George C. Russell

Comparative organization and function of the major histocompatibility complex of domesticated cattle

Summary: This review focuses on recent advances in research on the bovine major histocompatibility complex (BoLA), with specific reference to the genetic organization, polymorphism and function of the class II genes. The BoLA region is unlike the MHC of humans and mice in that a large inversion has moved several class II genes, including the TAP/LMP cluster, close to the centromere of bovine chromosome 23. Therefore, dose linkage of MHC genes and other genes associated with the MHC in humans and mice does not appear to be required for normal immunological function. In cattle, polymorphism in the class IIa genes influences both the magnitude and the epitope specificity of antigen‐specific T‐cell responses to foot‐and‐mouth disease virus peptides. Disease association studies have demonstrated that BoLA alleles affect the subclinical progression of bovine leukemia virus (BLV) infection. This association is strongly correlated with the presence of specific amino acid motifs within the DRB3 antigen‐binding domain. In addition to the practical significance of these findings, the association between BoLA and BLV provides a unique model to study host resistance to retrovirus infection in a non‐inbred species. These studies contribute to our understanding of the evolution of the MHC in mammals, to the development of broadly effective vaccines, and to breeding strategies aimed at improving resistance to infectious diseases.

Duplicated DQ Haplotypes Increase the Complexity of Restriction Element Usage in Cattle

The MHC of cattle encodes two distinct isotypes of class II molecules, DR and DQ. Unlike humans, cattle lack the DP locus and about half the common haplotypes express duplicated DQ genes. The number and frequency of DQA and DQB alleles means that most cattle are heterozygous. If inter- and/or intrahaplotype pairing of DQA and DQB molecules occurs, cattle carrying DQ-duplicated haplotypes may express more restriction elements than would be predicted by the number of expressed alleles. We are investigating whether duplicated haplotypes cause differences in immune response, particularly in terms of generating protective immunity. We have analyzed the Ag-presenting function of DQ molecules in two heterozygous animals, one of which carries a duplicated haplotype. We compared the class II isotype specificity of T cell clones recognizing a putative vaccinal peptide from foot-and-mouth disease virus (FMDV15). We show for the first time that bovine T cells can recognize Ag in the context of DQ molecules. We also present evidence that interhaplotype pairings of DQA and DQB molecules form functional restriction elements. Both animals showed distinct biases to usage of particular restriction elements. Mainly DQ-restricted clones were derived from the animal with duplicated DQ genes, whereas the majority of clones from the animal with a single DQ gene pair were DR restricted. Furthermore, haplotype bias was observed with both animals. These experiments show that understanding of class II chain pairing in addition to knowledge of the genotype may be important in vaccine design where effective epitope selection is essential.

T cell activation by Theileria annulata-infected macrophages correlates with cytokine production

A major feature of the pathology induced by Theileria annulata is acute lymphocytic proliferation, and this study investigates the mechanisms underlying the intrinsic ability of T. annulata‐infected monocytes to induce naive autologous T cells to proliferate. Different T. annulata‐infected clones expressed different but constant levels of MHC class II, varying from < 1.0 × 105 to 1.5 × 106 molecules/cell, as measured by saturation binding. However, no correlation was found between the level of MHC class II expression and levels of induced T cell proliferation. Theileria anmulata infected cell lines and clones were assayed for cytokine mRNA expression by reverse transcriptionpolymerase chain reaction (RT‐PCR). The infected cells assayed produced mRNA specific forIL‐lα, IL‐1β, lL‐6, IL‐10 and tumour necrosis factor‐alpha (TNF‐a). but not IL‐2 or lL‐4. One clone (clone G)did not produce mRNA for TNF‐α. The degree of T cell proliferation induced by infected cells was directly correlated with the amount of mRNA produced for the T cell stimulatory cytokines IL‐lα and IL‐6, as assessed by a semiquantitative technique. In contrast, cells infected with the related parasite T. parva produced mRNA for IL‐lα, IL‐2, IL‐4, IL‐10 and interferon‐gamma (IFN‐γ). Since T. parva‐infected cells also induce naive autologous T cell proliferation, it seems likely that the production of lL‐1α by cells infected with either parasite is a major signal for the induction of non‐specific T cell proliferation.

