Search

Deeper understanding of T-cell-mediated adaptive immune responses is important for the design of cancer immunotherapies and antiviral vaccines against pandemic outbreaks. T-cells are activated when they recognize foreign peptides that are presented on the cell surface by Major Histocompatibility Complexes (MHC), forming peptide:MHC (pMHC) complexes. 3D structures of pMHC complexes provide fundamental insight into T-cell recognition mechanism and aids immunotherapy design. High MHC and peptide diversities necessitate efficient computational modelling to enable whole proteome structural analysis. We developed PANDORA, a generic modelling pipeline for pMHC class I and II (pMHC-I and pMHC-II), and present its performance on pMHC-I here. Given a query, PANDORA searches for structural templates in its extensive database and then applies anchor restraints to the modelling process. This restrained energy minimization ensures one of the fastest pMHC modelling pipelines so far. On a set of 835 pMHC-I complexes over 78 MHC types, PANDORA generated models with a median RMSD of 0.70 Å and achieved a 93% success rate in top 10 models. PANDORA performs competitively with three pMHC-I modelling state-of-the-art approaches and outperforms AlphaFold2 in terms of accuracy while being superior to it in speed. PANDORA is a modularized and user-configurable python package with easy installation. We envision PANDORA to fuel deep learning algorithms with large-scale high-quality 3D models to tackle long-standing immunology challenges., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marzella, Parizi, Tilborg, Renaud, Sybrandi, Buzatu, Rademaker, ‘t Hoen and Xue.)

Two classes of major histocompatibility complex (MHC) molecules, MHC class I and class II, play important roles in our immune system, presenting antigens to functionally distinct T lymphocyte populations. However, the origin of this essential MHC class divergence is poorly understood. Here, we discovered a category of MHC molecules (W-category) in the most primitive jawed vertebrates, cartilaginous fish, and also in bony fish and tetrapods. W-category, surprisingly, possesses class II-type α- and β-chain organization together with class I-specific sequence motifs for interdomain binding, and the W-category α2 domain shows unprecedented, phylogenetic similarity with β 2 -microglobulin of class I. Based on the results, we propose a model in which the ancestral MHC class I molecule evolved from class II-type W-category. The discovery of the ancient MHC group, W-category, sheds a light on the long-standing critical question of the MHC class divergence and suggests that class II type came first., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

A 1983 Immunology Today rostrum hypothesized that each T cell has two recognition units: a T cell receptor (TCR) complex, which binds antigen associated with a polymorphic region of a MHC molecule (pMHC), and a CD4 or CD8 molecule that binds to a conserved region of that same MHC gene product (class II or I, respectively). Structural biology has since precisely revealed those bidentate pMHC interactions. TCRαβ ligates the membrane-distal antigen-binding MHC platform, whereas CD8 clamps a membrane-proximal MHCI α3 domain loop and CD4 docks to a hydrophobic crevice between MHCII α2 and β2 domains. Here, we review how MHC class-restricted binding impacts signaling and lineage commitment, discussing TCR force-driven conformational transitions that may optimally expose the co-receptor docking site on MHC., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

