A single autoimmune T cell receptor recognizes more than a million different peptides

Abstract

The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.

FIGURE 1.

Decamer CPL scan of the 1E6 CD8⁺ T cell clone. A, 6 × 10⁴ C1R-A2 cells were pulsed in duplicate with each mixture from a decamer CPL (100 μg/ml) at 37 °C. After 2 h, 3 × 10⁴ 1E6 CD8⁺ T cells were added and incubated overnight. Supernatant was harvested and assayed for MIP1β. B, data from A are displayed as a box plot summary. The index insulin peptide sequence is shown below the boxes in black.

FIGURE 2.

Recognition of 30 peptides sampled at random from a large peptide set (motif, RQWGPDP{A/C/D/F/H/I/K/L/M/N/P/R/S/V/Y}{A/C/G/H/I/K/L/M/N/P/Q/R/S/T/V}A; total set size = 225). 6 × 10⁴ C1R-A2 cells were pulsed with peptides at various concentrations. After 2 h, 3 × 10⁴ 1E6 CD8⁺ T cells were added and incubated overnight. Supernatant was harvested and assayed for MIP1β. Each panel displays titrations of five different peptides relative to index. A, titrations of peptides with the highest functional sensitivities. F, titrations of peptides with the lowest functional sensitivities. Error bars, S.D. from the mean of two replicates. pEC₅₀ values for each peptide are displayed in supplemental Table S3.

FIGURE 3.

Recognition of 30 peptides sampled at random from a large peptide set (motif,RQWGP{D/F}{P/F}XX{A/I/L/V}; total set size = 5,776). Details are as described for Fig. 2.

FIGURE 4.

Recognition of 30 peptides sampled at random from a large peptide set (motif, RQXGPDXXXA; total set size = 19⁴). Details are as described for Fig. 2.

FIGURE 5.

Recognition of 30 peptides sampled at random from a large peptide set (motif, XQXGPDXXXV; total set size = 19⁵). Details are as described for Fig. 2.

FIGURE 6.

Recognition of 30 peptides drawn from a CPL-based importance sampling set with effective size = 1. 66 × 10⁸ (calculated from the sampling entropy) (first set). Details are as described for Fig. 2.

FIGURE 7.

Recognition of 30 peptides drawn from a CPL-based importance sampling set with effective size = 1. 66 × 10⁸ (calculated from the sampling entropy) (second set). Details are as described for Fig. 2.

FIGURE 8.

1E6 CD8⁺ T cells can recognize more than one million different decamer peptides. I, RQWGPDP{A/C/D/F/H/I/K/L/M/N/P/R/S/V/Y}{A/C/G/H/I/K/L/M/N/P/Q/R/S/T/V}A (set size 225; 30 peptides sampled at random). II, RQWGP{D/F}{P/F}XX{A/I/L/V} (set size 5,776; 30 peptides sampled at random). III, RQXGPDXXXA (set size 19⁴; 30 peptides sampled at random). IV, XQXGPDXXXV (set size 19⁵; 30 peptides sampled at random). In the motifs, X denotes any one of the 19 amino acids excluding cysteine. Va and Vb, two replicates of a biased sampling set (effective set size 1.66 × 10⁸, calculated from the sampling entropy); each set of 30 peptides was sampled with bias toward strong agonists, where the bias weights were based on the primary CPL scan. Relative functional sensitivities (pEC₅₀ − pEC₅₀ index) are plotted as survivor curves. Gray dashed line, theoretical curve (supplemental Equation S2, with α = 4.5, β = 10, γ = 2, N₀ = 20⁸ anchorable peptides). The biased samples (black dashed and dotted lines) estimate the TCR degeneracy spectrum, whereas the motif-based samples (black solid lines) provide a lower boundary to the TCR degeneracy spectrum.