title: Detection of low avidity CD8(+) T cell populations with coreceptor-enhanced peptide-major histocompatibility complex class I tetramers.
abstract: 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) A0201 monomers engineered in the heavy chain alpha2 domain to enhance CD8 binding (K(D) approximately 85 microM) 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 A0201 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.
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: J Joseph
authors.initials: JJ
authors.lastname: Melenhorst
authors.forename: Phillip
authors.initials: P
authors.lastname: Scheinberg
authors.forename: Pratip K
authors.initials: PK
authors.lastname: Chattopadhyay
authors.forename: Anna
authors.initials: A
authors.lastname: Lissina
authors.forename: Emma
authors.initials: E
authors.lastname: Gostick
authors.forename: David K
authors.initials: DK
authors.lastname: Cole
authors.forename: Linda
authors.initials: L
authors.lastname: Wooldridge
authors.forename: Hugo A
authors.initials: HA
authors.lastname: van den Berg
authors.forename: Ethan
authors.initials: E
authors.lastname: Bornstein
authors.forename: Nancy F
authors.initials: NF
authors.lastname: Hensel
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Douek
authors.forename: Mario
authors.initials: M
authors.lastname: Roederer
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Sewell
authors.forename: A John
authors.initials: AJ
authors.lastname: Barrett
authors.forename: David A
authors.initials: DA
authors.lastname: Price
mesh_terms: D000595:Amino Acid Sequence; D016827:CD8 Antigens / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D005434:Flow Cytometry; D015234:HLA-A Antigens / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016827:CD8 Antigens; D015234:HLA-A Antigens; D011948:Receptors, Antigen, T-Cell