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. 2009 Mar 10;106(10):3889-94.
doi: 10.1073/pnas.0900411106. Epub 2009 Feb 20.

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

Sarah Nicholls  1 , Karen P Piper, Fiyaz Mohammed, Timothy R Dafforn, Stefan Tenzer, Mahboob Salim, Premini Mahendra, Charles Craddock, Peter van Endert, Hansjörg Schild, Mark Cobbold, Victor H Engelhard, Paul A H Moss, Benjamin E Willcox
Affiliations

Secondary anchor polymorphism in the HA-1 minor histocompatibility antigen critically affects MHC stability and TCR recognition

Sarah Nicholls et al. Proc Natl Acad Sci U S A. .

Abstract

T cell recognition of minor histocompatibility antigens (mHags) underlies allogeneic immune responses that mediate graft-versus-host disease and the graft-versus-leukemia effect following stem cell transplantation. Many mHags derive from single amino acid polymorphisms in MHC-restricted epitopes, but our understanding of the molecular mechanisms governing mHag immunogenicity and recognition is incomplete. Here we examined antigenic presentation and T-cell recognition of HA-1, a prototypic autosomal mHag derived from single nucleotide dimorphism (HA-1(H) versus HA-1(R)) in the HMHA1 gene. The HA-1(H) peptide is restricted by HLA-A2 and is immunogenic in HA-1(R/R) into HA-1(H) transplants, while HA-1(R) has been suggested to be a "null allele" in terms of T cell reactivity. We found that proteasomal cleavage and TAP transport of the 2 peptides is similar and that both variants can bind to MHC. However, the His>Arg change substantially decreases the stability and affinity of HLA-A2 association, consistent with the reduced immunogenicity of the HA-1(R) variant. To understand these findings, we determined the structure of an HLA-A2-HA-1(H) complex to 1.3A resolution. Whereas His-3 is accommodated comfortably in the D pocket, incorporation of the lengthy Arg-3 is predicted to require local conformational changes. Moreover, a soluble TCR generated from HA-1(H)-specific T-cells bound HA-1(H) peptide with moderate affinity but failed to bind HA-1(R), indicating complete discrimination of HA-1 variants at the level of TCR/MHC interaction. Our results define the molecular mechanisms governing immunogenicity of HA-1, and highlight how single amino acid polymorphisms in mHags can critically affect both MHC association and TCR recognition.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Detection and isolation of HA-1-specific cytotoxic T lymphocytes after SCT. (A) HLA-A2-VLH tetramers were used to detect HA-1-specific CD8 T-cell responses after donor lymphocyte infusion therapy for leukemic relapse following CML. DLI treatment was initiated 80 weeks after transplant (zero timepoint). Presence of tumor was assessed by quantitative RT-PCR for BCR-ABL transcripts (+/–). (B) The HA-1-specific T-cell clone KP7 stained with HLA-A2-VLH HA-1 tetramer. (C) The KP7 T-cell clone recognizes HA-1-positive lymphoblastoid cell line (LCL) cells presenting endogenously processed HLA-A2-VLH, and HA-1-negative HLA-A2+ LCL pulsed with VLH peptide. However, HA-1-negative LCL are not recognized in the absence of exogenously added peptide, or when pulsed with a control peptide (FIDS).
Fig. 2.
Fig. 2.
CD analysis of HA-1 variant complexes. (A) Wavelength spectrum of HLA-A2-VLH (solid line) and HLA-A2-VLR (dashed line) complexes. (B) Thermal stability measurement of HLA-A2-VLR (open circles) indicates an additional folding transition relative to HLA-A2-VLH (solid circles).
Fig. 3.
Fig. 3.
Crystallographic structure of HLA-A2-VLH at 1.3Å. (A) Overall structure of HLA-A2-VLH complex, with heavy chain (gray), β2m (cyan), and VLH peptide (blue) shown. (B) 2Fo-Fc electron density for the VLH peptide, with primary anchors and P3 to P5 highlighted. (C) Structure of the VLH mHag in the HLA-A2-antigen binding groove, with antigen-binding pockets A to F indicated, and VLH peptide surface indicated in green. The structure highlights relatively poor contacts with pockets E and F. (D) Orientation of H3 in and around the D pocket. H3 packs snugly against the walls of the D pocket, maintaining van der Waal's contacts with Tyr-159, Leu-156, and Gln-155, and also to Asp-4 of the peptide. It is also participates in a hydrogen-bonding network to Gln-155, and peptide residues Asp-4 and Asp-5, via ordered water molecules. Semitransparent peptide surface shown in green.
Fig. 4.
Fig. 4.
Surface plasmon resonance analysis of TCR/HLA-A2-HA-1 interaction. (A) Specific binding of KP7 TCR to HLA-A2-VLH (solid line), with control (HLA-B7-TPR) and HLA-A2-VLR signals also shown (dashed and dotted lines, respectively). (B) Equilibrium affinity analysis of TCR/HLA-A2-VLH interaction. Scatchard plot is shown inset.
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