IMGT(®), the international ImMunoGeneTics information system(®)(http://www.imgt.org) is the global reference in immunogenetics and immunoinformatics. By its creation in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS), IMGT(®) marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT(®) is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH) and proteins of the IgSF and MhSF superfamilies. IMGT(®) is built on the IMGT-ONTOLOGY axioms and concepts, which bridged the gap between genes, sequences and 3D structures. The concepts include the IMGT(®) standardized keywords (identification), IMGT(®) standardized labels (description), IMGT(®) standardized nomenclature (classification), IMGT unique numbering and IMGT Colliers de Perles (numerotation). IMGT(®) comprises 7 databases, 17 online tools and 15,000 pages of web resources, and provides a high-quality and integrated system for analysis of the genomic and expressed IG and TR repertoire of the adaptive immune responses, including NGS high-throughput data. Tools and databases are used in basic, veterinary and medical research, in clinical applications (mutation analysis in leukemia and lymphoma) and in antibody engineering and humanization. The IMGT/mAb-DB interface was developed for therapeutic antibodies and fusion proteins for immunological applications (FPIA). IMGT(®) is freely available at http://www.imgt.org., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The crystal structures of unliganded and liganded pMHC molecules provide a structural basis for TCR recognition yet they represent 'snapshots' and offer limited insight into dynamics that may be important for interaction and T cell activation. MHC molecules HLA-B*3501 and HLA-B*3508 both bind a 13 mer viral peptide (LPEP) yet only HLA-B*3508-LPEP induces a CTL response characterised by the dominant TCR clonetype SB27. HLA-B*3508-LPEP forms a tight and long-lived complex with SB27, but the relatively weak interaction between HLA-B*3501-LPEP and SB27 fails to trigger an immune response. HLA-B*3501 and HLA-B*3508 differ by only one amino acid (L/R156) located on α2-helix, but this does not alter the MHC or peptide structure nor does this polymorphic residue interact with the peptide or SB27. In the absence of a structural rationalisation for the differences in TCR engagement we performed a molecular dynamics study of both pMHC complexes and HLA-B*3508-LPEP in complex with SB27. This reveals that the high flexibility of the peptide in HLA-B*3501 compared to HLA-B*3508, which was not apparent in the crystal structure alone, may have an under-appreciated role in SB27 recognition. The TCR pivots atop peptide residues 6-9 and makes transient MHC contacts that extend those observed in the crystal structure. Thus MD offers an insight into 'scanning' mechanism of SB27 that extends the role of the germline encoded CDR2α and CDR2β loops. Our data are consistent with the vast body of experimental observations for the pMHC-LPEP-SB27 interaction and provide additional insights not accessible using crystallography.

Salmon have provided key insights into the relative influence of natural and sexual selection on major histocompatibility complex (MHC) variation. Natural selection on salmon MHC genes has been demonstrated in pathogen studies, and there is evidence of MHC-based mate choice (sexual selection). We tested whether parental MHC genes affect survivorship of juvenile Atlantic salmon (Salmo salar) by quantifying the influence of parental genome-wide relatedness and MHC genotype on survivorship to the swim-up stage. Thirteen microsatellite loci were used to estimate the influence of genome-wide relatedness between parents on offspring survivorship and MHC genotypes were determined by sequencing part of the class IIbeta gene. Our results revealed no significant relationship between early offspring survivorship and genome-wide relatedness, predicted MHC heterozygosity, or MHC allelic similarity. Overall, our data are consistent with the contention that excess MHC heterozygosity in Atlantic salmon juveniles is due to sexual selection as well as differential survival of offspring due to MHC genotype.

Sequence variability at three major histocompatibility complex (MHC) genes (DQB, DRA, and MHC-I) of cetaceans was investigated in order to get an overall understanding of cetacean MHC evolution. Little sequence variation was detected at the DRA locus, while extensive and considerable variability were found at the MHC-I and DQB loci. Phylogenetic reconstruction and sequence comparison revealed extensive sharing of identical MHC alleles among different species at the three MHC loci examined. Comparisons of phylogenetic trees for these MHC loci with the trees reconstructed only based on non-PBR sites revealed that allelic similarity/identity possibly reflected common ancestry and were not due to adaptive convergence. At the same time, trans-species evolution was also evidenced that the allelic diversity of the three MHC loci clearly pre-dated species divergence events according to the relaxed molecular clock. It may be the forces of balancing selection acting to maintain the high sequence variability and identical alleles in trans-specific manner at the MHC-I and DQB loci.

