Natural HLA class I polymorphism controls the pathway of antigen presentation and susceptibility to viral evasion - PubMed (original) (raw)

. 2004 Jul 5;200(1):13-24.

doi: 10.1084/jem.20031680. Epub 2004 Jun 28.

Anthony W Purcell, Whitney A Macdonald, Lars Kjer-Nielsen, Lauren K Ely, Nihay Laham, Tanya Crockford, Nicole A Mifsud, Mandvi Bharadwaj, Linus Chang, Brian D Tait, Rhonda Holdsworth, Andrew G Brooks, Stephen P Bottomley, Travis Beddoe, Chen Au Peh, Jamie Rossjohn, James McCluskey

Affiliations

Natural HLA class I polymorphism controls the pathway of antigen presentation and susceptibility to viral evasion

Danielle Zernich et al. J Exp Med. 2004.

Abstract

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion.

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Figures

Figure 1.

Figure 1.

HLA-B*4405 (Tyr 116) but not B*4402 (Asp 116) is expressed on the surface of cell lines deficient in human tapasin. (A) Mouse L cells expressing human β2m (J26) and either the human cell line 721.220 (.220) or transfectants expressing human tapasin (.220.hTsn) were stably transfected with genes encoding either B*4402 or B*4405 and then stained by indirect immunofluorescence with mAb W6/32 (anti-HLA class I) after culture overnight at 26°C (solid lines) or 37°C (bold dotted lines). Flow cytometric histograms also show background staining of untransfected J26 and 721.220 cells (thin dotted lines) and conjugated antibody alone (shaded). (B) Expression of human tapasin (hTsn) was verified in immunoblots of the indicated cell lysates probed with a specific rabbit antiserum.

Figure 2.

Figure 2.

No detectable incorporation of HLA-B*4405 into the PLC. (A) Cell lysates (lysate) or TAP immune complexes formed with anti-mAb 148.3 mAb (TAP IP) were immunoblotted for the presence of class I heavy chains using biotinylated HC-10 (anti–class I hc). Untransfected 721.220 cells (untrans) and cells transfected with the indicated genes were compared. (B) Class I–reduced (C1R) B-LCLs expressing either B*4402 or B*4405 were assayed as in A. A prolonged exposure (5 min) of the blot is shown to demonstrate the association of low levels of endogenous HLA-C class I heavy chain with the TAP complex (bottom). (C) Cell lysates (lysate) or class I heavy chain complexes formed with mAb HC-10 (hc IP) were then immunoblotted for the presence of TAP-1 using anti–TAP-1 rabbit antiserum. Immunoblotted lysates of the C1R cells and TAP-negative cell T2 are shown as controls (right). (D) Cell lysates (lysate) or class I heavy chain complexes (hc IP) from C were immunoblotted for the presence of tapasin using anti-hTsn rabbit antiserum. The parental 721.220 (.220) and 721.220.hTsn (.220.hTsn) transfectants are shown as controls.

Figure 3.

Figure 3.

Rapid maturation and transport of HLA-B*4405 compared with HLA-B*4402. Cellular proteins from the indicated 721.220 transfectants expressing or lacking membrane tapasin (A), C1R cells (B), and the indicated 721.220 transfectants expressing or lacking soluble tapasin (C) were labeled with 35S-Met/Cys for 5 min and chased for 20, 45, and 70 min before immunoprecipitating class I/β2M molecules with mAb W6/32. Immune complexes were treated with Endo H to determine the proportion of mature, Endo H–resistant (r), or post-Golgi proteins versus the immature, Endo H–sensitive (s), or ER proteins by SDS-PAGE and fluorography. In the C1R.B*4405 transfectants, there is an additional band of unknown origin migrating faster than the class I proteins. This appears to be a clone-specific background band that is not observed in anti–class I Western blots.

Figure 4.

Figure 4.

Overlapping peptide repertoires but altered P9 anchor preference in B*4402 and B*4405. Comparison of B*4402 and B*4405 peptide repertoires by MALDI-TOF mass spectrometry. Total peptide eluates from HLA-B*4402 (positive polarity spectra) and HLA-B*4405 (negative polarity spectra) after a single dimension chromatographic separation. The masses of prominent species are shown. Pool Edman sequence analysis of peptides eluted from HLA-B*4402 and B*4405 revealed dominant anchor residues at P2Glu and P9Tyr/Phe. Phe and Tyr were recovered in equal amounts from P9 of B*4402, but Phe was predominant at this position in B*4405 (Phe:Tyr = 4:1; see Table S1)

Figure 5.

