Simultaneous detection of many T-cell specificities using combinatorial tetramer staining

Abstract

The direct detection of antigen-specific T cells using tetramers of soluble peptide-major histocompatibilty complex (pMHC) molecules is widely used in both basic and clinical immunology. However, the number of specificities that can be assessed simultaneously has been a major limitation. Here we describe and validate a method using combinations of fluorescent pMHC tetramers to simultaneously detect and enrich for many (>or=15) T-cell specificities in a single human blood sample.

Figure 1

Combinatorial pMHC tetramer staining. Enriched CD8⁺ T cells from a CMV sero-positive donor were split into 15 aliquots and stained separately with the 15 CMV tetramer color combinations, washed, mixed together and run as a single sample. All panels show flow cytometry scatter plots. (a) Live CD8⁺ T cells were gated into four populations labeled b-e, based on threshold fluorescence of APC and PE-Cy7 as shown. Each population is represented in (b-e) and segregated based on PE and PE-Cy5 threshold fluorescence. The relative abundance of cells in each of the 15 tetramer-positive populations is represented in a bar chart in (f). In (g), cells from a different CMV sero-positive donor were stained with CMV tetramers labeled with PE, PE-Cy5, PE-Cy7, APC and all possible combinations of PE-Cy5.5 and APC-Cy7, washed, mixed and analyzed. A separate population was resolved for each possible color combination used. (h-l) Blood samples were processed and tetramer stained using a prepared cocktail of 15 different pMHC specificities represented by combinations of PE, PE-Cy5, PE-Cy7 and APC conjugated streptavidins (peptide scheme A, Supplementary Table 2 online). Cells were gated on PE and APC fluorescence (h) into populations labeled i-l, each of which is shown (i-l) further fractionated based on its PE-Cy5 and PE-Cy7 fluorescence.

Figure 2

Specificity of combinatorial pMHC tetramer staining. (a) The percentage of cells labeled with the six different pMHC tetramers using two different staining schemes (Supplementary Table 2 online). (b) The percentage of cells stained with the indicated pMHC tetramers in cells co-cultured with T2 cells in the presence of a control peptide (HCV peptide, black bars) or the peptide corresponding to the pMHC tetramer reagent (grey bars); representative of three independent experiments. (c) Tetramer staining from four donor samples, each stained separately with 15 different PE-labeled pMHC tetramers (scheme C; Supplementary Table 2 online) is plotted on the x axis. Staining of the same sample with three separate preparations of combinatorial pMHC tetramer cocktails (Supplementary Table 2 online) is plotted +/- SEM on the y axis (d) The percentage of cells detected for each pMHC specificity before (gray bars) and after tetramer enrichment (black bars) using peptide scheme D (Supplementary Table 2).