A TCR β-Chain Motif Biases toward Recognition of Human CD1 Proteins

Abstract

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1⁺ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

Figure 1.. TRBV4–1⁺ CD1b-restricted clones recognize a wide variety of antigens.

(A) PBMC from 49 healthy donors were pre-gated for CD3 expression and CD1b-GMM tetramer or CD1b-MA tetramer binding (Supplemental Fig. 1A). Within the tetramer⁻ or tetramer⁺ gates, the percentage TRBV4–1-expressing cells was determined. Box: median with interquartile range; whiskers: minimum to maximum value. (B) Previously published CD1b-restricted TCRs are shown according to variable (TRAV, TRBV) genes and the antigen they recognize: LDN5 (26); GEM1, GEM18, GEM21,GEM42 (27); clone 2, clone26, clone34, clone71, clone83 (27); Z5B71 (36); DN1 (37); DN.POTT (19, 38); PG10, PG90, BC8 Bru (33); YE2.14 (39); MT2.21 (40); C56SL37, C58SL37 (41); clone 11, clone 20, clone 28 (42), BC24A, BC24B, and BC24C (34). BC24C expresses two different α chains. (C) Lipid antigens illustrate the structural diversity of molecules, with polar head groups indicated in red, recognized by TRBV4–1 TCRs.

Figure 2:. CD1b recognition by TRBV4–1⁺ T cells.

(A) Cell lines sorted from blood bank donor D43 based on expression or absence of TRBV4–1 (Supplemental Figure 1B), were tested for TRBV4–1 expression. (B) Both cell lines were stained with CD1b-PG or CD1b-GMM tetramers, or mock loaded CD1b tetramers carrying diverse endogenous lipids (CD1b-endo). (C) TCR sequences were obtained by single cell TCR sequencing of CD1b-GMM tetramer⁺ and CD1b-PG tetramer⁺ cells are shown with germline (grey) and non-germline (white) residues encoded by the indicated variable and joining region genes. After pre-gating using anti-CD3 and anti-TRBV4–1 TCR antibodies (D), TRBV4–1⁺ and TRBV4–1⁻ T cells from PBMC from three random blood bank donors were analyzed for binding of CD1b-PG and CD1b-GMM tetramers directly ex vivo (E). Equal numbers of cells are shown in each plot. All acquired TRBV4–1⁻ cells are shown in Supplemental Figure 1B (top).

Figure 3:. TRBV4–1⁺ T cells are enriched among CD1b tetramer⁺ T cells.

(A) PBMC from three blood donors were stimulated with autologous monocyte-derived dendritic cells and mycolic acid (MA) for 18 days. The resulting cells were stained with CD1b-MA tetramers and an anti-CD3 followed by sorting of tetramer⁺ and tetramer⁻ cells as shown in Supplemental Figure 1D and subjected to high throughput TCR sequencing. The percentages of TRBV gene usage are shown. (B) Using the approach described above, we reanalyzed a publicly available dataset (48) of CD1b-glucose monomycolate (GMM) tetramer⁺ and tetramer⁻ cells from four donors. (C) Summary of TRBV4–1 percentage among tetramer⁺ and tetramer⁻ cells of the three donors shown in A and four donors shown in B.

Figure 4:. Structural analysis of TRBV4–1⁺ TCRs.

CD1b-specific TRBV4–1⁺ TCR conservation is limited to the germline encoded TRBV gene. (A) The schematic shows the role of TRB locus genes in encoding residues in the CDR1, CDR2 and CDR3 regions. CDRβ regions of new and previously sequenced TRBV4–1⁺ and TRBV4–1⁻ CD1b-specific clones were aligned. BbGL-II is 1,2-di-oleyl-a-galactopyranosyl-sn-glycerol; SL37 is synthetic di-acylated sulfoglycolipid analog (67). (B) Upper: Side view of clone 2, PG10, PG90 and GEM42 TCRs, with α-chains (grey), TRBV4–1 β-chains (cyan), and other β chains (green and pink) highlighted. Lower: Bottom-up view of TCR interface surface electrostatic potential. (C) In comparison, top-down view of the CD1b interface surface electrostatic potential are shown (right), with CD1b presenting GMM (brown, upper), and PG (blue, lower). Potential contours are shown on a scale from + 5.0 (positive charge, blue) to – 5.0 k_BT e⁻¹ (negative charge, red); white indicates value close to 0 k_BT e⁻¹ (neutral charge). (D) Overlay image shows CDR regions of clone 2 (green) and PG10 (Blue) TCRs. (E) Key interactions in the TRBV4–1⁺ CDRβ regions are shown, including positions of H29β and R30β on the CDR2β region of clone 2 (green, left) and PG10 (blue, right) TCRs. Amino acid residues involved in contacts are represented as sticks, with hydrogen bonds represented as black dashes. Nitrogen, oxygen, and phosphate are represented in blue, red, and orange respectively, and color coding of CDR regions are highlighted in the legend.

Figure 5.. Mutational analysis of the TCR-CD1b interaction.

(A) CD1b-reactive clone 2 TCR was expressed as a heterodimer encoded by wild type sequences or subjected to β chain point mutation with alanine substitutions in the TRBV4–1 encoded region at positions 29 or 30. Binding to GMM-loaded CD1b complexes was measured using surface plasmon resonance to generate steady state affinities, with binding curves (upper) and equilibrium curves (lower) shown here. (B) The steady state affinities of the wild type clone 2 TCR for wild type and mutant CD1b proteins loaded with GMM was determined. Equilibrium curves for CD1b-E80A, CD1b-Y151A, and CD1b-Y154A showed no observable binding. Error bars represent mean + standard error of the mean (SEM). (C) Surface representation of the CD1b-GMM surface (white), with residues, when mutated to alanine, demonstrate less than a 3-fold decrease in affinity (yellow), 3–5-fold decrease in affinity (orange), and greater than a 5-fold decrease (red), upon binding against the clone 2 TCR. Positions of residues E80, Y151, I154, and T157 are indicated.

Figure 6:. The effect of mutations in CD1b on interaction with T cells.

(A) T cell clones LDN5 and GEM42 were tested for binding of GMM-loaded CD1b tetramers with the indicated point mutations. (B) T cell clones PG10 and BC24B were tested for binding of PG-loaded CD1b tetramers with the indicated point mutations. (C) In addition, four additional T cell clone-CD1b-antigen combinations were tested (Supplemental Fig. 3A–D) and percentages of tetramer positive cells or MFI (Supplemental Fig. 3E) were Z score normalized per cell line and shown as a heatmap. The negative control (“neg”) is mock-loaded wild type CD1a tetramer and the positive control (“pos”) is the indicated antigen-loaded wild type tetramer.