The Changes Of Immunological Synapse Molecules Expressed By Porcine T Cells And APC Cells Stimulated By Staphylococcal Enterotoxins

Staphylococcal enterotoxins(SEs),produced by Staphylococcus,is a family of proteins with similar structure and virulence.Having good superantigenic activity,SEs stimulate the activation of mass T cells and release of inflammatory cytokines.In this process,immunological synapse(IS)is the basis of T cell activation,inflammatory cytokines storm and SE superantigenic activation.Thus,we foucused on the changes of the IS related molecular on the antigen presenting cells(porcine alveolar macrophages)and T cells,which related with enterotoxin superantigenic activation.The impact of different enterotoxins on the IS formation was further studied.(1)The clone and analysis of the major IS forming-related molecules:Three porcine immune cell surface molecules MHC?,MHC? and CD4 were amplified by PCR from porcine PBMC and 3D4/21.Then products were cloned into cloning vector pEASY-T1 or pEASY-blunt,and then sequenced.The results showed that sequences of these three molecules shared over 99% identity with those logged in GeneBank/NCBI.Primary structure of amino acids indicated that MHC? and CD4 molecules both included a transmembrance domain,and had Ig domain in the extracellular region.Besides,the glycosylation site,antigenic epitope and phosphorylation site of these three molecules were predicted for further study of cellular signal transduction.(2)The expression of major IS related molecules and the prepatation of polyclonal antibody:Expression and purity of CD28 and MHC?were done before infecting rabbits to gain polyclonal antibody.Then,ELISA was used to determine antibody titer,which were 1:3200 and 1:6400,respectively.The results of Western blot indicated CD28 and MHC? antibody had good reactivity with the corresponding molecules on immune cells.(3)The impact of the different enterotoxin on the IS formation:After the different enterotoxins stimulated PBMC and 3D4/21,the transcription level changes of IS related molecules(CD8-MHC?,CD4-MHC?,CD80/CD86-CD28/CTLA-4,ICAM-1-LFA-1)were detected by qPCR.The results showed SEs forced the transcription of IS related molecular above after the SEs stimulated the PBMC and mixed PBMC and 3D4/21,which could accelerate IS formation to activate T cells and increase the interaction between T cells and APC.ICAM-1 had a higher transcription level at 24 h,which facilitated the recognition of specific antigens in the early IS formation stage by TCR.The negative molecule CTlA-4 had the highest transcription level at 72 h,which could inhibit the excessive activation and proliferation of T cells.In intestinal tract,the transcription level of IS related molecules was increased after 5 hours of six enterotoxins stimulation,which might be related to inflammatory cells and T cell migration.Specially,higher transcription level of MHC?contributed to the superantigenic presenting.We analyzed the impact of core IS molecules on enterotoxin superantigenic activity through the sCD4-sMHC?,sTCR-SEs-sMHC? homology modeling and the intramolecular forces.The results showed that interamolecular interaction(hydrogen and saltbridge)between sCD4 and s MHC? could intensify the interaction between T cell and APC cell.SEA,SEK and SEQ formed different interface recognitions with cloned different TCRV?.So the stability of combination between diverse SEs and TCRV? was different.