Trevor Huyton

CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence

The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.

The impact of human leukocyte antigen (HLA) micropolymorphism on ligand specificity within the HLA-B*41 allotypic family

Background Polymorphic differences between human leukocyte antigen (HLA) molecules affect the specificity and conformation of their bound peptides and lead to differential selection of the T-cell repertoire. Mismatching during allogeneic transplantation can, therefore, lead to immunological reactions. Design and Methods We investigated the structure-function relationships of six members of the HLA-B*41 allelic group that differ by six polymorphic amino acids, including positions 80, 95, 97 and 114 within the antigen-binding cleft. Peptide-binding motifs for B*41:01, *41:02, *41:03, *41:04, *41:05 and *41:06 were determined by sequencing self-peptides from recombinant B*41 molecules by electrospray ionization tandem mass spectrometry. The crystal structures of HLA-B*41:03 bound to a natural 16-mer self-ligand (AEMYGSVTEHPSPSPL) and HLA-B*41:04 bound to a natural 11-mer self-ligand (HEEAVSVDRVL) were solved. Results Peptide analysis revealed that all B*41 alleles have an identical anchor motif at peptide position 2 (glutamic acid), but differ in their choice of C-terminal pΩ anchor (proline, valine, leucine). Additionally, B*41:04 displayed a greater preference for long peptides (>10 residues) when compared to the other B*41 allomorphs, while the longest peptide to be eluted from the allelic group (a 16mer) was obtained from B*41:03. The crystal structures of HLA-B*41:03 and HLA-B*41:04 revealed that both alleles interact in a highly conserved manner with the terminal regions of their respective ligands, while micropolymorphism-induced changes in the steric and electrostatic properties of the antigen-binding cleft account for differences in peptide repertoire and auxiliary anchoring. Conclusions Differences in peptide repertoire, and peptide length specificity reflect the significant functional evolution of these closely related allotypes and signal their importance in allogeneic transplantation, especially B*41:03 and B*41:04, which accommodate longer peptides, creating structurally distinct peptide-HLA complexes.

Position 156 influences the peptide repertoire and tapasin dependency of human leukocyte antigen B*44 allotypes

Background Polymorphic differences between donor and recipient human leukocyte antigen class I molecules can result in graft-versus-host disease due to distinct peptide presentation. As part of the peptide-loading complex, tapasin plays an important role in selecting peptides from the pool of potential ligands. Class I polymorphisms can significantly alter the tapasin-mediated interaction with the peptide-loading complex and although most class I allotypes are highly dependent upon tapasin, some are able to load peptides independently of tapasin. Several human leukocyte antigen B*44 allotypes differ exclusively at position 156 (B*44:02156Asp, 44:03156Leu, 44:28156Arg, 44:35156Glu). From these alleles, only the high tapasin-dependency of human leukocyte antigen B*44:02 has been reported. Design and Methods We investigated the influence of position 156 polymorphisms on both the requirement of tapasin for efficient surface expression of each allotype and their peptide features. Genes encoding human leukocyte antigen B*44 variants bearing all possible substitutions at position 156 were lentivirally transduced into human leukocyte antigen class I-negative LCL 721.221 cells and the tapasin-deficient cell line LCL 721.220. Results Exclusively human leukocyte antigen B*44:28156Arg was expressed on the surface of tapasin-deficient cells, suggesting that the remaining B*44/156 variants are highly tapasin-dependent. Our computational analysis suggests that the tapasin-independence of human leukocyte antigen B*44:28156Arg is a result of stabilization of the peptide binding region and generation of a more peptide receptive state. Sequencing of peptides eluted from human leukocyte antigen B*44 molecules by liquid chromatography-electrospray ionization-mass spectrometry (LTQ-Orbitrap) demonstrated that both B*44:02 and B*44:28 share the same overall peptide motif and a certain percentage of their individual peptide repertoires in the presence and/or absence of tapasin. Conclusions Here we report for the first time the influence of position 156 on the human leukocyte antigen/tapasin association. Additionally, the results of peptide sequencing suggest that tapasin chaperoning is needed to acquire peptides of unusual length.

