Tumors that recurred after TCRα transgenic T cell (TCRαTg) therapy harbored multiple mutations in the P1A epitope. P1A gene fragments were amplified from recurrent tumors and analyzed by either restriction enzyme mapping or by cloning followed by sequencing. (a) Restriction mapping by a panel of restriction enzymes that identify mutations P1A(1P) and P1A(7P) (HaeIII), P1A(6R) (Fnu4HI), and any alteration in nucleotides encoding P8 and P9 (BbsI). P1A fragments were amplified from tumor cells from mice that received TCRαTG wild-type T cell treatment (Ntg). Note the presence of mutation P1A(6R) and alterations in P8 or P9 in the TCR α chain–transgenic group, but not in the nontransgenic group. Similar results were observed in another recurrent tumor from mice that received transgenic T cells (data not shown). (b) Sequencing of the P1A fragment that resisted digestion by BbsI. Note that of the three clones analyzed, two had mutation P1A(8G) and one had mutation P1A(8L).