Albert Bensaïd

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5′ and 3′ termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3′ untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3UT regions of clones 2.1 and 5.1, respectively, Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of and homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the] w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.

Characterization of a bovine thymic differentiation antigen analogous to CD1 in the human.

Three monoclonal antibodies (MoAb), TH97A, CC13, and CC14, define a thymic differentiation antigen in cattle. The antigen is expressed on 50–60% of bovine thymocytes, located mainly in the cortical areas, but is not expressed on peripheral blood mononuclear cells (PBMC). In cryostat sections of lymph node, the antibodies read with large dendritic‐like cells in the paracortical regions. They also react with a proportion of the large ‘frilly’ cells in afferent lymph and with dendritic‐like cells in the dermis. The antibodies apparently do not react with cells in the epidermis. Biochemical analysis of the antigen recognized by MoAb TH97A reveals two bands of 44 kDa and 12 kDa under reducing conditions. These polypeptides are distinct from bovine class I major histocompatibility complex molecules reactive with the MoAb w6/32. The tissue distribution of positive cells together with results of biochemical analyses indicate that the antigen recognized by these MoAb is the bovine analogue of the human CD1.

Analysis of the reactivity of anti-bovine CD8 monoclonal antibodies with cloned T cell lines and mouse L-cells transfected with bovine CD8

Mouse L-cells transfected with bovine CD8 and two Theileria parva-infected cloned T cell lines expressing bovine CD8 were used to screen the panel of ten monoclonal antibodies (mAbs) submitted to the workshop. Eight of the ten mAbs reacted with the transfectant and both the cloned T cell lines. However, two mAbs CC58 and BAT82A did not recognise the transfectant and only reacted with one of the T cell lines. Further biochemical studies indicated that the eight mAbs react with both homo- and heterodimeric forms of bovine CD8 whilst the two mAbs CC58 and BAT82A react with only heterodimeric forms. These data suggest that bovine DC8 is encoded by two genes as is the case in mouse and man.

Recombinant bovine interferon gamma inhibits the growth of Cowdria ruminantium but fails to induce major histocompatibility complex class II following infection of endothelial cells

Recombinant bovine IFN gamma is a potent inhibitor of Cowdria ruminantium growth in vitro irrespective of the rickettsial stock, or the origin of the endothelial cells. These results suggest an important role for IFN gamma in protective immune responses against C. ruminantium infections. Here we also show that IFN gamma can induce the expression of MHC class II molecules on the surface of endothelial cells. However, treatment of endothelial cells with IFN gamma following infection with Cowdria fails to induce MHC class II expression. The implications of this pathogen-specific effect on class II expression by endothelial cells with regard to its recognition by the host immune system are discussed.

An immunochemical analysis of class I (BoLA) molecules on the surface of bovine cells

As in other species, bovine class I MHC molecules have been found to be cell surface heterodimers composed of a glycosylated heavy chain of approximate relative mass 44000, associated with B2m of relative mass 12000 (Hoang-Xuan et al. 1982). To date, serological definition of the BoLA system, using alloantisera, has proceeded through two international workshops (Spooner et al. 1979, Anon 1982)which have identified 17 apparent class I alleles at a single locus, BoLA-A. To gain further insights into the nature and heterogeneity of expressed bovine MHC products, we have attempted to obtain some measure of the diversity of cell surface-expressed BoLA class I molecules by establishing a structural relationship between polymorphic and monomorphic epitopes detectable on bovine class I MHC products. This was done by immunochemical analysis of detergent lysates of radiolabeled peripheral blood lymphocytes (PBL) immunoprecipitated with bovine alloantisera, mouse mAbs, and a rabbit antiserum specific for class I HLA heavy chains not complexed to B2m. BoLA typing was performed using a panel of alloantisera and monoclonal antibodies developed in our laboratory in a standard microlymphocytotoxicity assay (Teale et al. 1983). All immunochemical studies were performed with PBL of a four-year-old Boran (Bos indicus) steer, number B641. This animal is heterozygous at the BoLA-A locus with the phenotype Awl0/KN18. The KN18 specificity is defined by two alloantisera (KMA010 and KMA018) and mAb P3. The lead serum, KMA018, has a correlation coefficient with the specificity of 0.885. A population study involving more than 1600 cattle suggested that KN18 is encoded at the BoLA-A locus (Kemp 1985). Furthermore, in an extended survey of more than 2000 cattle,

Cell-mediated immune responses of cattle to Theileria parva.

