Importance of MHC class 1 alpha2 and alpha3 domains in the recognition of self and non-self MHC molecules

The importance of the species of different domains of class I MHC molecules in peripheral T cell recognition and positive and negative selection was evaluated in a single system. In transgenic mice expressing AAD (containing the alpha1+alpha2 domains of HLA-A2.1 and the alpha3 domain of H-2Dd), the CTL response to influenza peptide M1(58-66) in the context of the alpha1+alpha2 domains of HLA-A2.1 was as strong as the influenza-specific H-2Db-restricted response. However, this strong response was only discernible if the target cell MHC molecule also contained a murine alpha3 domain. In contrast, the response in HLA-A2.1 transgenic mice was about 30-fold weaker, and these CTL were indifferent to the origin of the target molecule alpha3 domain. Further analysis suggested that the major impact of the murine alpha3 domain of the transgene product was to enhance positive selection of a low affinity population of AAD-restricted T cells, presumably through species-specific interaction with CD8. Surprisingly, the response to non-self human class I MHC determinants was not augmented in AAD mice, indicating that the T cells selected are narrowly focused on AAD-related structures. Further analysis indicated that the alphal+alpha2 domains as well as the alpha3 domain influenced the magnitude of the response to non-self human class I MHC determinants, and this effect was mapped to alpha2. We suggest that the alpha2 domains of murine class I molecules contain conserved structural elements that augment the avidity of T cell-class I interactions, and this is particularly important in the recognition of non-self MHC molecules.     

The alpha2 domain of H-2Dd restricts the allelic specificity of the murine NK cell inhibitory receptor Ly-49A

Mouse NK lymphocytes express Ly-49 receptors, which inhibit cytotoxicity upon ligation by specific MHC I molecules on targets. Different members of the lectin-like mouse Ly-49 receptor family recognize distinct subsets of murine H-2 molecules, but the molecular basis for the allelic specificity of Ly-49 has not been defined. We analyzed inhibition of natural killing by chimeric MHC I molecules in which the alpha1, alpha2, or alpha3 domains of the Ly-49A-binding allele H-2Dd were exchanged for the corresponding domains of the nonbinding allele H-2Db. Using the Ly-49A-transfected rat NK cell line, RNK-mLy-49A.9, we demonstrated that the H-2Dd alpha2 domain alone accounts for allelic specificity in protection of rat YB2/0 targets in vitro. We also showed that the H-2Dd alpha2 domain is sufficient to account for the allele-specific in vivo protection of H-2b mouse RBL-5 tumors from NK cell-mediated rejection in D8 mice. Thus, in striking contrast to the alpha1 specificity of Ig-like killer inhibitory receptors for human HLA, the lectin-like mouse Ly-49A receptor is predominantly restricted by the H-2Dd alpha2 domain in vitro and in vivo.     

NK cells from human MHC class I (HLA-B) transgenic mice do not mediate hybrid resistance killing against parental nontransgenic cells

We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7kb (human-mouse hybrid consisting of the alpha1 and alpha2 domains of HLA-B7, and the alpha3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC "self" elements for mouse NK cells, even when present throughout ontogeny.     

Bone marrow fibrosis and radiological changes of the long bones in children with acute megakaryocytic leukaemia

Using polymerase chain reaction, bottle-nosed dolphin (Tursiops truncatus) major histocompatibility complex (DoLA) class I alpha chain cDNA was cloned and sequenced. Predicted amino acid sequences of DoLA class I alpha chain have cystein residues for intradomain disulfide bond formation and N-linked glycosylation sites, suggesting that DoLA class I alpha chain molecules construct alpha 1, alpha 2, and alpha 3 domain structures. Similarity of DoLA class I alpha chain cDNA to land mammal MHC class I alpha chain cDNA was 90.4% in cattle, 90.2% in horse, 89.4% in sheep, and 87.8% in human. This investigation suggests that DoLA is closely related to land mammals.     

Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection

The crystal structure of the mouse major histocompatibility complex (MHC) class I molecule H-2Dd with an immunodominant peptide, designated P18-I10 (RGPGRAFVTI), from human immunodeficiency virus envelope glycoprotein 120 was determined at 3.2 A resolution. A novel orientation of the alpha3 domain of Dd relative to the alpha1/alpha2 domains results in significantly fewer contacts between alpha3 and beta2-microglobulin compared with other MHC class I proteins. Four out of ten peptide residues (P2 Gly, P3 Pro, P5 Arg and P10 Ile) are nearly completely buried in the Dd binding groove. This is consistent with previous findings that Dd exploits a four-residue binding motif comprising a glycine at P2, a proline at P3, a positively charged residue at P5, and a C-terminal hydrophobic residue at P9 or P10. The side-chain of P5 Arg is directed toward the floor of the predominantly hydrophobic binding groove where it forms two salt bridges and one hydrogen bond with Dd residue Asp77. The selection of glycine at P2 appears to be due to a narrowing of the B pocket, relative to that of other class I molecules, caused by Arg66 whose side-chain folds down into the binding cleft. Residue P3 Pro of P18-I10 occupies part of pocket D, which in Dd is partially split by a prominent hydrophobic ridge in the floor of the binding groove formed by Trp97 and Trp114. Residues P6 through P9 form a solvent-exposed bulge, with P7 Phe protruding the most from the binding groove and thereby probably constituting a major site of interaction with T cell receptors. A comparison of H-2Dd/P18-I10 with other MHC class I/peptide complexes of known structure provides insights into the possible basis for the specificity of the natural killer cell receptor Ly-49A for several related class I molecules.     

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

Glu227-->Lys substitution in the acidic loop of major histocompatibility complex class I alpha 3 domain distinguishes low avidity CD8 coreceptor and avidity-enhanced CD8 accessory functions

Cytotoxic T lymphocyte (CTL) activation requires specific T cell receptor (TCR)-class I major histocompatibility complex (MHC) antigen complex interactions as well as the participation of coreceptor or accessory molecules on the surface of CTL. CD8 can serve as a coreceptor in that it binds to the same MHC class I molecules as the TCR to facilitate efficient TCR signaling. In addition, CD8 can be "activated" by TCR stimulation to bind to class I molecules with high avidity, including class I not recognized by the TCR as antigenic complexes (non-antigen [Ag] class I), to augment CTL responses and thus serve an accessory molecule function. A Glu/Asp227-->Lys substitution in the class I alpha 3 domain acidic loop abrogates lysis of target cells expressing these mutant molecules by alloreactive CD8-dependent CTL. Lack of response is attributed to the destruction of the CD8 binding site in the alpha 3 domain which is likely to disrupt CD8 coreceptor function. The relative importance of the class I alpha 3 domain acidic loop Glu227 in coreceptor as opposed to accessory functions of CD8 is unclear. To address this issue, we examined CTL adhesion and degranulation in response to immobilized class I-peptide complexes formed in vitro from antigenic peptides and purified class I molecules containing wild-type or Glu227-->Lys substituted alpha 3 domains. The alpha 3 domain mutant class I-peptide complexes were bound by CTL and triggered degranulation, however to much lower levels than wild-type class I-peptide complexes. In further experiments, it is directly demonstrated that the alpha 3 domain mutant class I molecules, which lack the Glu227 CD8 binding site, still serve as TCR-activated, avidity-enhanced CD8 accessory ligands. However, mutant class I peptide Ag complexes failed to effectively serve as CD8 coreceptor ligands to initiate TCR-dependent signals required to induce avidity-enhanced CD8 binding to coimmobilized non-Ag class I molecules. Thus the Glu227-->Lys mutation effectively distinguishes CD8 coreceptor and avidity-enhanced CD8 accessory functions.     

Orientation of the Ig domains of CD8 alpha beta relative to MHC class I

The cell surface glycoprotein CD8 functions as a coreceptor with the TCR for interaction with MHC class I. The cocrystal structure of the CD8 alpha alpha-MHC complex showed that one CD8 Ig domain provided the majority of the contact with MHC class I and that residue R4 of that domain contacted the alpha2 domain of MHC class I. We previously showed by mutational analysis that this residue was critical for binding to MHC class I. To determine which of the Ig domains for the CD8 alpha beta heterodimer would make the most contact with class I MHC, we expressed single-chain or dimeric forms of CD8 on COS-7 cells and measured the adhesion of MHC class I positive cells. We found that when one of the R4 residues was mutated in a CD8 alpha alpha homodimer binding comparable to that of wild type was observed, whereas a double R4 mutant severely impaired binding. However, when mutant CD8 alpha (R4K) was coexpressed with wild-type CD8 beta, binding was not observed. These results support the model in which it is CD8 alpha, not CD8 beta, that is making the most of the contact with MHC class I, including the alpha 2 domain. In addition, they demonstrate that a single-chain form of CD8 alpha alpha can bind to MHC class I.     

Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b)

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.     

Pattern ERGs from isoluminant gratings; poor selectivity compared with VEPS

The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (alpha 1), 3 (alpha 2), 4 (alpha 3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the alpha 2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes.     