Haplotype characterization of transcribed ovine major histocompatibility complex (MHC) class I genes

The ovine major histocompatibility complex (MHC) remains poorly characterized compared with those of other livestock species. Molecular genetic analysis of the bovine MHC has revealed considerable haplotype and allelic diversity that earlier serological analysis had not detected. To develop cellular and molecular tools to support development of vaccines against intracellular pathogens of sheep, we have undertaken a molecular genetic analysis of four distinct ovine MHC haplotypes carried by two heterozygous Blackface rams. We have identified 12 novel class I transcripts and used a class I sequence-specific genotyping system to assign each of these transcripts to individual haplotypes. Using a combination of phylogenetic analysis, haplotype and transcript expression data, we identified at least four distinct polymorphic class I MHC loci, three of which appear together in a number of combinations in individual haplotypes. The haplotypes were further characterized at the highly polymorphic Ovar-DRB1 locus, allowing selection of the progeny of the two founder rams for the establishment of an MHC-defined resource population.

Functional expression of a cattle MHC class II DR-like antigen on mouse L cells

CattleDRA andDRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfectedDRB gene clearly showed showed that it wasDRB3 alleleDRB3*0101, which corresponds to the 1D-IEF-determined alleleDRBF3. 1D-IEF analysis of the transfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigenspecific CD4+ T cells from both the original animal used to obtain the genes, and also from an unrelated DRBF3+ heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design.

Amplification and sequencing of expressed DRB second exons from Bos indicus

Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins found on the surface of antigen presenting cells. They bind processed peptides in an endosomal compartment before being transported to the cell surface where the class II peptide complex is recognized by the T-cell receptor of CD4 ÷ T cells, initiating the immune response (for reviews see Rothbard and Gefter 1991; Unanue 1992; Neejfes and Momburg 1993). Cattle MHC class II sequences have been obtained from genomic and cDNA clones of the DRA, three different DRB, DQA, DQB, DYA, and DIB genes (van tier Poel et al. 1990; Groenen et al. 1990; MuggliCockett and Stone 1988, 1989; Burke et al. 1991; Xu et al. 1991, 1993; Stone and Muggli-Cockett 1990). The DRB and DQB genes from a range of haplotypes have been characterized by cloning and sequencing of the polymorphic second exon from genomic DNA (Sigurdardottir et al. 1991, 1992; Ammer et al. 1992). Here we describe the cloning and sequencing of polymerase chain reaction (PCR)-amplified DRB exon 2 fragments from peripheral blood monocyte (PBM) RNA. A group of nine MHC-homozygous Kenya Boran cattle were used in this work to facilitate the interpretation of the results and to provide information on the DRB alleles expressed by Bos indicus cattle. The animals, generated by sire-daughter matings, were typed as homozygous for class I (BoLA-A; Spooner et al. 1978) and expressed a range of class II types (not shown). The RNA was reverse transcribed to produce firststrand cDNA, which served as the template for PCR

Sequence duplication at the 3′ end of BoLA-DQB genes suggests multiple allelic lineages

Abstract. Full-length cDNAs encoding the DQB genes expressed by three BoLA classxa0II haplotypes (DH8A, DH15B, and DH24A) were amplified by reverse-transcription polymerase chain reaction, cloned, and sequenced. The sequence data revealed that the DH8A haplotype expressed two DQB genes (DQB*1005 and DQB*1201) while the DH15B and DH24A haplotypes expressed the same single gene (DQB*0101). Comparison of the three alleles showed that the 3′ untranslated (3′UT) sequence of the DQB*1201 allele contained a duplication of about 200xa0bp. This repeat was also found in other DQB alleles from cattle and sheep, but only in haplotypes with duplicated DQB genes. This 200-bp repeat and other features of the 3′UT may provide useful markers of DQB evolution, allowing us to distinguish and selectively amplify the different DQB loci.

