Cloning and expression of the rat class I MHC gene RT1.A l

Correspondence to: T. J. Gill, III.     

Identical RT1 class II molecules are expressed by rat RT1m and RT1c haplotypes

The RT1m haplotype of MNR rats has been suggested to be a recombinant RT1 haplotype inheriting RT1.A (class I) alleles from RT1a (DA) and RT1.B (class II) alleles from RT1c (AUG). Additional serologic and biochemical assays, however, have suggested that RT1m and RT1c share a single identical RT1.B molecule, although differing in the expression of the second RT1.B molecule. To resolve this contradiction, RT1.B class II molecules, comparable to I-A and I-E molecules in mice, expressed by the RT1c and RT1m haplotypes were immunoprecipitated by cross-reactive mouse anti-Ia antibodies and were compared by two-dimensional gel electrophoresis and by high pressure liquid chromatographic separation of tryptic peptides. Respective subunits expressed by the two haplotypes co-migrate on two-dimensional gels and have identical tryptic peptide maps. The results at the protein level were confirmed at the DNA level by Southern blot analysis of MNR and AUG genomic DNA. Identical restriction fragments associated with the RT1m and RT1c haplotypes hybridized with each of the DC1 beta, DR alpha, and DR beta cDNA probes. The results at both the protein and DNA levels suggest that the RT1m and RT1c haplotypes share identical expressed alleles at the RT1.Ba, RT1.Bb, RT1.Bc, and RT1.Bd loci.     

Polymorphism and mapping of the class II genes in the rat: RT1.B,RT1.D,and RT1.H,a new DP-like region

The major histocompatibility complex of the rat (RTI) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the - and -chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has - and -chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1. B. The RT1. H and RT1.H genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H and D genes have high levels of polymorphism; B , B ,and H have intermediate levels of polymorphism, and D has a low level of polymorphism.     

Serological and biochemical characterization of rat (RT1) class II molecules with restricted mouse anti-Ia sera

Murine anti-Ia sera were tested for their reactivity with rat peripheral lymphocytes. Extensive cross-reactions were observed in a complement-dependent cytotoxicity assay. In the majority of strain combinations, these reactions were specific for B cells. We demonstrated by absorption analyses that (1) the reaction was specific, (2) B cells from all rat strains reacted with anti-I-E^k serum, and (3) the anti-I-A sera defined five new specificities. These antisera can also immunoprecipitate class II (Ia-like) molecules from radiolabeled, detergent-solubilized cell extracts. The rat equivalents of molecules encoded in the I-A and I-E subregions in mice can be isolated from all rat strains studied. The and subunits of the rat class II molecules were polymorphic when analyzed by sodium dodecyl sulfate (SIDS) polyacrylamide electrophoresis. This observation allows the characterization of rat alloantisera or monoclonals of unknown specificity by comparing them to the defined reactivity of the mouse anti-Ia sera.     

Peptide motif for the rat MHC class II molecule RT1.Da: similarities to the multiple sclerosis-associated HLA-DRB1*1501 molecule

Experimental autoimmune encephalomyelitis induced with myelin proteins in DA and LEW.1AV1 rats is a model of multiple sclerosis (MS). It reproduces major aspects of this detrimental disease of the central nervous system. MS is associated with the HLA-DRB1*1501, DRB5*0101, and DQB1*0602 haplotype. DA and LEW.1AV1 rats share the RT1^av1 haplotype. So far, no MHC class II peptide motif of RT1.D^a molecules has been described. Sequence alignment of the chain of the rat MHC class II molecule RT1.D^a with human HLA class II molecules revealed strong similarity in the peptide-binding groove of RT1.D^a and HLA-DRB1*1501. According to the putative peptide-binding pockets of RT1.D^a, after comparison with the pockets of HLA-DRB1*1501, we predicted the peptide motif of RT1.D^a. To verify the predicted motif, naturally processed peptides were eluted by acidic treatment from immunoaffinity-purified RT1.D^a molecules of lymphoid tissue of DA rats and subsequently analyzed by ESI tandem mass spectrometry. In addition, we performed binding studies with combinatorial nonapeptide libraries to purified RT1.D^a molecules. Based on these studies we could define a peptide-binding motif for RT1.D^a characterized by aliphatic amino acid residues (L, I, V, M) and of F for the peptide pocket P1, aromatic residues (F, Y, W) for P4, basic residues (K, R) for P6, aliphatic residues (I, L, V) for P7, and aromatic residues (F, Y, W) and L for P9. Both methods revealed similar binding characteristics for peptides to RT1.D^a. This data will allow epitope predictions for analysis of peptides, relevant for experimental autoimmune diseases.     

