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R. G. Matnani, M. Ward, J. Woodward; Cloning and Analysis of Retinal Autoantigen Specific T Cell Receptors. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4768. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Experimental autoimmune uveitis (EAU) can be induced in the B10.RIII mouse strain by immunization with retinal autoantigen interphotoreceptor retinoid binding protein (IRBP) or the immunodominant peptide sequence, IRBP 161-180. In order to develop T cell receptor (TCR) transgenic mice specific for the retinal antigen and to study the presentation of IRBP by antigen presenting cells (APCs), we have isolated and characterized four IRBP 161-180 specific T cell clones and molecularly cloned alpha and beta receptors specific for IRBP 161-180 from three T cell clones.
Four IRBP 161-180 specific T cell clones were obtained by limiting dilution cloning. Clonality was determined by FACS analysis using a panel of variable beta chain antibodies (Vß1 to Vß17). The antigen specificity and apparent affinity of the T cell clones was determined by IRBP 161-180 specific proliferation. The productively rearranged beta receptors were obtained using RT-PCR utilizing corresponding Vß sense and Cß anti-sense primers. The productively rearranged alpha receptors were identified and cloned utilizing 5' RACE (rapid amplification of cDNA ends) technique. The alpha and beta receptors obtained were confirmed by sequencing and were sub-cloned into a T cell expression cassette.
The four IRBP specific T cell clones express different Vα and Vß receptors but all are specific for IRBP 161-180. The T cell clones also exhibit different apparent affinities and different I-A restriction for IRBP 161-180. The T cell clones express different V-J and V-D-J gene segments and the amino acid sequence of the peptide binding (CDR3) region is unique for each T cell clone, indicating different properties of each T cell clone specific for the same retinal antigen.
We have isolated T cell clones specific for a retinal self-antigen IRBP. In addition, we have cloned and sequenced the productively rearranged alpha and beta T cell receptors from three clones. These receptors will be useful for the development of TCR transgenic mice and to detect rare IRBP expressing antigen presenting cells in the thymus and the periphery.
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