Figure 5.

The structure of HLA-B*4405 alters peptide specificity and increases hydrophobicity in the F pocket. (A) Overview of the structure of B*4405 highlighting Tyr116 and the electron density of the peptide (Dpα46-54, EEFGRAFSF). The structure of the F pocket in HLA-B*4402/EEFGRAFSF (B) (35) and the new structures B*4405/EEFGRAFSF (C) and HLA-B**4403/EEPTVIKKY (D) are shown as ball and stick representations of F pocket amino acids (gray), residue 116 (yellow), and the peptide ligand (green), including the COOH-terminal peptide residue P9 Phe in B and C and P9 Tyr in D. Dotted lines are H bonds. The α2-helix has been removed for clarity. In the B*4402 structure (B), Asp 116 is part of an intricate polar network involving Asp 114, Asp 156, Arg 97, and several conserved water molecules that additionally bridge contacts to the bound peptide. Asp 116 is orientated in the same direction as the aromatic ring of Tyr 116 in the B*4405 structure. A water molecule fills the “cavity” at position 116 of B*4402 such that it superposes closely to Tyr 116 Oη group in B*4405 and forms a similar role in that it bridges one H bond to Asp 114. In B*4405 (C), the P9 anchor residue Phe projects into a hydrophobic F pocket where it is surrounded by the aromatic rings of Tyr 74, Tyr 116, Tyr123, and Trp 147, as well as making van der Waals contacts with Ile 95 and the aliphatic moiety of Asn77. The main chain of this COOH-terminal residue is tethered by H bonds to Asn77, Tyr84, and Thr143. Tyr 116 forms the base of this pocket, where the aromatic ring points away from the F pocket, such that the Tyr 116 Oη group points toward the P7 pocket where it forms two H bonds with the Asp 114 carboxylate, an H bond to Arg 97 Nɛ, and additionally forms a water-mediated H bond to the backbone of the bound peptide (Phe 7N). The aromatic ring of Tyr 116 also packs against the long aliphatic side chain of Arg 97. The electrostatic surfaces of the area bounding the F pocket of B*4402 (E) and B*4405 (F) are depicted using the program GRASP(39). Electropositive (blue); electronegative (red).

Figure 6.

Figure 6.

Expression of the HSV TAP inhibitor ICP47 differentially impairs cell surface expression of HLA-B*4402 versus B*4405. (A) Cell surface expression HLA-B*4402 and B*4405 by C1R transfectants in the presence and absence of ICP47. Cells were stained by indirect immunofluorescence for the shared Bw4 determinant after culture at 37°C (bold dotted lines) or 26°C (solid lines). Flow cytometric histograms include staining of untransfected cells (thin dotted lines) and conjugate alone (filled histograms). (B) Steady-state total HLA–class I heavy chain expression in C1R transfectants ± ICP47. Immunoblots using mAb HC-10 were performed on graded cell numbers of transfectants. (C) Lack of detectable B*4405 within the TAP-associated PLC of C1R transfectants expressing ICP47. TAP complexes were immunoprecipitated from C1R transfectants expressing either B*4402 or B*4405 ± ICP47 using mAb 148.3. Immunoprecipitates were subsequently immunoblotted for the presence of class I hc using biotinylated HC-10 and developed as described in Fig. 2.

Figure 7.

Figure 7.

Infection of primary lymphocytes with HSV differentially down-regulates surface expression of B*4402 versus B*4405. Human PBMCs from B*4402- and B*4405-positive donors were inoculated with HSV-KOS at an multiplicity of infection of 4. PBMCs were then stained with biotinylated anti-Bw4 (solid lines) followed by streptavidin APC at the indicated time points after inoculation. Flow cytometric histograms include staining of mock-infected cells (thin dotted lines). One of two independent experiments is shown. The arrows show the population of infected cells with reduced class I expression. The experiment shown used WT HSV for infection to exclude artifacts from the β-gal–tagged virus that was used in other experiments to define the infected cells.

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