D. F. Antczak

ISAG/IUIS-VIC Comparative MHC Nomenclature Committee report, 2005

Nomenclature for Major Histocompatibility Complex (MHC) genes and alleles in species other than humans and mice has historically been overseen either informally by groups generating sequences, or by formal nomenclature committees set up by the International Society for Animal Genetics (ISAG). The suggestion for a Comparative MHC Nomenclature Committee was made at the ISAG meeting held in Göttingen, Germany (2002), and the committee met for the first time at the Institute for Animal Health, Compton, UK in January 2003. To publicize its activity and extend its scope, the committee organized a workshop at the International Veterinary Immunology Symposium (IVIS) in Quebec (2004) where it was decided to affiliate with the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS). The goals of the committee are to establish a common framework and guidelines for MHC nomenclature in any species; to demonstrate this in the form of a database that will ensure that in the future, researchers can easily access a source of validated MHC sequences for any species; to facilitate discussion on this area between existing groups and nomenclature committees. A further meeting of the committee was held in September 2005 in Glasgow, UK. This was attended by most of the existing committee members with some additional invited participants (Table 1). The aims of this meeting were to facilitate the inclusion of new species onto the database, to discuss extension, improvement and funding of the database, and to address a number of nomenclature issues raised at the previous workshop.

Report of the First International Workshop on Equine Leucocyte Antigens, Cambridge, UK, July 1991

The First International Workshop on Equine Leucocyte Antigens was organized and convened for the purposes of identifying immunologically relevant cell surface molecules of equine leucocytes and establishing a system of nomenclature for those molecules. Participating members of the workshop represented the majority of laboratories world-wide engaged in the tasks of production and characterization of equine leucocyte and lymphocyte markers using monoclonal antibodies. The workshop confirmed the identification of several equine CD molecules described previously by individual laboratories, and in addition recognized antibodies identifying new CD molecules. The workshop also succeeded in fostering co-operation between laboratories around the world which study equine immunobiology. Equine CD molecules identified by the current battery of monoclonal antibodies include EqCD2, EqCD4, EqCD5, EqCD8, EqCD11a/18, EqCD13 and EqCD44. Other antibodies are markers for MHC class I and class II molecules, for B cells, granulocytes, macrophages, T cell subsets distinct from those defined by CD4 and CD8, and other sub-populations of horse leucocytes that do not have obvious counterparts in humans, rodents, or other species. Despite the progress made in the first workshop, there are still substantial gaps in the armory of reagents available to study equine leucocyte biology, and further definition of the structure, function, and genetics of the antigens identified by the workshop clusters (WC1, WC2 etc.) and other molecules of immunological importance will be a goal of future workshops. The study of equine immunobiology and resistance to disease also urgently requires the development of tools to study equine immunoglobulins and cytokines, and these needs will provide ample scope for future studies.

Patterning and post-patterning modes of evolutionary digit loss in mammals

A reduction in the number of digits has evolved many times in tetrapods, particularly in cursorial mammals that travel over deserts and plains, yet the underlying developmental mechanisms have remained elusive. Here we show that digit loss can occur both during early limb patterning and at later post-patterning stages of chondrogenesis. In the ‘odd-toed’ jerboa (Dipus sagitta) and horse and the ‘even-toed’ camel, extensive cell death sculpts the tissue around the remaining toes. In contrast, digit loss in the pig is orchestrated by earlier limb patterning mechanisms including downregulation of Ptch1 expression but no increase in cell death. Together these data demonstrate remarkable plasticity in the mechanisms of vertebrate limb evolution and shed light on the complexity of morphological convergence, particularly within the artiodactyl lineage.

Identification of equine major histocompatibility complex haplotypes using polymorphic microsatellites

A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serological and microsatellite typing demonstrated a linkage to known MHC class I protein polymorphisms and validated this assay as a useful supplement to ELA serotyping, and in some applications, a feasible alternative method for MHC genotyping in horse families and in population studies.

