Wayne R. Hein

Prominence of γδ T cells in the ruminant immune system

Abstract The lymphoid systems of sheep and cattle contain a large number of γδ T cells, in striking contrast to the lymphoid systems of humans and mice. In neonatal animals particularly, these cells comprise the predominant fraction of T cells in the blood. Here Wayne Hein and Charles Mackay discuss what is currently known about the ontogeny, phenotype, tissue distribution and function of γδ T cells in ruminants. There are a number of interesting molecular features that characterize ruminant γδ T cells, but these do not entirely explain the high frequency of use of the γδ T-cell receptor in these animals. Studies on sheep, cattle or other animals that preferentially use γδ T cells should provide insights into the biological significance of the existence of two distinct forms of the T-cell receptor.

Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process

Somatic hypermutation of light chain V genes during development of B cells in sheep ileal Peyers patches was studied in three experimental conditions: in sterile fragments of the ileum surgically isolated from the gut during fetal life, in germ-free sheep, and in animals thymectomized during early fetal life. The somatic mutation pattern was found identical to control tissues in all three experiments. The same age-dependent amount of mutations, a higher than theoretical R/S ratio in complementarity-determining regions (CDRs), and a similar clustering of mutations in CDRs were observed. The mechanism, as estimated from the silent mutation pattern, appears to target mutations to CDRs; moreover, the major V lambda genes have a specific codon usage with a high purine content at the first two bases of the codons and a low content at the third position, which, together with a specific targeting of mutations to purines, favors replacement mutations in CDRs.

Effect of Molecular Weight on the Lymphatic Absorption of Water-Soluble Compounds Following Subcutaneous Administration

The lymphatic absorption of four water-soluble compounds with different molecular weights (MW) was determined by measuring their cumulative recovery in lymph draining from the site of s.c. administration in sheep. The cumulative recoveries (% of dose, mean ± SD; N = 3) were 4.0 ± 1.5 (5-fluoro-2′-deoxyuridine, MW 246.2), 21.0 ± 7.1 (inulin, MW 5200), 38.6 ± 6.7 (cytochrome c, MW 12,300), and 59.5 ± 7.7 [human recombinant interferon (rIFN) alpha-2a, MW 19,000], respectively. Our data show that in the investigated MW range, there is a linear relationship between the molecular weight and the proportion of the dose absorbed lymphatically. An increase in molecular weight results in an increased lymphatic absorption. Molecules with MW > 16,000 are absorbed mainly by the lymphatics which drain the application site. The knowledge gained in this investigation may help to improve the mode of administration and therapeutic efficacy of endogenous proteins whose targets are lymphoid cells (e.g., interferons, interleukins). Practical implications for the clinical use of such proteins are discussed.

A road less travelled: large animal models in immunological research

The main advances in immunology have been forged or underpinned by animal experiments. However, animal research now focuses excessively on one laboratory species, and there is too much redundant repetition and too few transfers from basic discovery to successful clinical application. These features can be improved markedly by placing more emphasis on biological relevance when evaluating animal models and by taking greater advantage of the unique experimental opportunities that are offered by large animals.

Increased Expression of Interleukin-5 (IL-5), IL-13, and Tumor Necrosis Factor Alpha Genes in Intestinal Lymph Cells of Sheep Selected for Enhanced Resistance to Nematodes during Infection with Trichostrongylus colubriformis