In vertebrates, the major histocompatibility complex (MHC) presents peptides to the immune system. In humans, MHCs are called human leukocyte antigens (HLAs), and some of the loci encoding them are the most polymorphic in the human genome. Different MHC molecules present different subsets of peptides, and knowledge of their binding specificities is important for understanding the differences in the immune response between individuals. Knowledge of motifs may be used to identify epitopes, to understand the MHC restriction of epitopes, and to compare the specificities of different MHC molecules. Algorithms that predict which peptides MHC molecules bind have recently been developed and cover many different alleles, but the utility of these algorithms is hampered by the lack of tools for browsing and comparing the specificity of these molecules. We have, therefore, developed a web server, MHC motif viewer, that allows the display of the likely binding motif for all human class I proteins of the loci HLA A, B, C, and E and for MHC class I molecules from chimpanzee (Pan troglodytes), rhesus monkey (Macaca mulatta), and mouse (Mus musculus). Furthermore, it covers all HLA-DR protein sequences. A special viewing feature, MHC fight, allows for display of the specificity of two different MHC molecules side by side. We show how the web server can be used to discover and display surprising similarities as well as differences between MHC molecules within and between different species. The MHC motif viewer is available at http://www.cbs.dtu.dk/biotools/MHCMotifViewer/ .

Background: The haplochromine cichlid species assemblages of Lake Malawi and Victoria represent some of the most important study systems in evolutionary biology. Identifying adaptive divergence between closely-related species can provide important insights into the processes that may have contributed to these spectacular radiations. Here, we studied a pair of sympatric Lake Malawi species, Pseudotropheus fainzilberi and P. emmiltos, whose reproductive isolation depends on olfactory communication. We tested the hypothesis that these species have undergone divergent selection at MHC class II genes, which are known to contribute to olfactory-based mate choice in other taxa., Methodology/principal Findings: Divergent selection on functional alleles was inferred from the higher genetic divergence at putative antigen binding sites (ABS) amino acid sequences than at putatively neutrally evolving sites at intron 1, exon 2 synonymous sequences and exon 2 amino acid residues outside the putative ABS. In addition, sympatric populations of these fish species differed significantly in communities of eukaryotic parasites., Conclusions/significance: We propose that local host-parasite coevolutionary dynamics may have driven adaptive divergence in MHC alleles, influencing odor-mediated mate choice and leading to reproductive isolation. These results provide the first evidence for a novel mechanism of adaptive speciation and the first evidence of adaptive divergence at the MHC in closely related African cichlid fishes.

Unlabelled: Normal adaptive immune responses operate under major histocompatibility complex (MHC) restriction by binding to specific, short antigenic peptides and presenting them to appropriate T-cell receptors (TcRs). Sequence-structure-function information is critical in understanding the principles governing peptide/MHC (pMHC) and TcR/pMHC recognition and binding. A new database for sequence-structure-function information on TcR/pMHC interactions, MHC-Peptide Interaction Database version T (MPID-T), is now available with the latest available Protein Data Bank (PDB) data and interaction parameters on TcR/pMHC complexes. MPID-T is a manually curated MySQL database containing experimentally determined structures of 187 pMHC complexes and 16 TcR/pMHC complexes available in the PDB. Each structure is manually verified, classified, and analysed for intermolecular interactions (i) between the MHC and its corresponding bound peptide and (ii) between TcR and its bound pMHC complex where TcR structural information is available. The MPID-T database retrieval system has precomputed interaction parameters that include solvent accessibility, hydrogen bonds, gap volume and gap index. Structural visualisation of the TcR/pMHC complex, pMHC complex, MHC or the bound peptide can be performed using freely available graphics applications such as MDL Chime or RasMol, while structural alignment (based on MHC class and peptide length) can be viewed using the Jmol molecular viewer or an MDL Chime-compatible web browser client. MPID-T contains structural descriptors for in-depth characterisation of TcR/pMHC and pMHC interactions. The ultimate purpose of MPID-T is to enhance the understanding of the binding mechanism underlying TcR/pMHC and pMHC interactions by mapping the TcR footprint on the MHC and its bound peptide, as this eventually determines T-cell recognition and binding., Availability: The MPID-T database retrieval system is available at http://surya.bic.nus.edu.sg/mpidt, Contact: Joo Chuan Tong (jctong@i2r.a-star.edu.sg).

