Use of "one-pot, mix-and-read" peptide-MHC class I tetramers and predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle.
Peptide-major histocompatibility complex (p-MHC) class I tetramer complexes have facilitated the early detection and functional characterisation of epitope specific CD8+ cytotoxic T lymphocytes (CTL). Here, we report on the generation of seven recombinant bovine leukocyte antigens (BoLA) and recombinant bovine β2-microglobulin from which p-MHC class I tetramers can be derived in ~48 h. We validated a set of p-MHC class I tetramers against a panel of CTL lines specific to seven epitopes on five different antigens of Theileria parva, a protozoan pathogen causing the lethal bovine disease East Coast fever. One of the p-MHC class I tetramers was tested in ex vivo assays and we detected T. parva specific CTL in peripheral blood of cattle at day 15-17 post-immunization with a live parasite vaccine. The algorithm NetMHCpan predicted alternative epitope sequences for some of the T. parva CTL epitopes. Using an ELISA assay to measure peptide-BoLA monomer formation and p-MHC class I tetramers of new specificity, we demonstrate that a predicted alternative epitope Tp229-37 rather than the previously reported Tp227-37 epitope is the correct Tp2 epitope presented by BoLA-6*04101. We also verified the prediction by NetMHCpan that the Tp587-95 epitope reported as BoLA-T5 restricted can also be presented by BoLA-1*02301, a molecule similar in sequence to BoLA-T5. In addition, Tp587-95 specific bovine CTL were simultaneously stained by Tp5-BoLA-1*02301 and Tp5-BoLA-T5 tetramers suggesting that one T cell receptor can bind to two different BoLA MHC class I molecules presenting the Tp587-95 epitope and that these BoLA molecules fall into a single functional supertype.
International Livestock Research Institute (ILRI), P,O, Box 30709, Nairobi 00100, Kenya. n.svitek@cgiar.org.
Nicholas
N
Svitek
Andreas Martin
AM
Hansen
Lucilla
L
Steinaa
Rosemary
R
Saya
Elias
E
Awino
Morten
M
Nielsen
Søren
S
Buus
Vishvanath
V
Nene
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006649:Histocompatibility Antigens; D008285:Major Histocompatibility Complex; D008969:Molecular Sequence Data; D016415:Sequence Alignment / Q000662:veterinary; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D016797:Theileria parva / Q000276:immunology*; D001613:beta 2-Microglobulin / Q000276:immunology*
D018984:Epitopes, T-Lymphocyte; D006649:Histocompatibility Antigens; D001613:beta 2-Microglobulin