Toll-like receptors: structural pieces of a curve-shaped puzzle.

The toll‐like receptors are responsible for recognizing invading pathogens and triggering a primary innate immune response by initiating a signalling pathway that ultimately leads to the activation of nuclear factor‐κB (NF‐κB). NF‐κB induces the transcription of many genes that regulate both the inflammatory response and interferons that help control the development of adaptive immunity. In this review, we concentrate on the structure and function of toll‐like receptors and our current understanding of the complexities of ligand binding, receptor dimerization and signal transduction.

The production and crystallization of the human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 complexed with deamidated gliadin peptides implicated in coeliac disease

The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4+ T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 alpha-I (PQPELPYPQ) and DQ8 alpha-I (EGSFQPSQE), respectively, are reported.

HLA-E: Presentation of a Broader Peptide Repertoire Impacts the Cellular Immune Response-Implications on HSCT Outcome.

The HLA-E locus encodes a nonclassical class Ib molecule that serves many immune functions from inhibiting NK cells to activating CTLs. Structural analysis of HLA-E/NKG2A complexes visualized fine-tuning of protective immune responses through AA interactions between HLA-E, the bound peptide, and NKG2A/CD94. A loss of cellular protection through abrogation of the HLA-E/NKG2A engagement is dependent on the HLA-E bound peptide. The role of HLA-E in posttransplant outcomes is not well understood but might be attributed to its peptide repertoire. To investigate the self-peptide repertoire of HLA-E∗01:01 in the absence of protective HLA class I signal peptides, we utilized soluble HLA technology in class I negative LCL cells in order to characterize HLA-E∗01:01-bound ligands by mass-spectrometry. To understand the immunological impact of these analyzed ligands on NK cell reactivity, we performed cellular assays. Synthesized peptides were loaded onto recombinant T2 cells expressing HLA-E∗01:01 molecules and applied in cytotoxicity assays using the leukemia derived NK cell line (NKL) as effector. HLA-E in complex with the self-peptides demonstrated a shift towards cytotoxicity and a loss of cell protection. Our data highlights the fact that the HLA-E-peptidome is not as restricted as previously thought and support the suggestion of a posttransplant role for HLA-E.

Overexpression of the pp32r1 (ANP32C) oncogene or its functional mutant pp32r1Y140H confers enhanced resistance to FTY720 (Finguimod).

pp32r1 (ANP32C) is oncogenic and has been shown to be overexpressed in tumors of the breast, prostate, and pancreas. In this work we show that pp32 family proteins are able to bind to the sphingosine analog FTY720 (Finguimod). Molecular docking studies highlight that a conserved residue F136 is likely to be a key determinant of the FTY720 binding site on the pp32 leucine-rich repeat domain. Transduction of the renal carcinoma cell line ACHN or cervical cancer cell line HeLa with lentivirus expressing the oncogenic family member pp32r1 or a pp32r1Y140H functional mutant illustrated an enhanced resistance to FTY720 induced apoptosis. These findings highlight that certain cancers overexpressing pp32r1 or pp32r1 mutants are likely to demonstrate enhanced resistance to FTY720 treatment.

The nature of peptides presented by an HLA class I low expression allele

Background The functional integrity of human leukocyte antigen low expression variants is a prerequisite for considering them as essential in the matching process of hematopoietic stem cell donors and recipients to diminish the risk of serious complications such as graft-versus-host disease or graft rejection. The HLA-A*3014L variant has a disulfide bridge missing in the α2 domain which could affect peptide binding and presentation to T cells. Design and Methods HLA-A*3014L and HLA-A*3001 were expressed as truncated variants and peptides were eluted and subjected to pool sequencing by Edman degradation as well as to single-peptide sequencing by mass spectrometry. Quantitative analysis of binding peptides presented in vivo was performed by a flow cytometric peptide-binding assay using HLA-A*3001 and HLA-A*3014L-expressing B-LCLs. Results The truncated HLA-A*3014L protein was secreted in the supernatant and it was possible to elute and sequence peptides. Sequence analysis of these eluted peptides revealed no relevant differences to the peptide motif of HLA-A*3001, indicating that the Cys164Ser substitution does not substantially alter the spectrum of presented peptides. Strong binding of one of the shared in vivo identified HLA-A*3001/3014L ligands was confirmed in the peptide-binding assay. Conclusions This study is the first to demonstrate that HLA low expression variants are able to present peptides and, thus, can be considered as functionally active. When comparing peptide motifs, it is likely that HLA-A*3014L and HLA-A*3001 represent a permissive mismatch with low allogenicity in hematopoietic stem cell transplantation. These results indicate that surface expression, as well as peptide-binding data of HLA variants with similar disulfide bridge variations (e.g. HLA-A*3211Q) need to be considered as functionally active in an allogeneic hematopoietic stem cell transplantation setting as long as the opposite has not been shown. Otherwise a relevant but not considered HLA mismatch could result in a severe allogeneic T-cell response and graft-versus-host disease.

