Expression and function of Qa-2 major histocompatibility complex class I molecules in transgenic mice

Qa-2 molecules are weak transplantation antigens encoded by class I genes of the major histocompatibility complex. When expressed in transgenic CBA mice, Qa-2 molecules provoke rapid rejection of skin grafts and strong, Qa-2 specific, cytotoxic T-cell responses. Efficient rejection of skin grafts from Qa-2 transgenic mice takes place when Qa-2 molecules are attached to the cell membrane with a glycophosphatidyl anchor or by a transmembrane protein domain, except that rejection times are slightly longer in the former case. These results demonstrate that Qa-2 molecules can behave as major transplantation antigens, as do closely related H-2 molecules. Failure of Qa-2 molecules to provoke strong T-cell responses in non-transgenic mice is probably due to the very low level of expression of Qa-2 molecules in skin keratinocytes from such mice since these cells express increased levels of Qa-2 molecules in all Qa-2 transgenic mice.     

Biosynthesis of membrane and secreted epsilon-chains during lipopolysaccharide-induced differentiation of an IgE+ murine B-lymphoma

A switch variant of the I.29 murine B-cell lymphoma expressing membrane IgE and inducible by lipopolysaccharide (LPS) to increase the rate of IgE secretion was characterized. The cells (I.29 epsilon +2) express membrane-bound IgE, and also secrete considerable amounts of IgE when grown in regular culture medium. Membrane and secreted IgE contain structurally different heavy chains. The former is constituted by a 93-kd molecule (epsilon m), while secretory chains (epsilon s) have an apparent mol. wt of 86,000. Both epsilon m and epsilon s are heavily glycosylated: in the presence of tunicamycin their apparent mol. wt is reduced by approx. 35% (61 kd for epsilon m and 56 kd for epsilon s). Glycosylation is necessary for membrane expression and for secretion of IgE molecules. Stimulation with LPS leads to the disappearance of IgE molecules from the cell surface (determined by radioiodination) although epsilon m-chains are still synthesized, suggesting a defective transport of membrane IgE in LPS-treated cells. The epsilon m:epsilon s ratio decreases upon LPS stimulation. A similar change can be observed in the messenger RNAs specific for epsilon m and epsilon s, possibly suggesting a major pretranslational control for epsilon m and epsilon s biosynthesis.     

H-2 class I gene analysis in 3-methylcholanthrene-induced murine fibrosarcomas

The objective of our study was to determine whether and how frequently MHC class I gene alterations occur in fibrosarcomas induced in BALB/c mice by the carcinogen 3-methylcholanthrene (3-MCA). Southern blot was performed to analyze the genomic organization of class I genes in a panel of twenty tumors of different immunogenic strength used at early in vivo passages. Our results rule out the presence of gross rearrangements or deletions in the H-2 class I genes of the tumors tested and indicate that the frequency of detectable class I alterations giving rise to restriction fragments length polymorphism (RFLP) should be very low.     

Molecular genotyping of the murine H-2K MHC class I allele

An increasing number of genetically modified mouse mutants are employed in immunological research. Many experiments require the crossbreeding of various mouse lines and screening for the genetic background of the offspring. Analysis for various immunological phenotypes such as the MHC class I haplotype is usually performed by FACS analysis of peripheral blood lymphocytes. Here, we report a PCR based technique for the differentiation of the MHC class I H-2K(k) and the H-2K(b) haplotype. Advantages of this method are cost efficiency and rapidity in particular when multiple genetic variables such as the disruption of a gene, transgenesis or the MHC haplotype are to be tested.     

Qa-1, a nonclassical class I histocompatibility molecule with roles in innate and adaptive immunity

Qa-1, a nonclassical class I histocompatibility molecule expressed in mice, predominantly assembles with a single nonameric peptide, Qdm, derived from the signal sequence of certain class Ia molecules. The Qa-1/Qdm complex is the primary ligand for CD94/NKG2A inhibitory receptors expressed on a major fraction of natural killer (NK) cells. Cells become susceptible to killing by NK cells under conditions where surface expression of the Qa-1/Qdm inhibitory ligand is reduced. The CD94/NKG2 "missing-self" recognition system serves as mechanism for removing cells that have abnormalities in the intracellular machinery required for assembly and expression of class I-peptides complexes, as a consequence of viral infection, for example. Despite its highly focused peptide-binding specificity, Qa-1 also has a capacity to act as an antigen-presentation molecule for CD8+ T cells. It appears that a small subpopulation of these T cells undergoes positive selection by interaction with Qa-1 in the thymus, and they maintain their specificity for Qa-1 after maturation. The role of these unusual T cells in adaptive immune responses remains to be defined.     

