Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.     

A shared MHC supertype motif emerges by convergent evolution in macaques and mice, but is totally absent in human MHC molecules

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the D(d) mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These "G2" alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the "G2" alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.     

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1–α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.     

Changes at the floor of the peptide-binding groove induce a strong preference for proline at position 3 of the bound peptide: molecular dynamics simulations of HLA-A*0217

We report on molecular dynamics simulations of major histocompatibility complex (MHC)-peptide complexes. Class I MHC molecules play an important role in cellular immunity by presenting antigenic peptides to cytotoxic T cells. Pockets in the peptide-binding groove of MHC molecules accommodate anchor side chains of the bound peptide. Amino acid substitutions in MHC affect differences in the peptide-anchor motifs. HLA-A*0217, human MHC class I molecule, differs from HLA-A*0201 only by three amino acid residues substitutions (positions 95, 97, and 99) at the floor of the peptide-binding groove. A*0217 showed a strong preference for Pro at position 3 (p3) and accepted Phe at p9 of its peptide ligands, but these preferences have not been found in other HLA-A2 ligands. To reveal the structural mechanism of these observations, the A*0217-peptide complexes were simulated by 1000 ps molecular dynamics at 300 K with explicit solvent molecules and compared with those of the A*0201-peptide complexes. We examined the distances between the anchor side chain of the bound peptide and the pocket, and the rms fluctuations of the bound peptides and the HLA molecules. On the basis of the results from our simulations, we propose that Pro at p3 serves as an optimum residue to lock the dominant anchor residue (p9) tightly into pocket F and to hold the peptide in the binding groove, rather than a secondary anchor residue fitting optimally the complementary pocket. We also found that Phe at p9 is used to occupy the space created by replacements of three amino acid residues at the floor within the groove. These findings would provide a novel understanding in the peptide-binding motifs of class I MHC molecules.     

Unique Pathogen Peptidomes Facilitate Pathogen-Specific Selection and Specialization of MHC Alleles

A key component of pathogen-specific adaptive immunity in vertebrates is the presentation of pathogen-derived antigenic peptides by major histocompatibility complex (MHC) molecules. The excessive polymorphism observed at MHC genes is widely presumed to result from the need to recognize diverse pathogens, a process called pathogen-driven balancing selection. This process assumes that pathogens differ in their peptidomes—the pool of short peptides derived from the pathogen’s proteome—so that different pathogens select for different MHC variants with distinct peptide-binding properties. Here, we tested this assumption in a comprehensive data set of 51.9 Mio peptides, derived from the peptidomes of 36 representative human pathogens. Strikingly, we found that 39.7% of the 630 pairwise comparisons among pathogens yielded not a single shared peptide and only 1.8% of pathogen pairs shared more than 1% of their peptides. Indeed, 98.8% of all peptides were unique to a single pathogen species. Using computational binding prediction to characterize the binding specificities of 321 common human MHC class-I variants, we investigated quantitative differences among MHC variants with regard to binding peptides from distinct pathogens. Our analysis showed signatures of specialization toward specific pathogens especially by MHC variants with narrow peptide-binding repertoires. This supports the hypothesis that such fastidious MHC variants might be maintained in the population because they provide an advantage against particular pathogens. Overall, our results establish a key selection factor for the excessive allelic diversity at MHC genes observed in natural populations and illuminate the evolution of variable peptide-binding repertoires among MHC variants.     

Tolerant and diverse natural killer cell repertoires in the absence of selection

Natural killer (NK) cells are innate lymphocytes that participate in the early control of viruses and tumors. The function of NK cells is under tight regulation by two complementary inhibitory receptor families that bind to classical and non-classical HLA class I molecules: the CD94/NKG2A receptors and the killer cell immunoglobulin-like receptors (KIRs). In this mini-review, recent data on the structure of human NK cell receptor repertoires and its relation to functional responses and tolerance to self are discussed. We propose that no active selection is required to generate diverse NK cell repertoires characterized by a dominant expression of receptors with specificity for self-HLA class I. Instead, the primary consequence of interactions with HLA class I molecules is a functional tuning of randomly generated NK cell repertoires.     

Although divergent in residues of the peptide binding site, conserved chimpanzee Patr-AL and polymorphic human HLA-A*02 have overlapping peptide-binding repertoires

Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ～50% of chimpanzee MHC haplotypes. Comparing Patr-AL(+) and Patr-AL(-) haplotypes showed Patr-AL defines a unique 125-kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5' part of human pseudogene HLA-Y, carried by ～10% of HLA haplotypes. Thus, the AL gene alternatively evolved in these closely related species to become classical, nonclassical, and nonfunctional. Although differing by 30 aa substitutions in the peptide-binding α(1) and α(2) domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable with that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α(2) helix. Stimulating PBMCs from Patr-AL(-) chimpanzees with B cells expressing Patr-AL produced potent alloreactive CD8 T cells with specificity for Patr-AL and no cross-reactivity toward other MHC class I molecules, including HLA-A*02. In contrast, PBMCs from Patr-AL(+) chimpanzees are tolerant of Patr-AL.     