Water-soluble form of RT1.A class I MHC molecules in the kidney and liver of the rat

The RT1.A (H-2K,D type) class I major histocompatibility complex (MHC) antigens of the rat are well recognized as membrane-bound glycoproteins. In this report, we demonstrate that liver and kidney in the DA rat strain contain large amounts of a water-soluble RTl. A class I molecule with a discrete heavy chain approximately 5 kd smaller than the membrane-bound form. An identical molecule could be identified in DA rat serum. This small class I molecule carries all of the polymorphic antigenic determinants of the RT1.A^av1 class I molecule. The water-soluble molecule is readily denatured in its pure form when frozen and thawed, but this does not occur when it is mixed with serum, presumably because of a stabilizing interaction with one or more carrier proteins. The half-life of the class I molecule in serum was measured to be approximately 1.5 h. The LEW rat strain produced detectable but substantially smaller amounts of water-soluble RT1.A molecules. Our studies indicate that RT1.A^av1 class I MHC antigens are synthesized and presumably secreted in a smaller water-soluble form by liver, kidney, and possibly other tissues under physiological conditions, a point of con-siderable interest in view of the immunoregulatory functions of the membrane-bound forms of these molecules.     

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.     

An immunochemical procedure for the purification of rat class I antigens (RT1.A/RT1.E) from detergent-solubilized erythrocytes

An immunochemical procedure using two immunoaffinity columns for the isolation of rat Class I antigens from detergent-solubilized DA erythrocytes is described. The protocol yields a pure Class I preparation as assessed by silver staining of sodium dodecyl sulfate-polyacrylamide gels and comparison of the amino acid composition with that previously determined for H-2K/D. The antigen preparation is capable of specifically blocking the hemagglutination of DA erythrocytes by Class I-specific alloantiserum.     

MHC II类分子表达调控的研究进展

MHCII类分子提呈经过加工的抗原给CD4 T淋巴细胞 ,在诱发免疫反应中起重要作用。MHCII类分子不正常表达会引起严重的免疫缺陷疾病 ,如裸淋巴细胞综合征 (BLS)等。目前已识别出四种不同的MHCII调控基因。这些基因分别编码RFXANK、RFX5、RFXAP和CIITA。其中 ,前三个是RFX复合物的亚基 ,RFX是一种结合于所有MHCII类基因启动子上的泛式表达的因子。CIITA是MHCII类分子表达的主要调控因子 ,其严密调控的表达模式决定了MHCII类分子表达的细胞特异性 ,及能否被诱导且在何种水平上表达。本文着重介绍近年来国内外对MHCII类分子表达及其调控研究的新进展     

The class II genes of the rat MHC

Genes that encode class II Ag from the MHC of the rat, the RT1 region, have been isolated as a series of cosmid clones. The cosmids define two clusters, each of which contains three identifiable sequences; one homologous to alpha-chain and two to beta-chain genes. Both the serologically identified rat class II Ag have been expressed in mouse L cell fibroblasts after the introduction of each alpha-chain gene along with a beta-chain gene from the same cluster. There are substantial homologies to the I region of the mouse H-2 complex in the presence, location, orientation, and expression of the six identified sequences from the rat RT1, supporting the view that the overall organization of the two gene complexes has remained conserved since the species separated.     

Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.