Patricia Riegert

Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a “minimal essential MHC” for chickens, in strong contrast to typical mammals.

Different evolutionary histories of the two classical class I genes BF1 and BF2 illustrate drift and selection within the stable MHC haplotypes of chickens

Compared with the MHC of typical mammals, the chicken MHC (BF/BL region) of the B12 haplotype is smaller, simpler, and rearranged, with two classical class I genes of which only one is highly expressed. In this study, we describe the development of long-distance PCR to amplify some or all of each class I gene separately, allowing us to make the following points. First, six other haplotypes have the same genomic organization as B12, with a poorly expressed (minor) BF1 gene between DMB2 and TAP2 and a well-expressed (major) BF2 gene between TAP2 and C4. Second, the expression of the BF1 gene is crippled in three different ways in these haplotypes: enhancer A deletion (B12, B19), enhancer A divergence and transcription start site deletion (B2, B4, B21), and insertion/rearrangement leading to pseudogenes (B14, B15). Third, the three kinds of alterations in the BF1 gene correspond to dendrograms of the BF1 and poorly expressed class II B (BLB1) genes reflecting mostly neutral changes, while the dendrograms of the BF2 and well-expressed class II (BLB2) genes each have completely different topologies reflecting selection. The common pattern for the poorly expressed genes reflects the fact the BF/BL region undergoes little recombination and allows us to propose a pattern of descent for these chicken MHC haplotypes from a common ancestor. Taken together, these data explain how stable MHC haplotypes predominantly express a single class I molecule, which in turn leads to striking associations of the chicken MHC with resistance to infectious pathogens and response to vaccines.

Identification of class I major histocompatibility complex encoded molecules in the amphibian Xenopus

Class I-like molecules have been immunoprecipitated from Xenopus leukocytes and erythrocytes with alloantisera directed against major histocompatibility complex (MHC)-linked antigens. The heavy chains, depending on the allele examined, have molecular weights of 40 000–44 000 of which 3000 daltons are asparagine-linked carbohydrates, probably present as one N-linked glycan. The presumed analogue of β2-microglobulin has a molecular weight of 13 000 and bears no asparagine-linked glycans. Family studies show that the heavy chains are encoded by genes residing in or closely linked to the MHC.

The mouse HFE gene.

Idiopathic hemochromatosis is believed to represent the most frequent hereditary condition in Caucasian populations (for review see Online Mendelian Inheritance in Man, OMIM 1997). Twenty years after the discovery of the disease association to the major histocompatibility complex (MHC) (Simon et al. 1976), the underlying genetic basis has been recently characterized as a point mutation in a new MHC class I gene (MHC-I), originally called HLA-H and subsequently designated HFE (Feder et al. 1996; OMIM 1997). The HFE transcript of approximately 4 kilobases (kb) (1.5 kb in mouse) contains a 1029 nucleotide (1080 in mouse) open reading frame encoding a putative polypeptide of 343 residues (359 in mouse) (Feder et al. 1996; Hashimoto et al. 1997). The human HFE gene is located 4 megabases telomeric to HLA-F on chromosome 6p21.3± 6p22.1 (Feder et al. 1996). The primary structure of HFE resembles that of a typical MHC-I molecule. Following a hydrophobic leader sequence, there are three potential extracellular domains (a1, a2, and a3) followed by transmembrane and cytoplasmic sequences (Feder et al. 1996; Hashimoto et al. 1997). A preliminary study of HFE expression by northern blotting indicated an apparently wide range of tissue expression, not unlike that of typical MHC-I genes (with the reported exception of fetal brain and lymphoblasts) (Feder et al. 1996). The availability of an antiserum led to the detection of the HFE gene product along almost the entire gastrointestinal tract with apparently specific subcellular localization within the crypts of the small intestine, the hypothetical site of iron absorption (Parkkila et al. 1997). The proposed etiological role of HFE in hemochromatosis comes from the identification of a point mutation, G845A, leading to a Cys282Tyr substitution, found at homozygosity in most if not all patients (Feder et al. 1996; OMIM 1997). The replacement of this cysteine residue, the proximal partner of the a3 domain immunoglobulin-like loop disulfide bond, causes evident structural damage to the heavy chain architecture, destroying its ability to engage b2-microglobulin (b2m) (Feder et al. 1997). A powerful indirect argument for the relevance of this defect is the development of hepatic iron overload in b2m-deficient mice (De Sousa et al. 1994). The purpose of the present investigation was to define the genomic structure and chromosomal localization of the mouse HFE orthologue, as well as to collect evidence on the extent of the evolutionary conservation and the potential lineage specific transcription of this peculiar MHC-I gene. A human HFE cDNA clone encompassing the entire coding sequence was obtained by reverse transcriptionpolymerase chain reaction (RT-PCR) using oligonucleotides derived from the published sequence (Feder et al. 1996) and human intestinal total RNA (Clontech, Palo Alto, Calif.) as template, employing standard procedures. The oligonucleotides used were as follows; RT: 39-GAGGCAGTGGAGTCTCTGTA-59, PCR: 59-ACGTGCGGCCAGAGCTGGG-39 and 39-GCGTCGGACGTCTGAGTGA59. This cDNA clone of 1.1 kb was subsequently used as a probe to screen approximately 7 ́ 105 phage (l FIX II) clones of a 129/SvJ mouse genomic library (Stratagene, La Jolla, Calif.) under conditions of low stringency. In brief, overnight hybridization was carried out at 42 °C in a solution containing 50% formamide, 5 ́ Denhart9s solution, 5 ́ saline sodium phosphate EDTA (SSPE) (20 ́ SSPE is 3.0 M NaCI, 0.2 M NaH2PO4H2O, 0.02 M ethylenediaminetetraacetate (EDTA) pH 7.4, 0.1% sodium dodecyl sulfate (SDS), and 100 mg/ml of denatured Salmon sperm DNA (Sigma, St. Louis, Mo.). Upon completion, the nitrocellulose filters were washed in a solution containing 2 ́ Standard sodium citrate (SSC) (20 ́ SSC is 3.0 M NaCI, 0.3 M sodium citrate, pH 7.0), 0.1% SDS at 45 °C. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession number AF007558

