@article{87247de7af74415b8de1e76f564a2621,
title = "Screening self-peptides for recognition by mouse alloreactive CD8+ T cells using direct ex vivo multimer staining",
abstract = "Here, we present a protocol to identify immunogenic self-peptide/allogeneic major histocompatibility complex (MHC) epitopes. We describe the generation of enriched alloreactive CD8+ T cells by priming mice with a skin graft expressing the allogeneic MHC class I molecule of interest, followed by boosting with a liver-specific AAV vector encoding the heavy chain of that donor MHC allomorph. We then use a peptide-exchange approach to assemble a range of peptide-MHC (pMHC) multimers for measuring recognition of the various epitopes by these alloreactive T cells. For complete details on the use and execution of this protocol, please refer to Son et al. (2021).1",
keywords = "Antibody, Cell Biology, Cell isolation, Immunology, Model Organisms, Molecular Biology",
author = "Son, {Eric T.} and Moumita Paul-Heng and Mario Leong and Chuanmin Wang and Hill, {Alexandra E.} and Martina Denkova and Purcell, {Anthony W.} and Mifsud, {Nicole A.} and Sharland, {Alexandra F.}",
note = "Funding Information: We thank Laboratory Animal Services, University of Sydney, for the provision of high-quality animal care, Sydney Cytometry for flow cytometry support, and the Vector and Genome Engineering Facility, CMRI for AAV vector production and advice. This work was funded by the National Health and Medical Research Council of Australia (NHMRC); Principal Research Fellowship (1137739) to A.W.P. and Ideas Grant (APP1183806) to A.F.S. and N.A.M. M.L. received a Research Training Program Postgraduate Award from the Department of Education and Training, Australian Federal Government, while M.P-.H. was supported by an Earl Owen Fellowship from the Sydney Medical School Foundation, and E.T.S. was supported by the Royal Prince Alfred Hospital Transplant Institute. This project was also supported by Grants-in-aid from Sydney Medical School Foundation and the Myee Codrington Medical Research Foundation (both to A.F.S.). E.T.S. A.W.P. N.A.M. and A.F.S. designed experiments. E.T.S. M.P-.H. M.L. C.W. A.E.H. and M.D. developed the methodologies described here. E.T.S. M.P-.H. and A.F.S. analysed data. E.T.S. M.P-.H. and A.F.S. wrote the manuscript. All authors read and approved the manuscript. The authors declare no competing interests. E.T.S. M.P-.H. N.A.M. A.W.P. and A.F.S. were named investigators on a patent previously lodged by the University of Sydney and Monash University. This patent is no longer current. Funding Information: We thank Laboratory Animal Services, University of Sydney, for the provision of high-quality animal care, Sydney Cytometry for flow cytometry support, and the Vector and Genome Engineering Facility, CMRI for AAV vector production and advice. This work was funded by the National Health and Medical Research Council of Australia (NHMRC); Principal Research Fellowship ( 1137739 ) to A.W.P. and Ideas Grant ( APP1183806 ) to A.F.S. and N.A.M. M.L. received a Research Training Program Postgraduate Award from the Department of Education and Training , Australian Federal Government , while M.P-.H. was supported by an Earl Owen Fellowship from the Sydney Medical School Foundation , and E.T.S. was supported by the Royal Prince Alfred Hospital Transplant Institute . This project was also supported by Grants-in-aid from Sydney Medical School Foundation and the Myee Codrington Medical Research Foundation (both to A.F.S.). Publisher Copyright: {\textcopyright} 2022 The Authors",
year = "2023",
month = mar,
day = "17",
doi = "10.1016/j.xpro.2022.101943",
language = "English",
volume = "4",
journal = "STAR Protocols",
issn = "2666-1667",
publisher = "Cell Press",
number = "1",
}