Theileria parva is a protozoan parasite that infects lymphocytes of cattle and African buffalo. As is the case with certain viruses, the parasite causes antigenic changes on the cell surface against which the host mounts cytotoxic T-cell. Precise definition of the cells participating in these response and their specificity has been facilitated by the recent identification of markers for bovine T-cell subpopulations and functional analyses of bovine lymphocytes at the clonal level. In this paper Ivan Morrison and his colleagues discuss current information on the parasite specificity and MHC restriction of anti-Theileria cytotoxic T cell, in relation to their role in protective immunity.

Transcriptional analysis of the major antigenic protein 1 multigene family of Cowdria ruminantium

The major antigenic protein 1 (MAP1) of the tick-borne rickettsial pathogen Cowdria ruminantium is encoded by a multigene family containing conserved and variable genes. The part of a locus containing the map1 multigene family that was characterized contained three homologous, but non-identical map1 genes, designated map1-2, map1-1, and map1. Reverse transcriptase-polymerase chain reaction was used to study the transcriptional activity of these genes in isolates of C. ruminantium grown in bovine endothelial cells, in two different tick cell lines, and in Amblyomma variegatum ticks. The map1 gene was always transcribed, whereas transcription of map1-2 was not detected under any of the tested conditions. The map1-1 gene transcript was detected in A. variegatum ticks, but was not found in virulent C. ruminantium Senegal grown in bovine endothelial cells at 30 or 37 degrees C. Interestingly, transcripts of map1-1 were also found in different passages of the in vitro attenuated Senegal isolate grown in bovine endothelial cells, as well as in the Gardel isolate grown in two tick cell lines. When transcribed, map1-1 was present on a polycistronic messenger together with map1.

Cell surface phenotype of two cloned populations of bovine lymphocytes displaying non-specific cytotoxic activity

Monoclonal antibodies specific for T cell differentiation antigens were tested on four cloned populations of lymphocytes derived from the peripheral blood mononuclear cells of an animal immunised with Theileria parva. The clones were defined functionally in terms of cytotoxic activity, MHC restriction and expression of messenger RNA for CD3 and T cell receptor (TCR). Two clones contained RNA transcripts for CD3, TCR-alpha and beta and were positive for CD2, CD5 and CD6; one of these was a typical CD4+ class II MHC-restricted non-cytotoxic clone while the other was a CD8+ class I MHC-restricted cytotoxic clone. By contrast, the remaining two clones had the characteristics of non-specific killer cells in that they exhibited moderate levels of non-MHC-restricted killing; they contained TCR-delta mRNA and a 1.2 kb truncated form of TCR-beta message, but they did not contain CD3 or TCR-alpha mRNA. One of these non-specific killer clones only expressed CD2 whereas the other clone only expressed CD8, but without the CD8 determinant recognised by monoclonal antibodies CC58 and BAT52. All four clones were negative for the WC1 antigen which is expressed on gamma/delta T lymphocytes.

A monoclonal antibody which reacts specifically with a population of bovine lymphocytes lacking B cell and T cell markers

In the last few years a number of monoclonal antibodies (MAb) specific for bovine T lymphocytes has been produced. These include antibodies which react with the sheep erythrocyte receptor (designated BoT2) (Davis et al., in press) and thus can be used as pan-T cell markers, and antibodies to the two major subset markers BoT4 and B0T8 (Baldwin et al., 1986; Ellis et al., 1986). When these antibodies are used, in conjunction with reagents that identify B lymphocytes and monocytes, to phenotype bovine leukocytes, there is a residual population of lymphocytes comprising 10–20% of peripheral blood mononuclear cells (PBM) that is unreactive with these reagents. Recently, we have produced a MAb that reacts with a proportion of these “null” lymphocytes. Herein, we will summarise our findings with this antibody.

Somatic cell mapping of T-cell receptor CD3 complex and CD8 genes in cattle

Bovine genes encoding T-cell receptor, CD3, and CD8 molecules have been mapped to syntetic groups using bovine × rodent hybrid somatic cells. T-cell receptor α and δ chains were assigned to bovine syntenic group U5, and the β and γ genes were syntenic with each other and with markers on U13. CD3E and CD3D genes were syntenic with each other and located to bovine syntenic group U19. CD8 was most concordant with markers of syntenic group U16, although the concordancy was only 85% and the assignment must be regarded as tentative. The comparative gene maps of human chromosome 7, bovine syntenic group U13, and mouse chromosomes 6 and 13 suggest extensive evolutionary conservation.