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) 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 wild type 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.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

“…Jurkat cells stably expressing the ILA1 TCR (called Jurkat-ILA1 hereafter), as well as monomeric human HLA-A2 complexed with the ILA peptide and several altered peptides (3G, 8T, 5Y, 8E) were used in this study. These peptide ligands have been characterized previously (31,66,67). Their K D values are as follows: 3G (2.9 μM), 8T (24 μM), ILA (32 μM), 5Y (250 μM), and 8E (>500 μM).…”

Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase

“…We have previously characterized a number of APLs that alter the T-cell activation profile and TCR binding affinity of the ILA1 T-cell clone (9, 15, 16). To investigate how these ligands adjust TCR interactions to tune affinity, we solved the structure of four APLs (A2-ILA3G8R, IL G KFLH R L; A2-ILA3G, IL G KFLHWL; A2-ILA8T,ILAKFLH T L; and A2-ILA8E, ILAKFLH E L), included our previously published APL structure (A2-ILA8R, ILAKFLH R L) (23), and used the ILA1-A2-ILA complex as a model.…”

“…We have previously characterized a limited number of altered peptide ligands (APLs) for the ILA1 T-cell clone that exhibit different potencies in terms of T-cell activation (9, 15), corresponding to a wide range of binding affinities with the ILA1 TCR (15–17). These previous findings clearly demonstrate that the ILA1 T-cell clone can recognize multiple different peptide ligands.…”

High avidity myeloid leukemia-associated antigen-specific CD8+ T cells preferentially reside in the bone marrow

“…[10][11][12] Tetrameric D227K/T228A pHLA A*0201 complexes selectively identify high avidity cognate CD8 ϩ T cells [13][14][15] ; in contrast, Q115E pHLA A*0201 tetramers enable the inclusive identification of low avidity cognate CD8 ϩ T cells. 16 Biotinylated monomeric D227K/T228A, wildtype (WT) and Q115E pHLAA*0201 complexes were produced for the following epitopes: (1) CMV pp65 495-503 (NLVPMVATV) 17 ; (2) WT1 126-134 (RMFPNAPYL) 5 ; and (3) PR3 169-177 /HNE 168-176 (PR1; VLQELNVTV). 3,6 All tetramers were prepared afresh from cryopreserved monomers to eliminate potential bias arising from differential protein stability.…”

“…Gating of antigen-specific CD8 ϩ T cells was performed after background assessment, which differs according to the coreceptor binding properties of each pHLA A*0201 tetrameric form as described previously. 11,16,43 Individual plots were examined visually by 2 independent observers; only consistent interpretations were included for further analysis. (B) Representative data showing the identification of PR1-specific (left columns) and CMV-specific (right columns) CD8 ϩ T-cell populations in the peripheral blood (PB) and bone marrow (BM) of a patient with CML pre-HSCT (patient 13; Table 1).…”