Figure 3.

TCR-Vβ13⁺ transcripts are antigen-focused in the LEW.1WR1 islet. A: CDR3 sequence variants in islet Vβ13⁺ transcripts were analyzed by direct sequence analysis. Vβ13⁺ transcripts were PCR-amplified with a Vβ13-specific forward primer and a Cβ1/2 reverse primer. Amplicons were cloned and sequenced, and sequences were aligned with a multiple sequence alignment program (ClustalW). The percentage occurrence of each unique CDR3 sequence cluster was calculated and is plotted above. Each black or white segment on the graph represents a unique CDR3 sequence. B: Frequent sequences are annotated on the graph (1–7) and listed. The naïve periphery (spleen untreated) does not display a Vβ13-specific antigen-focused response evident by the large number of sequences all observed at low frequency. Vβ13⁺ T-cell transcripts do not display an antigen-specific clonal expansion on day 4. Two separate experiments, of pooled islets from eight LEW.1WR1 rats each, indicate antigen-specific expansion of a limited number of CDR3 clonotypes in the islet 5 days after poly I:C. For sequences analyzed, N = 117 cultured islet T cells, 66 islet day 7, 74 islet day 5-experiment (expt) 1, 71 islet day 5-experiment 2, 64 islet day 4, and 127 spleen untreated. Sequences represent results from pooled LEW.1WR1 islets; N = 8 cultured T cells, 2 day 7, 8 day 5-experiment 1, 8 day 5-experiment 2, 2 day 4, 2 untreated spleen. B: The most frequent junctional sequences identified among Vβ13 transcripts are listed here. The entire junction sequence is shown starting with the end of the V region; CDR3 sequences in bold are up to the conserved phenylalanine (F), and in the remainder the Jβ segment is displayed. All sequences identified in the analysis are listed in Supplementary Table 2.