA were nearly undetectable,and were below that of PBMCs,in CD14~+monocytes,B cells,CD4~+T cells,CD8~+T cells,WC1~+??T cells and NK cells.Cattle bloodc DCswerefurtherdividedintoCD26~+CD172a~-CD11c~+MHC?~+lin~-DCand CD26~-CD172a~+CD11c~+MHC?~+lin~-DC,corresponding to c DC1 and cDC2.Cattle XCL1 was expressed in quiescent NK cells and activated CD8~+T cells.Cattle XCR1~+DCs can be chemotactically migrated by mouse XCL1,but not human XCL1,inducing a maximum migration of 5%of the input cells.In addition,chemically synthesized peptides of cattle XCL1 were used to check the critical region of XCL1 binding with XCR1.The results showed that peptides containing disulfided bond(C-C fragment),all could bind with XCR1,indicating the the pivotal region of XCL1 binding with XCR1.To evaluate the XCL1-mediated antigen targeting immune efficiency,the cattle XCL1,ligand of XCR1,was fused with multi-epitopes protein OB7(XCL-OB7)of type O foot-and-mouth disease virus(FMDV)to make a XCR1~+DC targeted molecular vaccine antigen,and a~?XCL-OB7 protein with mutation in XCL1 as control.XCL-OB7 protein can specific bind to XCR1 receptor detected by flow cytometry.The cattle vaccinated with XCL-OB7 showed significant higher antibody response than~?XCL-OB7 control(P<0.05).In contrast,when XCL-OB7 incorporated with poly(I:C)to prepare the vaccine,the FMDV specific antibody response of immunized cattle were significantly lower than the~?XCL-OB7 group.FMDV challenge results showed cattle immunized with the XCL-OB7(0.1 mg or 1mg)alone or the~?XCL-OB7 plus poly(I:C)obtained 80%(4/5)clinical protective rate.The~?XCL-OB7and XCL-OB7plus poly(I:C)vaccinated cattle had two and three cattle with clinical signs,and the corresponding protection rates were 60%and 40%,respectively.In contrast,cattle vaccinated with PBS were all unprotected;Cattle vaccinated with whole virus inactivated vaccinewereall completely protected.However,cattle vaccinated with~?XCL-OB7 plus poly(I:C)showed more effective inhibition on virus replication than XCL-OB7 group(P<0.01)after virus challenge according to the antibody level against non-structural protein of FMDV at 10 DPC.This indicate that T cell reponse activated by mature DC cell are vital important for cleaning up the virus circulation.In addition,three strain monoclonal antibodies were screened out by immunization with peptide of Clec9A and could capture Clec9A~+DC according to the results of magnetic separation and qRT-PCR.This provided useful tools for evaluation of immune efficacy via antibody-mediated antigen targeted delivery.In conclusion,the phenotype of XCR1~+/Clec9A~+DC in PBMC of cattle is summarized as following:CD26~+CADM1~+CD205~+CD11c~+CD11b~-CD4~-CD8~-CD163~-lin~-cDC.The C-C fragment of XCL1 is the key region for binding to XCR1.XCL1 mediated antigen targeting to XCR1~+DC can enhance antibody response in cattle,which will be valuable for developing vaccines mainly relying on antibody to provide protective immunity against some infectious disease in cattle.