Unlabelled: The accurate computational prediction of T-cell epitopes can greatly reduce the experimental overhead implicit in candidate epitope identification within genomic sequences. In this article we present MHCPred 2.0, an enhanced version of our online, quantitative T-cell epitope prediction server. The previous version of MHCPred included mostly alleles from the human leukocyte antigen A (HLA-A) locus. In MHCPred 2.0, mouse models are added and computational constraints removed. Currently the server includes 11 human HLA class I, three human HLA class II, and three mouse class I models. Additionally, a binding model for the human transporter associated with antigen processing (TAP) is incorporated into the new MHCPred. A tool for the design of heteroclitic peptides is also included within the server. To refine the veracity of binding affinities prediction, a confidence percentage is also now calculated for each peptide predicted., Availability: As previously, MHCPred 2.0 is freely available at the URL http://www.jenner.ac.uk/MHCPred/, Contact: Darren R. Flower (darren.flower@jenner.ac.uk).

Summary: EPIMHC is a relational database of MHC-binding peptides and T cell epitopes that are observed in real proteins. Currently, the database contains 4867 distinct peptide sequences from various sources, including 84 tumor-associated antigens. The EPIMHC database is accessible through a web server that has been designed to facilitate research in computational vaccinology. Importantly, peptides resulting from a query can be selected to derive specific motif-matrices. Subsequently, these motif-matrices can be used in combination with a dynamic algorithm for predicting MHC-binding peptides from user-provided protein queries., Availability: The EPIMHC database server is hosted by the Dana-Farber Cancer Institute at the site http://immunax.dfci.harvard.edu/bioinformatics/epimhc/

IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr) provides a common access to expertly annotated data on the genome, proteome, genetics and structure of immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC), and related proteins of the immune system (RPI) of human and other vertebrates. The NUMEROTATION concept of IMGT-ONTOLOGY has allowed to define a unique numbering for the variable domains (V-DOMAINs) and constant domains (C-DOMAINs) of the IG and TR, which has been extended to the V-LIKE-DOMAINs and C-LIKE-DOMAINs of the immunoglobulin superfamily (IgSF) proteins other than the IG and TR (Dev Comp Immunol 27:55--77, 2003; 29:185--203, 2005). In this paper, we describe the IMGT unique numbering for the groove domains (G-DOMAINs) of the MHC and for the G-LIKE-DOMAINs of the MHC superfamily (MhcSF) proteins other than MHC. This IMGT unique numbering leads, for the first time, to the standardized description of the mutations, allelic polymorphisms, two-dimensional (2D) representations and three-dimensional (3D) structures of the G-DOMAINs and G-LIKE-DOMAINs in any species, and therefore, is highly valuable for their comparative, structural, functional and evolutionary studies.

Genes encoding MHC class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in teleost immune responses. This has in part been due to a lack of appropriate MHC class I and II matched and mismatched animals. To identify such animals, MHC segregation and linkage studies in the channel catfish were undertaken. The results of restriction fragment length polymorphism and fluorescent in situ hybridization studies showed that all the MHC class II genes are linked and most if not all MHC class I genes are linked. These studies also demonstrated that in catfish, as in other teleosts, MHC class I and II genes are not linked. Consequently, catfish matched and mismatched for MHC class I and II genes were identified and preliminary functional studies indicate that spontaneous non-specific allogeneic cytotoxic responses are likely mediated by differences in MHC class I, but not class II, region molecules.

MHCBN is a comprehensive database of Major Histocompatibility Complex (MHC) binding and non-binding peptides compiled from published literature and existing databases. The latest version of the database has 19 777 entries including 17 129 MHC binders and 2648 MHC non-binders for more than 400 MHC molecules. The database has sequence and structure data of (a) source proteins of peptides and (b) MHC molecules. MHCBN has a number of web tools that include: (i) mapping of peptide on query sequence; (ii) search on any field; (iii) creation of data sets; and (iv) online data submission. The database also provides hypertext links to major databases like SWISS-PROT, PDB, IMGT/HLA-DB, GenBank and PUBMED.

Summary: Binding of short antigenic peptides to Major histocompatibility complex (MHC) proteins is the first step in T-cell mediated immune response. To understand the structural principles governing MHC-specific peptide recognition and binding, we have developed the MHC-Peptide Interaction Database (MPID), containing sequence-structure-function information. MPID (version 1.2) contains curated x-ray crystallographic data on 86 MHC peptide complexes, with precomputed interaction parameters (solvent accessibility, hydrogen bonds, gap volume and gap index). A user-friendly web interface and query tools will facilitate the development of predictive algorithms for MHC-peptide binding from a structural viewpoint., Availability: Freely accessible from http://surya.bic.nus.edu.sg/mpid.

