Peptide-Major Histocompatibility Complex Class I Tetramers with Enhanced Coreceptor Binding Properties Enable Visualization of Low Avidity Leukemia-Associated Antigen-Specific CD8⁺ T Cells.

There is substantial evidence to indicate that CD8⁺ T cells specific for epitopes derived from overexpressed antigens form part of the immune response to leukemia in patients both prior to and after stem cell transplantation (SCT) and following adoptive immunotherapy with donor leukocyte infusion. However, the immunobiology of these CD8⁺ T cell populations has proven difficult to study directly ex vivo due to technical obstacles that preclude the reliable detection of low frequency CD8⁺ T cells that bind antigen poorly. We engineered a mutation (Q115E) in the heavy chain a2 domain of HLA-A*0201 that moderately enhances monomeric CD8 binding without impacting the TCR binding platform. Combined flow cytometric studies and biophysical analysis of recombinant TCR/peptide-major histocompatibility complex class I (pMHCI) interactions showed that tetrameric complexes of these coreceptor-enhanced HLA-A*0201 molecules were more sensitive reagents for the detection of specific CD8⁺ T cells at the clonal level compared to corresponding tetramers produced with wildtype HLA-A*0201 complexes; thus, the threshold of detection was enhanced approximately twofold relative to the monomeric TCR/pMHCI cut-off (K_D=80mM) observed with the wildtype tetramer. These novel reagents were incorporated in a polychromatic flow cytometric panel to detect CD8⁺ T cell populations specific for HLA-A*0201-restricted epitopes derived from Wilm’s tumor-1 (WT1) and proteinase 3 (PR1) in the bone marrow and peripheral blood of patients with acute myeloid leukemia before SCT. The HLA-A*0201-restricted CMV pp65-derived epitope NLVPMVATV, which induces high avidity cognate CD8⁺ T cell populations, was used as a control. Both wildtype and coreceptor-enhanced tetramers consistently identified populations of NLVPMVATV-specific CD8⁺ T cells that were identical in magnitude; molecular analysis of TCR usage in these populations after high resolution flow cytometric sorting confirmed the equivalent cognate binding properties of these reagents. In contrast, coreceptor-enhanced tetramers enabled the visualization and simultaneous immunophenotyping of CD8⁺ T cell populations specific for PR1 and WT1 in patients with AML that were not detectable with the corresponding wildtype reagent. These results provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8⁺ T cell populations by flow cytometry; such coreceptor-enhanced tetrameric reagents are likely to be useful in a multitude of settings for the detection of tumor-specific and autoreactive CD8⁺ T cells.