Establishment And Application Of A Flow Cytometric Assasy For The Detection Of Chicken Cytotoxic T Lymphocytes Based On CD107a Marker

Cytotoxic T-lymphocytes（CTL）and play critical roles in the immunity against intracellular pathogens and tumors.To detecte the frequency and function of CTL is very important for the evaluation of vaccine efficacy and elucidating the mechanisms of immune protection.CD 107a（Lysosome-associated membrane protein 1,LAMP-1）,as a major lysosomeassociated membrane protein,is mostly distributed on lysosomal and endosomal membranes.CD 107a protein is transiently trafficked onto the surface of CTL,thus surface expression of CD 107a represents the killing activity of CTL.The detection of CD 107a expression on lymphocytes by flow cytometry is widely used to evaluate the functions of human and mouse CTL,overcoming the disadvantages of traditional killing assays（e.g.chromium release assay）that require MHC match,cumbersome procedures and special experimental conditions.However,this method has not been ideally applied on chickens so far,the key reason is because of the lack of effective antibodies against chicken CD 107a.In this study,a truncated chicken CD 107a protein（chCD107a）was expressed in E.coli and used to immunize mice.Through hybridoma technique,monoclonal antibodies（mAb）recognizing natural chCD107a were prepared.Using one mAb,a flow cytometric assay was established and used to detect the surface expression of chCD107a on chicken cytotoxic T cells during avian viral infections.The details of this study are as follows.1.The cloning and recombinant expression of the truncated fragment of chicken CD107aBased on a chCD107a reference gene sequence in GenBank,the coding region of 6001245 bp was codon-optimized for E.coli and cloned into the expression vectors pET-32a（+）and pCAGGS,respectively,to construct recombinant plasmids pET-32a-sh-chCD107a and pCAGGS-sh-chCD107a.By transforming the pET-32a-sh-chCD107a into the Rosetta（DE3）strain and induction with IPTG,a fusion protein Trx-His-chCD107a was expressed,with a molecular weight of 39 kDa,consistent with the predicted size.The full-length chCD107a gene was amplified and cloned into pCAGGS to construct recombinant plasmid pCAGGSchCD107a.The plasmids pCAGGS-co-chCD107a and pCAGGS-chCD107a were transfected into DF-1 cells,respectively.The results from indirect immunofluorescence（IFA）and Western-Blotting indicated that recombinant chCD107a was successfully expressed in DF-1 cells.2.The generation of monoclonal antibodies against chicken CD107a and its preliminary application in a flow cytometric assay for the detection of chicken cytotoxic T lymphocytes.Through hybridoma technique,nine mAbs,named as 2E7,2E10,2F8,2A10,2A8,3C10,3B7,3F5 and 4F11 were generated using the recombinant Trx-His-chCD107a protein as immunogen.IFA results showed that all nine mAbs recognized the truncated and full-length chCD107a expressed in the transfected DF-1 cells.However,only five mAbs（2E7,2E10,4F11,3F5,2F8）recognized the full-length chCD107a protein in WB.Further identification by WB showed that four monoclonal antibodies（2E10,2E7,3C10,4F11）recognized the glycosylated chCD107a protein expressed in chicken splenocytes.Round coverslip results showed that five monoclonal antibodies（2E7,2E10,2F8,3F5,4F11）recognized natural chCD 107a on chicken lymphocytes.Through intracellular staining combined with flow cytometry,the reactivity of each mAb with intracellular chCD107a was identified,the results showed that six mAb（2E7,2E10,2F8,2A10,3C10,4F11）stained the CD107a inside the cells strongly,3F5 reacted moderately and 2A8 and 3B7 did not react.Cell surface staining indicated that mAb 2E10 detected the surface expression of chCD107a on chicken splenocytes after activation with PMA and ionomycin and 3C10 had weak reactivity.Using mAb 2E10 to detect CD 107a expression on the surface of chicken lymphocytes after infection with Newcastle disease virus（NDV）,it was found that about 2.3%total T lymphocytes was CD107a-positive at 3 days after infection.After immunization with attenuated infectious laryngotracheitis disease virus（ILTV）,there were 12.7%、7%CD107a-positive CD3+and CD3+CD8α+T lymphocytes,significantly different from the percentages of those cells in the unimmunized group.These results indicated that immunization with ILTV activiated cytotoxic T lymphocytes.In summary,mAbs against chCD107a that recognizes natural chCD107a on the surface of chicken lymphocytes were generated in this study and can be applied for the detection of chicken cytotoxic T lymphocytes by flow cytometry,providing a powerful tool for investigating the function of chicken T cells in disease and immunity.