Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers.
Antigen-specific CD4+ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4+ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4+ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4+ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4+ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4+ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4+ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.
Norimine
Junzo
J
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA. nori@vetmed.wsu.edu
Han
Sushan
S
Brown
Wendy C
WC
D000595:Amino Acid Sequence; D042322:Anaplasma marginale / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004912:Erythrocytes / Q000276:immunology / Q000382:microbiology; D005802:Genes, MHC Class II; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000235:genetics; D016056:Immunodominant Epitopes / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D014162:Transfection
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; C423282:BoLA-DRB3 antigen; D000949:Histocompatibility Antigens Class II; D016056:Immunodominant Epitopes; C449089:major surface protein 1a, Anaplasma; C103348:msp2 protein, Anaplasma marginale; D004247:DNA