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5′ untranslated regions (5′UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

The Initiation of B Cell Clonal Expansion Occurs Independently of Pre-B Cell Receptor Formation

Current models of B cell development posit that clonal expansion occurs as a direct result of Ig H chain expression. To test this hypothesis, we isolated a population of early B cells in which H chain recombination is initiated and assessed VHDJH rearrangements in both cycling and noncycling cells. We found that actively dividing cells within this population are enriched for H chain rearrangements that are productive when compared with their counterparts in G0/G1, apparently supporting a role for H chain expression in initiating early B cell division; entrance into the cell cycle was accompanied by VH gene-dependent H chain selection. However, we also identified a phenotypically identical population of actively cycling early B cells in the absence of H chain expression in recombination activating gene knockout mice. In addition, actively cycling early B cells could be detected in pre-B cell receptor (pBCR)-negative λ5 knockout mice, but we found no evidence for VH-dependent H chain selection in this population. Given these results, we suggest that the initiation of clonal expansion, at this early stage in B cell development, occurs independently of H chain expression. Although the cycling cell pool is enriched for pBCR-positive cells in mice expressing surrogate L chain, pBCR formation is not required for the initiation of cell division.

Detection of functional VH81X heavy chains in adult mice with an assessment of complementarity-determining region 3 diversity and capacity to form pre-B cell receptor

Recent reports have indicated that up to 50% of all H chain proteins formed cannot associate with the surrogate L chain complex and therefore fail to form a pre-B cell receptor (pBCR), which is required for allelic exclusion and, in most cases, verifies that the H chain can assemble with the L chain to form an Ab molecule. Certain VH genes, such as VH81X, appear to be particularly prone to encoding for nonpairing (dysfunctional) H chains. It has been suggested that sequence variability at complementarity-determining region 3, especially when increased by the enzyme TdT, often precludes the ability of VH81X-using H chains to form pBCR. To determine whether a motif exists that accounts for the ability of H chains to pair with surrogate L chain complex/L chain, we have bred a mouse line in which H chain recombination can only occur on one allele, allowing us to compile a pool of H chains capable of forming Ab molecules in the absence of dysfunctional H chains. Somewhat unexpectedly, we have found VH81X H chains capable of Ab formation and cell surface expression in the presence of TdT. Scrutiny of these H chains has revealed that, although highly prone to encode for dysfunctional H chains, sequence variability is not severely limited among functional VH81X H chains. We also demonstrate that surface Ig expression is highly indicative of the capacity of a H chain to form pBCR.