The immunogenicity of a viral cytotoxic T cell epitope is controlled by its MHC-bound conformation - PubMed (original) (raw)

. 2005 Nov 7;202(9):1249-60.

doi: 10.1084/jem.20050864.

Diah Elhassen, Anthony W Purcell, Jacqueline M Burrows, Natalie A Borg, John J Miles, Nicholas A Williamson, Kate J Green, Judy Tellam, Lars Kjer-Nielsen, James McCluskey, Jamie Rossjohn, Scott R Burrows

Affiliations

The immunogenicity of a viral cytotoxic T cell epitope is controlled by its MHC-bound conformation

Fleur E Tynan et al. J Exp Med. 2005.

Abstract

Thousands of potentially antigenic peptides are encoded by an infecting pathogen; however, only a small proportion induce measurable CD8(+) T cell responses. To investigate the factors that control peptide immunogenicity, we have examined the cytotoxic T lymphocyte (CTL) response to a previously undefined epitope ((77)APQPAPENAY(86)) from the BZLF1 protein of Epstein-Barr virus (EBV). This peptide binds well to two human histocompatibility leukocyte antigen (HLA) allotypes, HLA-B*3501 and HLA-B*3508, which differ by a single amino acid at position 156 ((156)Leucine vs. (156)Arginine, respectively). Surprisingly, only individuals expressing HLA-B*3508 show evidence of a CTL response to the (77)APQPAPENAY(86) epitope even though EBV-infected cells expressing HLA-B*3501 process and present similar amounts of peptide for CTL recognition, suggesting that factors other than peptide presentation levels are influencing immunogenicity. Functional and structural analysis revealed marked conformational differences in the peptide, when bound to each HLA-B35 allotype, that are dictated by the polymorphic HLA residue 156 and that directly affected T cell receptor recognition. These data indicate that the immunogenicity of an antigenic peptide is influenced not only by how well the peptide binds to major histocompatibility complex (MHC) molecules but also by its bound conformation. It also illustrates a novel mechanism through which MHC polymorphism can further diversify the immune response to infecting pathogens.

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Figures

Figure 1.

Figure 1.

Identification of a CTL epitope from the BZLF1 antigen of EBV that is immunogenic in HLA-B*3508 + individuals. (A) Cytotoxicity by a CTL line raised against the APQP peptide of autologous PHA blast target cells (SB) and PHA blasts sharing one HLA allele with the CTLs (underlined). The target cells were tested with or without the addition of 0.1 μM of the APQP peptide. E/T ratio = 10:1. (B) Peptide dose-response cytotoxicity assay using a CTL line from donor SB (HLA-B*3508+) raised against the APQP peptide, and PHA blast target cells also from donor SB. E/T ratio = 10:1. (C) CTL recognition at a variety of E/T ratios by polyclonal T cell lines raised against the APQP peptide from PBMCs from eight HLA-B*3501+ donors, three HLA-B*3501+ donors, and one HLA-B*3501/-B*3508 coexpressing donor. The target cells were autologous PHA blasts that were pretreated with 0.1 μM of the APQP peptide or left untreated.

Figure 2.

Figure 2.

The APQP peptide binds efficiently to both HLA-B*3501 and HLA-B*3508. (A) Peptide–MHC binding assay comparing APQP with other peptides for their ability to stabilize HLA-B*3501 or HLA-B*3508 expression on T2 cells that had been transfected with these HLA genes. These other peptides included EPLPQGQLTAY, an EBV epitope immunogenic in HLA-B*3501+ individuals (tested only with T2.B*3501), and LPEPLPQGQLTAY, an EBV epitope immunogenic in HLA-B*3508+ individuals (tested only with T2.B*3508). As negative controls, the HLA-A24/-A23–binding EBV epitope PYLFWLAAI and a truncated version of the APQP peptide (APQPAPENA_) were also included. The T2.B*3501 data were calculated relative to the EPLPQGQLTAY peptide used at 100 μM, and the T2.B*3508 data were calculated relative to the LPEPLPQGQLTAY peptide used at 100 μM. (B) Peptide–MHC dissociation rates were examined for the APQP and the EPLPQGQLTAY peptides using T2.B*3501. Data were calculated relative to EPLPQGQLTAY at time 0.

Figure 3.

Figure 3.

