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 UNCCDK4-1/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 b (TCRb) sequencing, we identified recurrent UNC-CDK4-1 tetramer-associated TCRb clonotypes in a patient with aUNC-CDK4-1 tetramer⁺ population, suggestingin vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRb repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer-associated TCRb clonotypes represented >17% of the entire TCRb repertoire - far in excess of the UNCCDK4-1 tetramer⁺ frequency - indicating that the recurrent TCRb 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 TCRb clonotype sequences onto TCRb repertoires can help confirm or refute antigen-specific T-cell expansion.