Seitz et al. 10.1073/pnas.0604247103. |
Supporting Figure 4
Supporting Table 2
Supporting Figure 5
Supporting Data Set 1
Fig. 4. Relative positions of the primers for amplification of TCR a- and b-chains. The organization of TCR a- and b-chains is shown. Both chains are composed of variable (V), joining (J), and constant (C) regions. Between the V and J regions are the hypervariable N(D)N regions that contribute particularly to antigen recognition (the regions are not drawn to scale). We present the relative positions of the reverse transcription and PCR primers used in steps 3-6 in Fig. 1. After preamplification step 3, we tested which cells expressed the correct, expanded b-chain (step 4). Then, the unknown a-chains were amplified by using pools of primers (step 5) and verified by clone-specific primers (step 6). Because of their higher sensitivity, these steps may reveal further a-chain-positive cells. Because the b-chains were known from CDR3 spectratyping, clone-specific b-chain primers could be used. Twenty-four partly degenerate primers were used in one pool in preamplification step 3. In the nested a-chain PCR, step 5, we used in total 36 primers in five aliquots. Primers used for preamplification (step 3) bear the suffix "-out". All primers used in steps 4 and 5 are nested to the primers used in preamplification step 3 (suffix "-in").
Fig. 5. CDR3 spectratype analysis of TCR b-chains from muscle-biopsy samples of patients PM16488 and IBM15551. Muscle tissue from biopsy specimens was homogenized, and the cDNA was isolated and analyzed for clonal T cell expansions. Dominant single peaks were detected for Vb1-Jb1.2 in PM16488 (A) and Vb23-Jb1.1 in IBM15551 (B). Sequencing of the PCR products revealed that both peaks consist of one single sequence and are not superpositions of several sequences of identical CDR3 length, proving that T cell clones with the respective TCR b-chains are expanded. In both patients, several additional clonal expansions were also detectable (data not shown). This finding is in accordance with previous reports that in inflammatory myopathies the T cell infiltrates are often oligoclonal rather than monoclonal.
Table 2. Yields of TCR α- and β-chains of single TCR-transfected T hybridoma cells
No. of cells | Vβ17 chains | Vβ17 and Vα2.3 chains | |
Unlabeled | 89 | 49 | 48 |
Anti-Vβ17- and | |||
AP-stained | 90 | 14 | 6 |
58α―β― T hybridoma cells were transfected with TCR Vα2.3 and Vβ17 chains, immobilized by Cytospin on membranes, and isolated by microdissection. We compared acetone-fixed but unlabeled cells with acetone-fixed cells that were labeled with an anti-Vβ17 antibody and stained by alkaline phosphatase (AP). Single
cells were analyzed by RT-PCR for the expression of TCR α- and β-chains. We determined the β-chains by using clone-specific primers. Only β-chain-positive cells were subjected to α-chain amplification. We used a universal primer set that allows amplification of all α-chains irrespective of their V or J elements. The numbers of cells investigated and the yields of β- and α-chains identified are listed. As expected, we found significantly higher yields of β- and α-chains with unlabeled cells.