The accurate prediction of T cell epitopes is one of the key aspirations of immunoinformatics. We have developed a partial least squares-based, robust multivariate statistical method for the quantitative prediction of peptide binding to major histocompatibility complexes (MHCs), the principal checkpoint on the antigen presentation pathway. As a service to the immunobiology community, we have made a Perl implementation of the method available as a World Wide Web server.

The international ImMunoGeneTics database (IMGT) (http://imgt.cines.fr), is a high quality integrated information system specializing in Immunoglobulins (IG), T cell Receptors (TR) and Major Histocompatibility Complex (MHC) of human and other vertebrates, created in 1989, by the Laboratoire d'ImmunoGénétique Moléculaire (LIGM), at the Université Montpellier II, CNRS, Montpellier, France. IMGT provides a common access to standardized data which include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, specificities, 2D and 3D structures. IMGT includes three sequence databases (IMGT/LIGM-DB, IMGT/MHC-DB, IMGT/PRIMER-DB), one genome database (IMGT/GENE-DB) with different interfaces (IMGT/GeneSearch, IMGT/GeneView, IMGT/LocusView), one 3D structure database (IMGT/3Dstructure-DB), Web resources comprising 8000 HTML pages ('IMGT Marie-Paule page') and interactive tools for sequence analysis (IMGT/V-QUEST, IMGT/JunctionAnalysis, IMGT/Allele-Align, IMGT/PhyloGene). IMGT data are expertly annotated according to the rules of the IMGT Scientific chart, based on IMGT-ONTOLOGY. IMGT tools are particularly useful for the analysis of the IG and TR repertoires in physiological normal and pathological situations. IMGT has important applications in medical research (autoimmune diseases, AIDS, leukemias, lymphomas, myelomas), biotechnology related to antibody engineering (phage displays, combinatorial libraries) and thera-peutic approaches (graft, immunotherapy). IMGT is freely available at http://imgt.cines.fr.

The IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla) has provided a centralized repository for the sequences of the alleles named by the WHO Nomenclature Committee for Factors of the HLA System for the past four years. Since its initial release the database has grown and is the primary source of information for the study of sequences of the human major histocompatibilty complex. The initial release of the database contained a limited number of tools. As a result of feedback from our users and developments in HLA we have been able to provide new tools and facilities. The HLA sequences have also been extended to include intron sequences and the 3' and 5' untranslated regions in the alignments and also the inclusion of new genes such as MICA. The IMGT/MHC database (http://www.ebi.ac.uk/imgt/mhc) was released in March 2002 to provide a similar resource for other species. The first release of IMGT/MHC contains the sequences of non-human primates (apes, new and old world monkeys), canines and feline sequences. Further species will be added shortly and the database aims to become the primary source of MHC data for non-human sequences.

Pre-graft priming of heart allograft recipients with donor strain blood induces tolerance in 100% of adult rats in the congenic LEW.1W to LEW.1A combination. This tolerant state is specific for donor MHC antigens as third-party blood transfusions fail to induce tolerance, and third-party skin grafts are promptly rejected by tolerant graft recipients. In this study we have characterized the immunodominant donor (RT1u) class I and II allogenic peptides which elicit an in vitro proliferative response to splenocytes from recipients (RT1a) undergoing acute rejection or tolerant to a LEW.1A cardiac allograft. Paradoxically, splenocytes from tolerant animals responded more vigorously to a broader set of donor peptides than splenocytes from rejecting animals. In addition, several of these peptides were observed to be stimulatory only for tolerant splenocytes. These findings suggest that regulatory cells may be involved in tolerance induction or maintenance and are selected by specific motifs, which could be utilized for manipulating the immune system of graft recipients.