Classification of A1- and A24-supertype molecules by analysis of their MHC-peptide binding repertoires

At the functional level, the majority of human leukocyte antigen (HLA) class I MHC variants can be classified into about ten different major groups, or supertypes, characterized by overlapping peptide binding motifs and repertoires. Previous studies have detailed the peptide binding specificity of the HLA A2, A3, B7, and B44 supertypes, and predicted, on the basis of MHC pocket structures, known motifs, or the sequence of T cell epitopes, the existence of the HLA A1 and A24 supertypes. Direct experimental validation of the A1 and A24 supertypes, however, has been lacking. In the current study, the peptide-binding repertoires and main anchor specificities of several common HLA A molecules (A*0101, A*2301, A*2402, A*2601, A*2902, and A*3002) predicted to be members of the A1 or A24 supertypes were analyzed and defined using single amino acid substituted peptides and a large peptide library. Based on the present findings, the A1 supertype includes A*0101, A*2601, A*2902, and A*3002, whereas the A24 supertype includes A*2301 and A*2402. Interestingly, A*2902 is associated with a motif and peptide binding repertoire that overlaps significantly with those of all of the A1- and A24-supertype molecules studied, representing—to our knowledge—the first report of significant cross-reactivity among molecules belonging to different supertypes.     

The 45 pocket of HLA-A2.1 plays a role in presentation of influenza virus matrix peptide and alloantigens

Amino acid substitutions were introduced into the 45 pocket of HLA-A2.1 to determine the potential role of this structurally defined feature of class I molecules in viral peptide and alloantigen presentation. The 45 pocket lies below the alpha 1-domain alpha-helix and is composed of five amino acids, three of which differ between HLA-A2.1 and HLA-B37. These two class I molecules have previously been shown to have largely non-overlapping peptide-binding specificities. Site-directed mutagenesis was used to replace the hydrophobic residues at positions 24, 45, and 67 in the 45 pocket of HLA-A2.1 with the hydrophilic amino acids found in these positions in HLA-B37. Thus, three single amino acid mutants were produced: 24A----S, 45 M----T, and 67V----S. These mutants were transfected into HMy2.C1R cells and assessed for their ability to present influenza virus matrix M1 57-68 peptide and HTLV-I Tax-1 2-25 peptide to HLA-A2.1-restricted, peptide-specific CTL and to present alloantigens to HLA-A2-allospecific CTL lines. Each of these substitutions in the 45 pocket produced a molecule that failed to present the M1 peptide to most M1 peptide-specific CTL lines. In contrast, none of these mutations affected presentation of the Tax-1 peptide to Tax-1-specific CTL lines, which indicates that these mutant HLA-A2 molecules can function in viral peptide presentation. Two of the three substitutions in the 45 pocket resulted in lack of recognition by a subset of HLA-A2 allospecific CTL lines. These results demonstrate that the amino acid side chains in the 45 pocket can strongly influence peptide presentation and suggest that the 45 pocket may play a role in determining peptide-binding specificity.     

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.     

MHC polymorphism can enrich the T cell repertoire of the species by shifts in intrathymic selection

The murine class I molecule H-2Kb and its natural gene conversion variant, H-2Kbm8, which differs from H-2Kb solely at 4 aa at the bottom of the peptide-binding B pocket, are expressed in coisogenic mouse strains C57BL/6 (B6) and B6.C-H-2bm8 (bm8). These two strains provide an excellent opportunity to study the effects of Mhc class I polymorphism on the T cell repertoire. We recently discovered a gain in the antiviral CTL repertoire in bm8 mice as a consequence of the emergence of the Mhc class I allele H-2Kbm8. In this report we sought to determine the mechanism behind the generation of this increased CTL diversity. Our results demonstrate that repertoire diversification occurred by a gain in intrathymic positive selection. As previously shown, the emergence of the same Mhc allele also caused a loss in positive selection of T cell repertoire specific for another Ag, OVA-8. This indicates that a reciprocal loss-and-gain pattern of intrathymic selection exists between H-2Kb and H-2Kbm8. Therefore, in the thymus of an individual, a new Mhc allele can select new T cell specificities, while abandoning some T cell specificities selected by the wild-type allele. A byproduct of this repertoire shift is a net gain of T cell repertoire of the species, which is likely to improve its survival fitness.     

The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 A) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, DeltaTm = 16 degrees C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a "closed conformation" of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes.     

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.     