CHARACTERIZATION OF CATTLE CDNA SEQUENCES FROM TWO DQA LOCI

The class II region of the major histocompatibility complex (MHC) of cattle encodes antigen-presenting molecules of two isotypes, DR and DQ. These highly polymorphic cell-surface glycoproteins bind peptide fragments from mainly exogenous antigens and present them to CD4 T cells to initiate an immune response. Each DR or DQ molecule can bind a range of antigenic peptides, defined by the shape and charge properties of the antigen binding cleft (Brown et al. 1993). Thus, the expression of a wide range of different class II molecules could increase the range of antigens presented to the immune system. Each class II haplotype expresses a single DR molecule, encoded by the DRA and DRB3 genes, but one or more DQ products because of the duplication which occurs in about half of the common class II haplotypes (Andersson and Rask 1988). TheDQ locus is duplicated in primates, but the DQA2andDQB2genes are transcriptionally silent (Kappes and Strominger 1988). In contrast, the DQB genes on duplicated cattle haplotypes are expressed (Bissumbhar et al. 1994; Xu et al. 1994; Marello et al. 1995). In order to correlate class II gene expression and polymorphism with immune function, we are cloning and transfecting the class II genes expressed by a pair of immunologically characterized Holstein-Friesian cattle. The animals (numbers 10795 and 10814) have well-characterized responses to immunization with a model peptide antigen, FMDV15, derived from foot-and-mouth-disease virus (Glass et al. 1991, 1992; Glass and Millar 1994), and had been extensively typed as part of the fifth BoLA workshop (Davies et al. 1994). TheMHC types carried by the animals were: 10795 BoLA-A11,DRB3*0102, DQA1A, DQB1 (class II haplotypeDH24A); BoLA-A36, DRB3*1201, DQA12, DQB12 (class II haplotypeDH8A). 10814 BoLA-A11,DRB3*0102, DQA1A, DQB1 (class II haplotypeDH24A); BoLA-A32, DRB3.2*15, DQA1E, DQB1 (class II haplotypeDH15B). Presentation of the FMDV15 antigen by mouse L cells transfected with theDRA-DRB3gene pair from the shared DH24A haplotype has been described previously (Fraser et al. 1996). Here we report the DQA sequences expressed by these animals, determined from polymerase chain reaction (PCR)-amplified cDNA clones. The lack of extensive DNA sequence data for the cattle DQAgenes led us to use the available sequences from cattle and sheep to design primers which could amplify fulllength DQA genes from cattle cDNA preparations. To improve the chances of amplifying all possible DQA sequences, one forward and two reverse primers were designed. All three primers contained degenerate bases to take into account positions which were polymorphic in the DQA sequences used. The forward primer DQAFWD (59-CCA CCT TGA GAA SAG GAT GRT CCT G-39) annealed at the 5 9 end of theDQA gene and included the start codon (underlined). The reverse primers DQAREV1 (59-ACT TKG SCA GAA AMT AGY TCT AGG-39) and DQAREV2 (59-TGA GAT GAT AYA GCA AYC TTA AGT CC-39) annealed in the 3 9 untranslated region, approximately 70 and 140 base pairs (bp), respectively, beyond the termination codon. Full-length DQA sequences were amplified from firststrand cDNA from both animals using a high-fidelity PCR system (Expand High Fidelity, Boehringer Mannheim, Lewes, UK) to reduce the frequency of PCR artefacts. Amplifications using the DQAFWD-DQAREV1 and DQAFWD-DQAREV2 primer pairs produced clean products of the expected sizes (880 and 950 bp, respectively) from animal 10795, but only the larger product was obtained from animal 10814 (Fig. 1). Despite several experiments using different RNA and cDNA preparations, The nucleotide sequence data reported in this paper have been submitted to the EMBL, GenBank, and DDBJ nucleotide sequence databases and have been assigned the accession numbers Y07819, Y07820, and Y07898

Transfection, expression, and DNA sequence of a gene encoding a BoLA-A11 antigen.