This review focuses on recent advances in research on the bovine major histocompatibility complex (BoLA), with specific reference to the genetic organization, polymorphism and function of the class II genes. The BoLA region is unlike the MHC of humans and mice in that a large inversion has moved several class II genes, including the TAP/LMP cluster, close to the centromere of bovine chromosome 23. Therefore, close linkage of MHC genes and other genes associated with the MHC in humans and mice does not appear to be required for normal immunological function. In cattle, polymorphism in the class IIa genes influences both the magnitude and the epitope specificity of antigen-specific T-cell responses to foot-and-mouth disease virus peptides. Disease association studies have demonstrated that BoLA alleles affect the subclinical progression of bovine leukemia virus (BLV) infection. This association is strongly correlated with the presence of specific amino acid motifs within the DRB3 antigen-binding domain. In addition to the practical significance of these findings, the association between BoLA and BLV provides a unique model to study host resistance to retrovirus infection in a non-inbred species. These studies contribute to our understanding of the evolution of the MHC in mammals, to the development of broadly effective vaccines, and to breeding strategies aimed at improving resistance to infectious diseases.

Studies of the major histocompatibility complex (MHC) of cattle over the past twenty years have revealed a reasonably detailed picture of the genetic organisation and function of the genes within this genetic system. Serological and biochemical analysis of lymphocyte cell surface antigens provided the first evidence for highly polymorphic MHC genes in cattle and other ruminant species. The MHC of cattle was thus named the bovine leucocyte antigen (BoLA) system. During the past 10 years, tools of molecular biology have been used to characterise the number of MHC genes, their sequence and fine structure in a number of ruminant species. Although individual MHC genes were found to have clear orthologues among ruminants and other mammalian species, the MHC of cattle, and probably that of sheep and goats, has a unique genetic organisation. Cattle have a class II gene cluster (class IIb region) which is physically distant from all the other MHC genes on the same chromosome. Moreover, genes involved in antigen processing, such as the proteosome subunit locus LMP2, are also found in the class IIb region, demonstrating that these genes need not be in close proximity to other MHC genes to function normally. The MHC class I and class II gene products of ruminants present processed peptides to T lymphocytes which mediate helper and cytotoxic functions. Identification of peptide binding motifs of cattle MHC class I molecules indicates that ruminant MHC molecules function in a similar manner to those of mice and humans. These functional studies provide a firm molecular basis for a number of well-documented associations with infectious diseases, although a detailed understanding of the immunogenetic mechanisms underlying these associations has yet to be elucidated.

The ontogeny and evolution of the major histocompatibility complex (MHC) are blossoming fields that grant insight into the origins of the adaptive immune system and into the strategies adopted by particular groups of vertebrates for expression of MHC during development. This review surveys general topics concerning MHC evolution, with special emphasis on the significance of linkage of gene families within the MHC; a model is proposed in which the MHC class III region is the "primordial immune complex" with its members giving rise to classical MHC molecules. The developmental expression of MHC, both of the classical and non-classical genes, is described in detail with a concentration on differential expression by extraembryonic tissues in mammals and by tissues in "transition" during metamorphosis in amphibians.

Serology and biochemistry were used to identify major histocompatibility complex (MHC) types in chicken lines selected for high and low antibody response to sheep red blood cells. Serological typing was performed by direct haemagglutination, using antisera obtained by erythrocyte alloimmunization within the lines. Four serotypes were identified, called preliminarily B114, B119, B121 and B124. Subsequently, these types were characterized for their B-G and B-F products by biochemical analysis, using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and one-dimensional IEF (isoelectric focusing) respectively. The B114, B119 and B121 serotypes displayed each characteristic banding patterns for both B-G and B-F. No additional types or subtypes were identified by biochemistry within these serotypes. The B124 serotype however could be subtyped into three different haplotypes with specific banding patterns for B-G and B-F. None of the haplotypes in the selection lines were identical for both B-G and B-F with the tested reference B haplotypes. Comparison of B-G alleles revealed similar, but not identical B-G patterns for B114 and B-G14, whereas B124C and B-G23, as well as B119 and B-G19 displayed indistinguishable patterns. For B-F, only B121 and B-F21 banding patterns were indistinguishable by IEF. All other B-F types differed from the reference types.