Possible assortment of a1 and a2 region gene segments in human MHC class I molecules

Using pair-wise comparison of aligned nucleotide sequences of distinct and complete human MHC class I molecules, we have constructed triangular tables to study the similarities and differences of various a1 (exon 2) and a2 (exon 3) region sequences. There are two HLA-A (A*6901 and A*6601) and 13 HLA-B (B*4201, B*8101, B*4102, B*4801, B*4007, B*4001, B*4802, Dw53, B*4406, B*4402, B*3901, B*1514 and B*3702) sequences that have identical a1 sequences with other known MHC class I molecules, while their a2 sequences are the same as those of different ones. Of these 15, A*6901, B*4001 and B*4802 have previously been suggested as the results of recombination between A*6801 and A*0201, B*4101 and B*8101, and B*4801 and B*3501, respectively. However, many other sequences can also be used to generate them by recombination. Furthermore, their reciprocal products have never been identified. Thus, gene conversion has subsequently been suggested as an alternative. Another possible genetic mechanism for generating these nucleotide sequence similarities can be assortment, or that some gene segments can be duplicated or multiplicated to be used in different human MHC class I molecules. Interestingly, this genetic mechanism is probably absent for the generation of different mouse MHC class I molecules.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

A case of congenital mumps infection complicated with persistent pulmonary hypertension

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.     

When is domestic violence a hidden face of addiction?

The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-gamma (IFN-gamma)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 alpha- and beta-subunits. The deduced amino acid sequences of the alpha- and beta-subunits were 49% identical. We also determined the primary structure of the mouse PA28 gamma-subunit (Ki antigen), a protein of unknown function structurally related to the alpha- and beta-subunits. The amino acid sequence identity of the gamma-subunit to the alpha- and beta-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the alpha- and beta-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-gamma-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the gamma-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a gamma-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-gamma-inducible alpha- and beta-subunits emerged by gene duplication from a gamma-subunit-like precursor.     

Role of conserved glycosylation site unique to murine class I MHC in recognition by Ly-49 NK cell receptor

The recognition of class I MHC molecules on target cells by the Ly-49 family of receptors regulates NK cytotoxicity. Previous studies have suggested that carbohydrates are involved in the recognition of class I MHC by Ly-49, although their precise role remains unclear. Here, we examined the role of asparagine-linked carbohydrates of the murine class I MHC in the binding to Ly-49A and Ly-49C. We have generated H-2Dd mutants that lack the highly conserved glycosylation sites at amino acid residues 86 in the alpha1 domain and 176 in the alpha2 domain, respectively. These mutant Dd cDNAs were transfected into leukemic cell lines, and the binding of the transfected cells to COS cells expressing Ly-49A or Ly-49C, as well as their susceptibility to lysis by Ly-49A+ NK cells, was examined. Only the mutation of the alpha2 domain glycosylation site significantly reduced the binding of Dd to Ly-49A and Ly-49C. Cells expressing Dd with the mutation at this site were partially resistant to killing by Ly-49A+ NK cells. These results suggest that, while carbohydrates linked to residue 176 seem to function as a part of the ligand structure for the Ly-49 family of NK receptors, there are additional structural features involved in this recognition. This glycosylation site is highly conserved among murine class I MHC but is not found among those of other species, suggesting that its role is unique to the murine immune system. It further suggests that murine class I MHC and Ly-49 gene families may have evolved in concert.     

Conservation of cytotoxic T lymphocyte (CTL) epitopes as a host strategy to constrain parasite adaptation: evidence from the nef gene of human immunodeficiency virus 1 (HIV-1)

Host cytotoxic T lymphocytes (CTLs) that recognize specific viral peptides (epitopes) are thought to provide the most effective control of viral replication and spread. However, viruses may escape this recognition through mutations in CTL epitopes. We tested the hypothesis that, as an adaptation on the part of the host to constrain parasite escape from immune control, class I major histocompatibility complex (MHC) molecules present peptides that are derived from conserved regions of foreign proteins to CTLs. We did this by estimating the relative conservation of CTL epitopes of the functionally important Nef protein of human immunodeficiency virus 1 (HIV-1) and relating this to the structure and function of the protein. In comparisons among sequences from several HIV-1 subtypes and both major groups, CTL epitopes had lower rates of nonsynonymous nucleotide substitution per site than did the remainder of the protein, indicating the relative conservation of these epitopes. In contrast, helper T-cell epitopes were as conserved as, and monoclonal antibody epitopes less conserved than, the remainder of the protein. The conservation of CTL epitopes is apparently due to their derivation from functionally important domains of Nef, since CTL epitopes coincide with these domains and these domains are conserved relative to the remainder of the protein, in contrast to secondary structural elements, which are not. Recent studies provide evidence of CTL selection on HIV-1 epitopes, but the variational range of viral escape mutants appears to be limited by functional constraints on the protein regions from which epitopes are derived. The presentation of conserved foreign peptides to CTLs by class I MHC molecules may be a general adaptation of vertebrate hosts to constrain the adaptation of their intracellular parasites.