Cattle major histocompatibility complex [(MHC) (BoLA)] class I molecules are heterodimeric glycoproteins which present endogenous antigenic peptides to CD8{sup +} T lymphocytes, initiating a cellular immune response. The MHC-encoded heavy chains are highly polymorphic and, in cattle, have been characterized mainly by using allo-antisera raised by reciprocal calf/dam immunizations. This has enabled the identification of about 50 serlogical specificities, most of which behave as alleles of a single highly polymorphic class I locus. However, evidence from biochemical and molecular biological studies suggest that more than one BoLA class I locus is expressed. These loci are apparently in linkage disequilibrium, making them difficult to distinguish by conventional methods. In order to investigate the expression and function of individual class I locus products, we are correlating BoLA class I gene sequences with the expressed products by the transfection and characterization of genomic class I clones. Shotgun transfection and expression of BoLA class I molecules has been described previously, but the genes involved were not isolated. In this paper we report the isolation, DNA sequencing, transfection, and expression of a genomic clone encoding a BoLA-A11 determinant from an animal expressing A10 and A11 serological specificities. 23 refs., 3 figs.

Molecular cloning and sequencing of a cattle DRA cDNA.

To date there is full-length sequence information for only two cattle MHC class 11 genes, the DQB gene on the genomac clone Y1 (Groenen et al. 1990) and the DRB3 cDNA clone (Burke et al. 1991). It is known that most sequence polymorphisms are located m the peptide binding domain, especially in exon 2 of the chain. However, proper correlanon of immune fanctaon with sequence polymorphimn necessarily involves studying the complete expressed molecule. In thas commumcation we report the cloning and sequencing of the entire coding region of a cattle DRA gene. Peripheral blood monneytas were purified from whole blood of Fries~an (Bos taurus) cattle with class 1I types DRBF 6,3. Reverse mmscriptmn-polymerase chain reaction (RT-PCR) was carried out on total RNA, using oligo-dT and primers derived from the pubhshed full-length sheep DRA cDNA sequence (Fabb et al 1993), m a reacuon volume of 50 gl comprising 10 mM Trts-Hcl pH 8.3, 50 mM KC1, 100 pg/ml gelatin, 2 mM MgCh, and 1 umt Taq polymerase (Boehrmger Mannhetm~ Lewes, UK). Amplificataon was primed wath 25pmolas of forward (5-CACCAAAGAAGAAAATGGCC-Y) and reverse (5-TGAGACCCACTI~AAGTITACTGTATTC-Y) primers and conststed of 20 cycles of 94 ° C for ] man, 55 ° C for 2 min, and 72 ° C for 3 mm, followed by a 7 mm extension at 72 ° C. ~ products were cloned into the TA vector pCR 11 (Invitrogen, San Dtego, CA) and sequenced in both directions, using Sequenase Vermon 2 0 enzyme (US Biochemacah, Cleveland, OH). We present here (Fig. 1) the sequence of a cailie DRA cDNA. The 1195 base pairs (bp) sequence contains a 762 bp reDon coding for a 253 residue polypeptade. Companr, on with the sequences of exons 2, 3, and 4 from the truncated genomic clone W3 (van der Pcel et al. 1990) shows that the two genes are idenUcal over the regton aligned, thus confirming the idenUty of the DRA cDNA clone and transcription of the W3-encoded gone. The degree of identaty between the translated cattle DRo~ chain sequence and the human DRa sequence ts 80%, and conserved residues of functional sigmficance are highhghted in Figure 1. Interestingly, these residues include E88 and K l l l wlmch may be important m the observed dimerisanon of I-ILA DRI ct/~ he~erodimers (Brown et al. 1993). However, It IS unclear whether this dimerisafion occurs m wvo or if It is ftmclaonally significant.