Fig. 1.

Strategy to identify paired TCR α- and β-chains from microdissected tissue-infiltrating T cells. In the first step, we investigated clonal T cell expansions by CDR3 spectratyping of the TCR β-chains. Then, in step 2, we stained sections from frozen biopsy specimens with appropriate anti-Vβ antibodies and isolated positive cells by microdissection. After cDNA preparation and a preamplification PCR for α- and β-chains (step 3), we tested the microdissected cells for expression of the expanded β-chain by PCR using clone-specific β-primers (step 4). Cells that expressed the correct sequence, i.e., the sequence identified by CDR3 spectratyping, were examined for the matching α-chains with a universal primer set that allows amplification of all TCR α-chain sequences (step 5). The preamplification product served as template. Based on the α-chain sequences identified in step 5, we designed clone-specific α-chain primers and reexamined all β-chain-positive cells (step 6), which allowed identification of additional αβ-pairs because clone-specific primers are more efficient than the universal primer set. For functional studies, the corresponding α- and β-chains were expressed on the surface of a T hybridoma cell line (step 7).