T cell receptor signaling is limited by docking geometry to peptide-major histocompatibility complex

Abstract

T cell receptor (TCR) engagement of peptide-major histocompatibility complex (pMHC) is essential to adaptive immunity, but it is unknown whether TCR signaling responses are influenced by the binding topology of the TCR-peptide-MHC complex. We developed yeast-displayed pMHC libraries that enabled us to identify new peptide sequences reactive with a single TCR. Structural analysis showed that four peptides bound to the TCR with distinct 3D and 2D affinities using entirely different binding chemistries. Three of the peptides that shared a common docking mode, where key TCR-MHC germline interactions are preserved, induced TCR signaling. The fourth peptide failed to induce signaling and was recognized in a substantially different TCR-MHC binding mode that apparently exceeded geometric tolerances compatible with signaling. We suggest that the stereotypical TCR-MHC docking paradigm evolved from productive signaling geometries and that TCR signaling can be modulated by peptides that are recognized in alternative TCR-pMHC binding orientations.

Figure 1. The 42F3 and 2C TCRs and their interactions with H2-L^d–QL9.

(A) Primary sequence alignment of the six CDR loops of 42F3 and 2C TCRs. (B) Ribbon representations of the 42F3 and 2C TCRs in complex with QL9-H2-L^d aligned on the MHC (left), Top-down perspective of the six CDR loops of 2C and 42F3 (loops) on the surface of H2-L^d (right). 2C is lighter shade, 42F3 is darker shade (C) Contacts made by the CDR3 loops to the QL9 peptide for both 2C (lighter shaded colors) and 42F3 (darker shaded colors) TCRs are depicted. (D) Contacts made by the Vα and Vβ CDR1 and CDR2 loops to the MHC helices. The MHC is colored green, QL9 is colored yellow, TCRα is colored firebrick and salmon and TCRβ is colored slate and palecyan for 42F3 and 2C, respectively. See also Figures S1 and S4.

Figure 2. Yeast surface display of peptide libraries presented by H2-L^d

(A) Schematics of yeast displayed pMHC libraries. Inset is the stick representation of QL9 from the structure shown in Figure 1, showing the ‘up-facing’ recognition epitope of the 2C and 42F3 TCRs. (B) Two dimensional plot of the final enriched yeast populations of the random, TCR contact and MHC contact libraries enriched by FACS. Residues varied in each library are indicated in red where X represents 32 codons (NNK) encoding all 20 amino acids. Positions with fixed sequence are indicated in black. (C) Sequences of 42F3 peptides isolated from individual yeast colonies. Selected K_D measurements are from SPR equilibrium values. See also Figure S1.

Figure 3. T cell activation by peptide antigens

IL-2 release for (A) CD8-negative and (B) CD8-positive 42F3 T cells stimulated by peptide (10μM) loaded APCs with an effector:target ratio of 1:1. Full dose-response curves are shown in Figure S4 (C) IL-2 release for CD8⁺ 42F3 T cells stimulated with peptide loaded H2-L^d-Ig dimers. (D) p3A1 antagonist assay where 42F3 T cells stimulated with 100nM p4B3 were assayed for IL-2 production over a range of p3A1 concentrations. The stimulatory anti-CD3 (2C11) antibody and/or the endogenous peptide QL9 were used to assay IL-2 sensitivity in each ELISA. The addition of no peptide and the irrelevant H2-L^d restricted MCMV epitope (YPHFMPTNL) were used to assay peptide independent IL-2 production in each experiment. Peptides showing CD8 dependence were colored cyan, peptides showing CD8 independence were colored green,. See also Figure S2.

