Novel soluble HLA-A2/MELAN-A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma

Multimeric MHC I-peptide complexes containing phycoerythrin-streptavidin are widely used to detect and investigate antigenspecific CD8+ (and CD4+) T cells. Because such reagents are heterogeneous, we compared their binding characteristics with those of monodisperse dimeric, tetrameric and octameric complexes containing linkers of variable length and flexibility on Melan-A-specific CD8+ T cell clones and peripheral blood mononuclear cells (PBMC) from HLA-A*0201⁺ melanoma patients. Striking binding differences were observed for different defined A2/Melan-A_26-35 complexes on T cells depending on their differentiation stage. In particular, short dimeric but not octameric A2/Melan-A_26-35 complexes selectively and avidly stained incompletely differentiated effector-memory T cells clones and populations expressing CD27 and CD28 and low levels of cytolytic mediators (granzymes and perforin). This subpopulation was found in PBMC from all six melanoma patients analyzed and proliferated on peptide stimulation with only modest phenotypic changes. By contrast influenza matrix_58-66 -specific CD8+ PBMC from nine HLA-A*0201⁺ healthy donors were efficiently stained by A2/Flu matrix_58-61 multimers, but not dimer and upon peptide stimulation proliferated and differentiated from memory into effector T cells. Thus PBMC from melanoma patients contain a differentiation defective sub-population of Melan-A-specific CD8+ T cells that can be selectively and efficiently stained by short dimeric A2/Melan- A_26-35 complexes, which makes them directly accessible for longitudinal monitoring and further investigation.