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T-cell Receptor Repertoire in Matched MART-1 Peptide-stimulated Peripheral Blood Lymphocytes and Tumor-infiltrating Lymphocytes

Department of Surgery and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425 [D. J. C., M. C. W.]; National Tumor Institute of Milan, Milan, Italy 20133 [L. R.]; and Surgery Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. C., M. I. N.]

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic 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_(27–35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1_(27–35)). All cultures had a human leukocyte antigen A2-restricted, MART-1_(27–35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR ß clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8–16 subfamilies were used) and a predominant usage of a different variable ß 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 expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in 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 clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.

¹ To whom requests for reprints should be addressed, at Medical University of South Carolina, Department of Surgery, 171 Ashley Avenue, Charleston, SC 29425. Phone: (803) 792-1387; Fax: (803) 792-2048.

Received 6/30/97. Accepted 10/ 3/97.

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