Figure 4. 2D and 3D measurements of peptide-MHC interactions with 42F3

(A) 2D affinities (A_cK_A) derived from T cell adhesion frequency and thermal fluctuation assays using pMHC coated RBCs. (B) SPR derived 3D affinities (expressed as K_A for comparison to 2D) using sc H2-L^d constructs (p3A1 has the highest affinity, QL9 the lowest). Direct comparison of 2D and 3D kinetics also shown in Figure S5. (C) Fluorescent 42F3-tetramer staining of peptide loaded APCs. (D) Fluorescent sc H2-L^d-tetramer staining of 42F3 T cells. Peptides presented by H2-L^d were CD8 dependent (cyan) agonists, CD8 independent agonists (green), or the non-stimulatory peptide p3A1 (red). (E) Correlation between TCR tetramer staining and the 3D affinity of the pMHC ligand is compared to the correlation between pMHC tetramer staining and the 2D affinity (F). Open bars represent tetramer staining, and closed bars represent 2D and 3D affinities shown in panel A. See also Figure S3.

Figure 5. Cross-reactivity through distinct peptide recognition chemistries by the 42F3 TCR

(A) Left column, shows ribbon diagrams of the VαVβ domains of 42F3 bound to QL9, p5E8, p4B10 and p3A1 presented by the α1α2 of H2-L^d. Center column shows accompanying interactions of the respective TCR CDR3 loops with the presented peptides (MHC is colored green, peptides are colored yellow). Right column shows two-dimensional contact maps of 42F3 CDR3α and CDR3β loop contacts. The TCR structures in the p3A1, p4B10, and p5E8 complexes included Constant domains, but these are not depicted in the figures. (B) Aligned structures of the 42F3 TCR Variable domains shows superpositions of CDR loop structures from the four different complexes. 42F3α is colored firebrick (QL9), lightpink (p5E8), raspberry (p4B10) and magenta (p3A1). The 42F3β is colored slate (QL9), lightblue (p5E8), cyan (p4B10) and blue (p3A1). See also Figure S4.

Figure 6. Stimulatory TCR/pMHC docking geometries mediated by a Vα3 germline ‘codon.’

(A) The TCR docking footprints of 42F3 recognizing three stimulatory and one non-stimulatory peptide. Dashed lines connecting the Cα of Y50α and Y50β carbons are used to illustrate the relative orientations of the respective complexes 42F3-QL9-H2-L^d (black) and 42F3-p3A1-H2-L^d (red). Black and red dots indicate the center of masses of the 42F3 TCR in the QL9 versus p3A1 complexes, respectively. (B) The convergent docking footprints of seven different Vα3Vβ8 TCR complexes with H2-L^d-peptide agonists including four previous 2C-complexes and three 42F3 complexes recognizing stimulatory peptides. Inset (top) is a color-coded legend for each TCR and the corresponding peptide recognized in each H2-L^d complex. Inset (bottom) is a contact map depicting conserved contacts between the Vα3.1 or Vα3.3 and the α2-helix in seven agonist complexes. (C) Detailed contacts of CDR1 and CDR2 of the Vα3.1 and Vα3.3 to the α2 helix (green) in seven agonist complexes. van der Waals contacts are depicted as sticks while hydrogen bonds and salt bridges are depicted with dashed lines. See also Figure S5.

Figure 7. Altered TCR-pMHC docking geometry in a non-stimulatory complex

(A) The docking footprint of 42F3 recognizing the p3A1 non-stimulatory peptide. As in Figure 6, a dashed line connects the Cα of Y50α and Y50β carbons (red, black for stimulatory complexes) to illustrate the binding orientation. (B) The germline contacts made by either the Vα (left panel) or the Vβ (right panel) to the MHC helices of H2-L^d presenting p3A1. Residues making van der Waals contacts are represented as sticks and dashed lines represent hydrogen bonds. (C) Range of docking footprints in 40 Class I TCR-pMHC agonist complexes (firebrick and slate) aligned on the MHC together with the non-stimulatory p3A1 peptide footprint (magneta and blue). The CDR loops of 42F3 in the p3A1 complex are highlighted as thick tubes, and the docking angle is shown as a dashed yellow line, with the center of mass between the CDR3α and CDR3β of 42F3 denoted by the yellow dot. (D) Theoretical models for how TCR-pMHC docking geometry in the p3A1 (red peptide) complex could influence signaling compared to the productive agonist (green peptide).