ABSTRACT Cytokine gene expression in cells migrating in afferent and efferent intestinal lymph was monitored for extended time periods in individual sheep experimentally infected with the nematode Trichostrongylus colubriformis. Animals from stable selection lines with increased levels of either genetic resistance (R) or susceptibility (S) to nematode infection were used. Genes for interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha (TNF-α), but not for IL-4, IL-10, or gamma interferon (IFN-γ), were consistently expressed at higher levels in both afferent and efferent lymph cells of R sheep than in S sheep. However, only minor differences were observed in the surface phenotypes and antigenic and mitogenic responsiveness of cells in intestinal lymph between animals from the two selection lines. The IL-4 and IL-10 genes were expressed at higher levels in afferent lymph cells than in efferent lymph cells throughout the course of the nematode infection in animals of both genotypes, while the proinflammatory TNF-α gene was relatively highly expressed in both lymph types. These relationships notwithstanding, expression of the IL-10 and TNF-α genes declined significantly in afferent lymph cells but not in efferent lymph cells during infection. Collectively, the results showed that R-line sheep developed a strong polarization toward a Th2-type cytokine profile in immune cells migrating in lymph from sites where the immune response to nematodes was initiated, although the IFN-γ gene was also expressed at moderate levels. Genes or alleles that predispose an animal to develop this type of response appear to have segregated with the R selection line and may contribute to the increased resistance of these animals.

TCR gamma delta+ cells are prominent in normal bovine skin and express a diverse repertoire of antigen receptors.

More than 80% of T cells in bovine skin localized in the superficial 0·5 mm of the dermis. Only 3% occurred within the epidermis or made contact with the stratum basale while the remainder occupied deeper dermal sites. The γδ T‐cell receptor (TCR) was expressed by 44% of T cells in skin and 39% and 35% expressed, respectively, the CD4 and CD8 markers. Some cells co‐expressed CD8 and the γδ TCR. A highly diverse repertoire of γδ TCR was expressed in skin due mainly to the usage of multiple Vδ segments and to extensive sequence variation at the junctions of both TCRγ and TCRδ chains. However, a single receptor isotype was used. Transcripts encoding several new components of the bovine γδ TCR were identified, including three new Vγ segments, the Cγ5 region and 13 new functional Vδ segments. Taken together with earlier findings, these results emphasize that ruminant γδ T cells express exceptionally diverse antigen receptors and suggest they may have a more elaborate recognitive capacity than do their counterparts in other species.

The intestinal habitat for organized lymphoid tissues in ruminants; comparative aspects of structure, function and development.

Unlike the Peyers patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyers patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyers patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyers patch is of a special type that sheds carbonic anhydrase-rich, 50-nanometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyers patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyers patches with respect to their lymphocyte population and the ileal Peyers patch with respect to their follicle-associated epithelium.

A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle.

In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.

Organization of Mucosal Lymphoid Tissue

Mucous membranes form interfaces between internal microenvironments that are subject to homeostatic regulation and external environments that vary widely and are frequently contaminated with microbes and other potentially injurious agents. To offset their inherent vulnerability, mucosal surfaces have therefore been endowed with specialized defense mechanisms, many of which have an immune component.

Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis

Allergens were identified from the gastrointestinal nematode of sheep, Trichostrongylus colubriformis, by probing Western blots of infective larvae (third stage) somatic antigen with IgE purified from the serum of sheep grazed on worm contaminated pasture. A 31 kDa allergen was frequently recognised by sera from immune sheep, particularly those deriving from a line that has been genetically selected over 23 years for parasite resistance. Using a proteomic approach, the 31 kDa allergen was identified as an aspartyl protease inhibitor homologue. The entire coding sequence of T. colubriformis aspartyl protease inhibitor (Tco-api-1) was obtained and the mature protein expressed in Escherichia coli. Anti-Tco-API-1 antibodies revealed that a commonly observed 21 kDa T. colubriformis allergen species is a truncated form of Tco-API-1. Specific IgE responses to T. colubriformis aspartyl protease inhibitor were significantly correlated with the degree of resistance to nematode infection as measured by faecal egg count in sheep. Surprisingly, IgE responses to Tco-API-1 were not correlated with breech soiling (dag score), which is thought to be caused, in part, by allergic hypersensitivity to worms. Therefore, a specific IgE response to this allergen may be a suitable marker for identifying lambs at an early age that will develop strong immunity to gastrointestinal nematodes.