A single-chain murine class I major transplantation antigen

Single-chain mouse Kd molecules (SC-Kd) were engineered by connecting residue 276 of Kd heavy chain to the first residue of beta 2-microglobulin through spacers of various lengths, and expressed intracellularly in monkey COS-1 cells. Labeled SC-Kd molecules were found to react with several monoclonal antibodies which recognize native Kd molecules. SC-Kd-15 (with a spacer of 15 residues) was studied in more details. It could be purified and shown to regain a native-like structure after treatment with denaturing agents. Purified SC-Kd-15 could bind certain peptides in a manner qualitatively similar to the Kd.     

Increased Con-A-induced thymocyte proliferation and de novo Qa-2 antigen expression in Qa-2-depleted thymocyte cultures

We have recently shown that thymocyte activation is associated with a strong increase of Qa-2 antigen expression. Contrary to what could be predicted from these results, the initial depletion of Qa-2+ cells from PNA- thymocytes increases the Con-A-induced proliferation. Qa-2+ cells not only reappeared after activation, but were enhanced in Qa-2-depleted PNA- cells compared to the control. The newly formed Qa-2+ population contained a higher percentage of Lyt 1+2- cells and thus seems to be different from the Qa-2+ pool initially present.     

Molecular weight diversity among murine class I antigens: both the mature cell surface forms and the unglycosylated polypeptides vary significantly in molecular weight

The molecular weights of the fully glycosylated cell surface form and the unglycosylated polypeptide of five murine class I antigens (H-2Kb, Db, TL, Qa-1.1, and Qa-2) were compared by SDS-PAGE. Significant molecular weight diversity was observed for both forms among these molecules. The size of the fully glycosylated forms ranged from approximately 52,000 daltons (H-2Db) to 41,000 daltons (Qa-2), whereas the unglycosylated polypeptides ranged from 43,000 daltons (H-2Kb and TL) to 33,000 daltons (Qa-2). The magnitude of the size variation observed in the unglycosylated polypeptides implies that there are differences in the gene organization, RNA processing or post-translational modifications of various class I glycoproteins.     

Structural polypeptides of the murine coronavirus DVIM

Summary The structural polypeptides of the murine coronavirus DVIM (diarrhoea virus of infant mice) have been analysed in comparison with other strains MHV-2, MHV-3, MHV-4 (JHM) and MHV-S by SDS-PAGE. In the presence of 2-mercaptoethanol, three major glycopolypeptides, gp180, gp69, gp25 (as a group of similar species) and one major non-glycosylated polypeptide p58 were detected. The gp69 is a DVIM specific glycopolypeptide, in which the glycosidic moieties are linked to the core polypeptide through N-glycosidic bonds, and hence may be correlated with the short projections of the viral envelope. Further gp140, which appears in the absence of reducing agents, is apparently a dimer of gp69 held together by disulfide linkages. The gp25 family, on the other hand, consists of four polypeptides, two of which are not metabolically inhibited by tunicamycin suggesting that they are O-linked glycopolypeptides. DVIM seems to be serologically closely related to the MHV-S strain as shown by neutralization.With 3 Figures     

Identification of naturally processed peptides bound to the class I MHC molecule H-2Kd of normal and TAP-deficient cells

This report details the biochemical features of natural peptides selected by the H-2Kd class I MHC molecule. In normal cell lines, the length of the naturally processed peptides ranged from 8 to 18 amino acids, although the majority were 9-mers (16% were longer than nine residues). The binding motif for the 9-mer peptides was dominated by the presence of a tyrosine at P2 and an isoleucine/leucine at the P9 position. The P2 residue contributed most towards binding; and the short peptides bound better and formed longer-lived cell surface complexes than the long peptides, which bound poorly and dissociated rapidly. The longer peptides did not exhibit this strictly defined motif. Trimming the long peptides to their shorter forms did not enhance binding and conversely, extending the 9-mer peptides did not decrease binding. The long peptides were present on the cell-surface bound to H-2Kd (Kd) and were not intermediate products of the class I MHC processing pathway. Finally, in two different TAP-deficient cells the long peptides were the dominant species, which suggested that TAP-independent pathways selected for long peptides by class I MHC molecules.     

