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Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers

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Abstract

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.

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Acknowledgements

We are thankful to Kimberly Kegerreis, Shelley Whidbee, and Emma Karel for excellent technical assistance, Waithaka Mwangi for providing 293-F cells, and Guy Palmer for helpful comments on the manuscript. This work was supported by grants R01-AI44005 and R01-AI053692 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) and by the US Department of Agriculture Cooperative Agreement 58-5348-044. These experiments are in compliance with USDA and NIH guidelines for vertebrate animal use.

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  1. Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, 99164-7040, USA

    Junzo Norimine, Sushan Han & Wendy C. Brown

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  1. Junzo Norimine
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  2. Sushan Han
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  3. Wendy C. Brown
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Correspondence to Junzo Norimine.

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Norimine, J., Han, S. & Brown, W.C. Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers. Immunogenetics 58, 726–739 (2006). https://doi.org/10.1007/s00251-006-0140-3

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