Differential Impact of HLA-B*44 Allelic Mismaches at Position 156 on Peptide Binding Specificities and T-Cell Diversity

The molecular understanding of how we can mismatch patients and donors and still have successful clinical outcomes will help to guide the future of unrelated bone-marrow transplantation. Single amino acid mismatches at position 156 on the alpha 2 helix of B*44 variants have been described to cause immunological episodes. The magnitude of permissivity between B44/156 variants differs from peptide presentation independent of the peptide loading complex (B*44:28) to influencing clinical episodes (B*44:02 vs. B*44:03). We here investigated if the single exchange of an Asp>Glu as occurring in B*44:35 at residue 156 would force an immune response in vitro. We developed an in vitro system by recombinant co-expression of a single membrane-bound allogeneic HLA class I molecule in donor cells and co-incubating these cells with autologous T-cells. This strategy enables the study of single HLA class I mismatches and excludes the influence of minor antigens. We found these T-cells to be able to differentially discriminate between mismatched B*44 subtypes and their micropolymorphism. To understand how certain pHLA landscapes shaping the alloreactive immune response, we sequenced the individual peptides derived from B*44/156 subtypes using LC-ESI-MS/MS technology. Based on the peptide data we modeled the structure of the B*44:35 variant and can describe the unexpected immunological reaction of the mismatched B*44 subtypes through structural manipulation of the heavy chain. The meticulous characterization of peptide-binding profiles for key alleles, as well as the evaluation of T-cell responses and structural analysis in the context of one-allele mismatches will open the door to a new era in bonemarrow transplantation.

Understanding the obstacle of incompatibility at residue 156 within HLA-B*35 subtypes

Defining permissive and non-permissive mismatches for transplantation is a demanding challenge. Single mismatches at amino acid (AA) position 156 of human leucocyte antigen (HLA) class I have been described to alter the peptide motif, repertoire, or mode of peptide loading through differential interaction with the peptide-loading complex. Hence, a single mismatch can tip the balance and trigger an immunological reaction. HLA-B*35 subtypes have been described to evade the loading complex, 156 mismatch distinguishing B*35:01 and B*35:08 changes the binding groove sufficiently to alter the sequence features of the selected peptide repertoire. To understand the functional influences of residue 156 in B*35 variants, we analyzed the peptide binding profiles of HLA-B*35:01156Leu, B*35:08156Arg and B*35:62156Trp. The glycoprotein tapasin represents a target for immune evasions and functions within the multimeric peptide-loading complex to stabilize empty class I molecules and promote acquisition of high-affinity peptides. All three B*35 subtypes showed a tapasin-independent mode of peptide acquisition. HLA-B*35-restricted peptides of low- and high-binding affinities were recovered in the presence and absence of tapasin and subsequently sequenced utilizing mass spectrometry. The peptides derived from B*35 variants differ substantially in their features dependent on their mode of recruitment; all peptides were preferentially anchored by Pro at p2 and Tyr, Phe, Leu, or Lys at pΩ. However, the Trp at residue 156 altered the p2 motif to an Ala and restricted the pΩ to a Trp. Our results highlight the importance of understanding the impact of key micropolymorphism and how a single AA mismatch orchestrates the neighboring AAs.