A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition

Abstract

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the alpha2 helix (B*4402 Asp156-->B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this "minimal" mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Figure 1.

Differential T cell recognition of HLA-B*4402 and HLA-B*4403 by CD8⁺ T cells. CTL clones, LC13 (A), and DD1 (B) lyse HLA-B*4402 but not HLA-B*4403 targets; untransfected C1R (closed triangles); and C1R-transfected APCs expressing either B*4402 (open squares), B*4403 (closed circles), B*4405 (closed squares), or B*4407 (closed triangles). (C) The CTL clone 5101.1999.23 is alloreactive with HLA-B*4402 and HLA-B*4403. IFN-γ production was assayed after 4 h. APCs: HERLUFF, B*4403 + LCL; PITOUT, B*4402 + LCL; and auto HSV-2, autologous cells infected with HSV-2. (D) PBMCs from donors mismatched for B*4402 and B*4403 were cocultured in a 13-d MLR supplemented with IL-2. The specificity of the responding cells was evaluated by restimulation for 6 h with defined APCs. Vertical axis, IFN-γ staining; horizontal axis, CD8 staining. Gates are shown for IFN-γ–producing CD8⁺ T cells. The percentage of positive cells is shown in each histogram. (E) The percentage of allospecific CD8⁺ T cells was assayed as in Fig. 4 D for 28 independent MLR reactions between B*4403 responders and B*4402 stimulators and 13 independent MLR reactions between B*4402 responders and B*4403 stimulators.

Figure 2.

Subtle differences in ligand selection by B*4402 and B*4403 despite a shared dominant anchor motif and overlapping peptide repertoires. Pool Edman sequence analysis of peptides eluted from HLA-B*4402 and B*4403. Comparison of B*4402 and B*4403 peptide repertoires by MALDI-TOF MS. Total peptide eluates from HLA-B*4402 (positive polarity spectra) and HLA-B*4403 (negative polarity spectra) after a single-dimension chromatographic separation.

Figure 3.

High resolution peptide mapping of B*4402 and B*4403 ligands reveals ∼5% bound peptides are unique or preferentially bound by one allotype. (A) After an initial RP-HPLC separation of HLA-B*4402 bound peptides from C1R transfectants, a second dimension RP-HPLC separation was performed (right) and mass spectra determined for the selected fractions (left). (B–G) MALDI-TOF mass spectra of ligands from selected fractions after a 2nd round of RP-HPLC (A, right). Peptides from HLA-B*4402 (positive polarity spectra) and HLA-B*4403 (negative polarity spectra).

Figure 4.

Structures of bound DPα ligand complexed to two HLA-B44 alleles, highlighting polymorphic amino acids and the electron density of the peptide. The structures of the DPα_46–54 (EEFGRAFSF) complexed to (A) HLA-B*4402 and (B) B*4403 highlighting the polymorphism at position 156. (C) Superposition of the two antigen-binding clefts B*4402 (green) and B*4403(yellow) showing the shift in the α1 helix around residues 70–77. (D) Superposition of the two peptide ligands (side view) highlighting the solvent-accessible amino acids.

Figure 5.

Novel structure of the B pocket of HLA-B*4403 and B*4402 determines the selection of dominant anchor residues P2 Asp/Glu. The structure of the B pocket of HLA-B*4403 is compared with previously reported structures for HLA-B*2705 and HLA-B*3501. The upper panels are ball and stick representations of B pocket amino acids 9, 24, 45, 66, 67, 70, and 99 (gray) and the P2 amino acid of the ligand (green); B*4403 (P2 Glu, left), HLA-B*2705 (P2 Arg, middle), and B*3501 (P2 Pro, right). The lower panels represent electrostatic surfaces of the area bounding the B pocket using the program GRASP (70) and showing the bound peptide ligand for the structures in ball and stick format (blue, electropositive; red, electronegative). The B pocket of B*4402 is virtually identical to B*4403.

Figure 6.

The F pocket of B*4402. The main chain of the peptide carboxy-terminal residue is tethered by H-bonds to Asn 77, Tyr 84, and Thr 143. The P9 anchor residue phenylalanine projects into a hydrophobic F pocket where it stacks between the aromatic ring of Tyr123 and the aliphatic moiety of Asn 77. The walls of the pocket are bounded by Trp 147, Tyr 74, and Ile 95. Peptide residues in green, HLA heavy chain in gray and H-bonds shown as dashed lines.

Figure 7.

The structural interactions around residue 156 constrain the antigen-binding cleft of HLA-B*4402 (156 Asp) relative to HLA-B*4403 (156 Leu) and alter the orientation of peptide side chains. The side chain of residue 156 is shown in yellow and forms a unique H-bond with Asp 114 in B*4402 (A) that is missing in B*4403 (B), where instead Asp 114 is reoriented to maximize its H-bond network with Arg 97, creating a wider cavity in this region of the cleft. Peptide, green; HLA heavy chain, gray; H-bonds, dotted lines; W, water.