CTL recognition of the APQP peptide in the context of HLA-B*3508 or HLA-B*3501. (A) Peptide dose-response cytotoxicity assay using five APQP-specific CTL clones from donor SB (HLA-B*3508+), and PHA blast target cells expressing either HLA-B*3501 or HLA-B*3508. E/T ratio = 2:1. (B) Multiple CTL microcultures were established from the HLA-B*3508+ donor SB and the HLA-B*3501/-B*3508 coexpressing donor MB by stimulating PBMCs at limiting dilution with the APQP peptide. On day 13, the microcultures were screened for cross-recognition of the peptide (used at 0.02 μg/ml) presented on either HLA-B*3501+ or HLA-B*3508+ PHA blasts. The data are from CTL microcultures raised from responder PBMC concentrations from which <50% of the wells produced CTLs specific for the stimulator EBV epitope; thus, most were likely to have been generated from a single peptide-specific CTL. Data from any CTL microcultures that killed the PHA blast target cells without peptide addition was discarded, and data are only shown for microcultures that displayed considerable lysis of one or both of the peptide-coated target cells. (C) The CTL microculture 2D6 that could efficiently recognize exogenously added APQP peptide in the context of HLA B*3501 was used in an IFN-γ ELISPOT assay to determine if this epitope is naturally presented on EBV-infected cells expressing this HLA allele. The target cells were HLA B*3501+ LCLs carrying either the WT EBV genome (BZLF1+ LCL) or an EBV genome that had been rendered incapable of lytic cycle entry by disruption of the BZLF1 gene (BZLF1− LCL). The number of spots per well is shown.

Figure 4.

Figure 4.

EBV-infected cells expressing HLA-B*3501 or HLA-B*3508 present similar levels of the APQP peptide after endogenous processing. Approximately 8 × 108 LCLs from the HLA-B3501+ donor MW or the HLA-B3508+ donor CA were irradiated and incubated overnight to enhance expression of BZLF1 (donor MW, 12.3% of cells BZLF1+; donor CA, 6.5% of cells BZLF1+). Cells were then lysed in 0.5% TFA, homogenized, and subjected to ultrafiltration and HPLC fractionation. Fractions 30–33 from (A) the HLA-B*3501+ LCL and (B) the HLA-B*3508+ LCL that were predicted to include the APQP peptide (based on a parallel HPLC run with synthetic APQP peptide) were tested at varying dilutions for their ability to sensitize HLA-B*3508+ target cells to lysis by an APQP-specific CTL clone (see panel D for the graph legend; E/T ratio = 2:1). The broken lines at the points of inflection on the dose-response curves mark the fraction dilution that led to 37% lysis. Toxicity controls consisting of target cells incubated with fractions in the absence of CTLs were negative (not depicted). (C) To allow an estimate of the concentration of the APQP peptide in each fraction, synthetic APQP was tested in parallel for CTL recognition at varying concentrations. Half-maximum lysis (37%) was measured at a synthetic peptide concentration of 48 pg/ml. (D) The concentration of synthetic peptide that led to 37% lysis (48 pg/ml) was divided by the fraction dilution that also led to 37% lysis to give an estimate of the amount of APQP in each fraction and a total amount of the peptide eluted from each cell populations.

Figure 5.

Figure 5.

High resolution structures of HLA-B*3501 and HLA-B*3508 presenting APQPAPENAY show that the peptides are presented in different conformations. Structures of APQP complexed to (A) HLA-B*3508 and (B) HLA-B*3501. For clarity, the α2 helix has been removed. 2Fo–Fc electron density, displayed in mesh format, clearly shows the accurate modeling of peptide residues. (C and D) Superposition of the APQP peptides presented by HLA-B*3501 (yellow) and HLA-B*3508 (green) show a dramatic difference in peptide presentation, including (C) a switch from the P5-Ala Cβ group pointing toward the α1 helix (HLA-B*3508) to the α2 helix (HLA-B*3501) and (D) a change from cis–P6-Pro to trans–P6-Pro.

Figure 6.

Figure 6.

Local impact of the 156 polymorphism on peptide presentation. (A) An extensive network of H bonding is seen involving Arg 156 in HLA-B*3508. Particularly important is the interaction with P3-Gln. (B) The polymorphic residue, Leu 156, makes no direct contacts with the peptide in HLA-B*3501. The side chain of P3-Gln interacts instead by pushing P4-Pro toward the α1 helix and pulling P5-Ala and P6-Pro in the direction if the α2 helix. Particularly evident is the switch from cis–P6-Pro to trans–P6-Pro. Residues are in ball-and-stick format. Polar interactions are depicted as dotted lines. The polymorphic residue is green, the peptide is yellow, and other MHC heavy chain residues are shown in gray.

Figure 7.

Figure 7.

The impact of single amino acid substitutions within the APQP peptide on CTL recognition and HLA-B*3508 binding. The APQP-specific CTL clones SB8 (A), SB16 (B), and SB12 (C) were tested for recognition of a panel of altered peptide ligands into which single amino acid substitutions were introduced (E/T ratio = 2:1). A range of peptide concentrations were used in these chromium release assays, and the concentration required for half-maximum lysis was calculated from this dose-response data. (D) MHC–peptide binding assays were also conducted by testing each peptide at a range of concentrations for its ability to stabilize HLA-B*3508 expression on the surface of the antigen-processing mutant T2 cell line. The concentration of peptide required for half-maximum HLA-B*3508 stabilization was calculated. Peptides that were well recognized by a CTL clone were not tested (NT) for MHC binding.

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