Peptide/MHC tetramer-based sorting of CD8⁺ T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.

Testing of T cell-based cancer therapeutics often involves measuring cancer antigen-specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8(+) T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer(+) populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor beta (TCR beta) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCR beta clonotypes in a patient with a UNC-CDK4-1 tetramer(+) population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCR beta repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCR beta clonotypes represented >17% of the entire TCR beta repertoire-far in excess of the UNC-CDK4-1 tetramer(+) frequency-indicating that the recurrent TCR beta clonotypes identified from UNC-CDK-4-1 tetramer(+) cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1-driven clonal T-cell expansion. Mapping recurrent TCR beta clonotype sequences onto TCR beta repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. (C) 2015 AACR.