Polypeptides of mammalian oncornaviruses. I. Isolation and serological analysis polypeptides from murine and feline C-type viruses

Agents representative of murine and feline C-type viruses contain four major polypeptides of molecular weights 10,000 (P1), 12,000 (P2), 15,000 (P3), and 31,000 (P4). Each was shown to contain distinct antigenic determinants. P1 of Friend leukemia virus (FLV) demonstrates species-specific reactivity different from that present in FLV P4. It also seems to be associated with a host cell component. FLV P2 revealed type or subgroup specificity and was shown to be associated with carbohydrate. FLV P3 was not investigated in as much detail. Feline leukemia virus P4, which is known to contain both species and interspecies determinants, revealed also a type-or subgroup-specific reactivity. Finally, an antiserum prepared against murine P4 was shown to contain antibodies directed against distinguishable interspecies determinants.     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

Description of a Qa-2 like alloantigen (Qa-m2)

Several new aspects of the chemistry, genetics and cellular distribution of the murine Qa-2 alloantigen were apparent in an analysis of this antigen using monoclonal antibodies recognizing a Qa-2-like antigen called Qa-m2. Immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis identified the Qa-m2 alloantigen as a two-chain structure composed of a 39 000 dalton heavy chain and a 12000 light chain which is probably β₂-microglobulin; the heavy chain was readily distinguished from that of H-2. In addition, the expression of Qa-m2 alloantigens on the cell surface was found to be controlled by the H-2D gene with H-2D^b strains carrying approximately 8-10 times greater amounts of Qa-m2 than strains carrying the H-2D^d or H-2D^q alleles. Finally, the Qa-m2 antigen, found predominantly on peripheral T cells, was present on only 10% of thymus cells. However, subpopulations of B cells (approximately 25% of all B cells) and bone marrow cells (15-20%) were also reactive. The monoclonal anti-Qa-m2 antibodies differed in their reactions from that reported for the conventional anti-Qa-2 sera, which must, therefore, be complex. The monoclonal antibodies may be useful reagents for functional analysis of T and B cell subpopulations.     

A single T cell receptor bound to major histocompatibility complex class I and class II glycoproteins reveals switchable TCR conformers

Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T?cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα β strand, switching Vα's position relative to Vβ. In several other structures, either Vα or Vβ undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands.     

Biosynthesis and processing of class II histocompatibility antigens

The class II major histocompatibility antigens at the cell surface exist as heterodimers of alpha and beta subunits. During biosynthesis, these subunits are associated with a third chain, the invariant (I) or I chain. Association with the I chain occurs early in biosynthesis in the rough endoplasmic reticulum and persists during transport through the Golgi apparatus. One of the two alpha subunit N-linked oligosaccharides and the single beta subunit N-linked oligosaccharide are converted to the complex form during Golgi transit. In the human system, both I chain N-linked oligosaccharides can also be processed to the complex form, and at least two O-linked oligosaccharides can be added to the I chain. At some point during transit to the cell surface, class II antigens associate with a proteoglycan bearing chondroitin sulfate side chains. Complexes containing alpha, beta and I chain subunits and the associated proteoglycan accumulate in human B-cell lines treated with the ionophore monensin, an inhibitor of Golgi transport, suggesting that this may be a biosynthetic intermediate in class II antigen transport and assembly. Prior to cell surface expression of class II antigens, the exocytic pathway which they follow intersects the endocytic route, followed by certain ligands internalized by receptor-mediated endocytosis. The I chain appears to dissociate from mature class II alpha, beta dimers prior to their cell surface expression but following the intersection of the exocytic and endocytic pathways.     

HLA class I genes integrated into murine cells are inducible by interferon

In human cells treated with interferon, there is an increase in the amount of HLA-A, B, C mRNA and, to a lower extent, membrane-bound antigen. However, the mechanism of this mRNA enhancement is still unknown. Using mouse L cells transfected with a unique class I HLA gene, we were able to show that both the related HLA mRNA and protein are increased after murine but not human interferon treatment. Moreover, the discrepancy between interferon-directed HLA mRNA and protein enhancement is also observed. The mouse transfected cells allowed us to study more precisely the origin of this discrepancy.