T-cell Receptor Repertoire in Matched MART-i Peptide-.stimulatedPeripheral Blood Lymphocytes and Tumor-infiltrating Lymphocytes

Characterizationof tumor-associatedantigens (TAAs) recognized by CTLSmakes the considerationof therapeuticstrategiesbased on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendntic cells, and direct vaccination with TAA derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLS are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27..35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(V@S)-reactlve PBL and TIL cultures were generated from three patients by in vitrostimulation with an immunodominant peptide of MART-i (MART-1(fl@,,)).AUcad tures had a human leukocyte antigen A2-restricted, MART-1(27@,)-spe cific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR fi clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8—16subfamilies were used) and a predominant usage of a different variable @J chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansionwas presentfor both PBLsand TILs; the relativeusageof each prevailingBV was more markedin TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLS, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared(8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypesderived from predominantTCR subfamiliesin the PBLs and TILs were present in each patient, suggesting peptide stimulatedexpansionin both biologicalcompartments.These studies sug