Detection of low avidity CD8+ T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers

The development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) molecules conjugated in multimeric form to fluorescent labels has enabled the physical quantification and characterization of antigen-specific CD8+ T cell populations by flow cytometry. Several factors determine the binding threshold that enables visualization of cognate CD8+ T cells with these reagents; these include the affinity of the T cell receptor (TCR) for pMHCI antigen. Here, we show that multimers constructed from peptide-human leukocyte antigen (pHLA) A⁎0201 monomers engineered in the heavy chain α2 domain to enhance CD8 binding (KD ≈ 85 μM) without impacting the TCR binding platform can detect cognate CD8+ T cells bearing low affinity TCRs that are not visible with the corresponding wildtype pHLA A⁎0201 multimeric complexes. Mechanistically, this effect is mediated by a disproportionate enhancement of the TCR/pMHCI association rate. In direct ex vivo applications, these coreceptor-enhanced multimers exhibit faithful cognate binding properties; concomitant increases in background staining within the non-cognate CD8+ T cell population can be resolved phenotypically using polychromatic flow cytometry as a mixture of naïve and memory cells. These findings provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8+ T cell populations; such coreceptor-enhanced multimeric reagents are likely to be useful in a multitude of settings for the detection of auto-immune, tumor-specific and cross-reactive CD8+ T cells.