Comparative modeling of marsupial MHC class I molecules identifies structural polymorphisms affecting functional motifs

Major histocompatibility complex (MHC) class I molecules are transmembrane glycoproteins that present antigenic peptides to CD8+ T cells and are subsequently important for the initiation of an immune response. In this study novel MHC class I sequences from the tammar wallaby (Macropus eugenii) have been characterized. Analysis and comparative modeling of these and existing marsupial molecules reveals potential functional polymorphisms within peptide-binding grooves, MHC assembly motifs and the T cell receptor recognition interface. In addition, we show that a previously identified marsupial-specific insertion is within a region, which is known as a putative NK cell receptor (Ly49A) binding site in the mouse, suggesting that this site may be functionally active in marsupials. Further, the analysis highlighted differences in structural and sequence based grouping of marsupial MHC class I molecules.     

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.     

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota)

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.     

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8⁺ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.     

The crystal structure of H-2D(b) complexed with a partial peptide epitope suggests a major histocompatibility complex class I assembly intermediate

In the absence of bound peptide ligands, major histocompatibility complex (MHC) class I molecules are unstable. In an attempt to determine the minimum requirement for peptide-dependent MHC class I stabilization, we have used short synthetic peptides derived from the Sendai virus nucleoprotein epitope (residues 324-332, 1FAPGNYPAL9) to promote its folding in vitro of H-2D(b). We found that H-2D(b) can be stabilized by the pentapeptide 5NYPAL9, which is equivalent to the C-terminal portion of the optimal nonapeptide and includes both the P5 and P9 anchor residues. We have crystallized the complex of the H-2D(b) molecule with the pentamer and determined the structure to show how a quasi-stable MHC class I molecule can be formed by occupancy of a single binding pocket in the peptide-binding groove.     

Distinctive interactions at multiple site 2 subsites by allele-specific rat and mouse ly49 determine functional binding and class I MHC specificity

Rodent Ly49 exhibit allele-specific MHC I recognition, yet the interaction site, site 2, encompassing the area below the MHC peptide-binding groove, the alpha3 domain, and associated beta(2) microglobulin, is highly conserved among rat and mouse MHC I alleles. We previously demonstrated that allele-specific Ly49 recognition can be affected by polymorphisms specifically in the peptide anchor-binding and supertype-defining B pocket of MHC I, possibly through differential conformations assumed by solvent-exposed interaction residues when articulating with this pocket. Through mutagenesis of RT1-A1(c) and H-2D(d), we map for the first time the interaction site(s) on rat MHC I mediating rat Ly49i2 recognition and the previously unexamined Ly49G(BALB/c) interaction with H-2D(d). We demonstrate that rat Ly49i2 and mouse Ly49G use both unique and common interactions at three MHC I H chain subsites to mediate functional binding and allele-specific recognition. We find that the F subsite, formed by solvent-exposed residues below the more conserved C-terminal anchor residue-binding F pocket, acts as an anchoring location for both Ly49i2 and Ly49G, whereas these receptors exhibit distinctive reliance on solvent-exposed residues articulating with the polymorphic anchor-binding and supertype-defining pocket(s) at subsite B, as well as on interaction residues at subsite C in the MHC I alpha3 domain. Our findings, combined with previous Ly49A/H-2D(d) and Ly49C/H-2K(b) cocrystal data, suggest how allele-specific MHC I conformations and Ly49 polymorphisms may affect Ly49 placement on MHC I ligands and residue usage at site 2, thereby mediating allele-specific recognition at the highly conserved MHC I interface.     

Molecular cloning and characterization of a novel CD1 gene from the pig.

Much effort is underway to define the immunological functions of the CD1 multigene family, which encodes a separate lineage of Ag presentation molecules capable of presenting lipid and glycolipid Ags. To identify porcine CD1 homologues, a cosmid library was constructed and screened with a degenerate CD1 alpha3 domain probe. One porcine CD1 gene (pCD1.1) was isolated and fully characterized. The pCD1.1 gene is organized similarly to MHC class I and other CD1 genes and contains an open reading frame of 1020 bp encoding 339 amino acids. Expression of pCD1.1 mRNA was observed in CD3- thymocytes, B lymphocytes, and tissue macrophages and dendritic cells. The pCD1.1 cDNA was transfected into Chinese hamster ovary cells, and subsequent FACS analysis demonstrated that mAb 76-7-4, previously suggested to be a pig CD1 mAb, recognizes cell surface pCD1.1. Structurally, the pCD1.1 alpha1 and alpha2 domains are relatively dissimilar to those of other CD1 molecules, whereas the alpha3 domain is conserved. Overall, pCD1.1 bears the highest similarity with human CD1a, and the ectodomain sequences characteristically encode a hydrophobic Ag-binding pocket. Distinct from other CD1 molecules, pCD1.1 contains a putative serine phosphorylation motif similar to that found in human, pig, and mouse MHC class Ia molecules and to that found in rodent, but not human, MHC class-I related (MR1) cytoplasmic tail sequences. Thus, pCD1.1 encodes a molecule with a conventional CD1 ectodomain and an MHC class I-like cytoplasmic tail. The unique features of pCD1.1 provoke intriguing questions about the immunologic functions of CD1 and the evolution of Ag presentation gene families.