Horse cDNA clones encoding two MHC class I genes

Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

Detection of equine and bovine T- and B-lymphocytes in formalin-fixed paraffin-embedded tissues

Formalin-fixed paraffin-embedded sections of equine and bovine lymph nodes, spleen, thymus, and Peyers patches were incubated with monoclonal antibodies to B-lymphocyte markers BLA.36, B29, and mb-1 and T-lymphocyte markers CD3 and CD5. The monoclonal antibody BLA.36 reacted with 80-90% of lymphocytes in the germinal centers and mantle zones of follicles in lymph nodes, spleen, and Peyers patches. In addition, 90% of lymphocytes in the marginal zone of the spleen, and variable numbers of lymphocytes within lymph node medullary cords were immunopositive for BLA.36. Antibodies to B29 and mb-1 produced similar staining patterns as BLA.36 with fewer positive cells in the germinal centers and medullary cords. BLA.36, B29, and mb-1 reacted with 30-50% of lymphocytes in the medulla of the thymus and with 5-10% of lymphocytes in the cortex. CD3 and CD5 reacted with 90% of lymphocytes in the paracortex and parafollicular zones of lymph nodes, spleen, and Peyers patches; 40-50% of lymphocytes in the medullary cords of lymph nodes, and scattered positive cells within follicles. Anti-CD3 antibody reacted with 95% of lymphocytes in the splenic red pulp, but antibodies directed against CD5 reacted only faintly with approximately 5-10% of lymphocytes in the red pulp. CD3 and CD5 reacted with 50-60% of cells in the medulla of the thymus and with 40-80% of lymphocytes in the thymic cortex. The biochemical characterization of the antibodies by Western blotting against lysates of equine and bovine peripheral blood mononuclear cells confirmed that antibodies to BLA.36, mb-1, B29, CD3, and CD5 detected molecules of the same approximate molecular mass as found on lymphoid cells of human beings and rats.

IMMUNOLOGICAL STUDIES ON FETO-MATERNAL RELATIONSHIPS IN EQUINE PREGNANCY

This chapter discusses immunological studies on feto–maternal relationships in equine pregnancy. The mechanisms that protect the equine fetus or an intrauterine allograft are of particular interest because of the development of the endometrial cups. These structures are unique to the Equidae and comprise a series of small, ulcer-like, and endometrial outgrowths that form a circle around the conceptus in the gravid uterine horn between 40 and 130 days of gestation. The endometrial cups are the source of the equine chorionic gonadotrophin, formerly known as pregnant mare serum gonadotrophin, which is present in varying concentration in the blood of equids during the first half of pregnancy. The high incidence of cytotoxic antibody production in primigravid mares and its temporal relationship to the accumulation of maternal leucocytes around the endometrial cups suggest that these two phenomena represent the humoral and cell-mediated arms of the same maternal response to paternally-inherited major histocompatibility complex (MHC) antigens. The chapter examines this hypothesis and its necessary corollary that endometrial cup cells do, in fact, express paternal MHC antigens. The chapter also discusses the influence of MHC compatibility on endometrial cup development.

An equine B cell surface antigen defined by a monoclonal antibody

A surface antigen of equine B lymphocytes was identified using the Equine Leucocyte Antigen Workshop antibody WS 65. This marker was expressed on almost all equine B cells, but not on T cells, granulocytes or thymocytes. WS 65 strongly stained cells in the follicular areas of lymph nodes and cells in the splenic nodules when tested on frozen tissue sections by immunohistochemistry. Equine leukemic T cells were not labeled by WS 65, and neither were the cells from a horse with B cell leukemia, although these latter cells carried surface immunoglobulin. Immunoprecipitation of lymphocyte membrane molecules with the antibody produced a band at 85-90 kDa under reducing conditions. The equine B cell antigen defined by WS 65 appears to be different from surface immunoglobulin by its molecular characteristics and its lack of expression on malignant B cells.

Donor-derived equine mesenchymal stem cells suppress proliferation of mismatched lymphocytes

REASONS FOR PERFORMING STUDY Recently, it has been shown that mesenchymal stem cells (MSCs) do not express the major histocompatibility complex (MHC) II antigen and are able to inhibit proliferation of MHC-mismatched stimulated lymphocytes, enabling their use as in vivo allogeneic transplants. However, prior to clinical application of allo-MSCs, in vitro tests are required to confirm the safety of treatment protocols. OBJECTIVES To evaluate the immunosuppressive capabilities of equine bone-marrow-derived MSCs (BM-MSCs) on MHC-mismatched lymphocytes. STUDY DESIGN In vitro experiment. METHODS Phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) from 3 Thoroughbreds (recipients) were co-cultured with mismatched BM-MSCs from 3 Connemara ponies (donors). Proliferation of lymphocytes was monitored by carboxyfluorescein succinimidyl ester labelling and analysed by flow cytometry. In total, 6 horses were haplotyped using microsatellites to confirm mismatching. Optimisation of the conditions to stimulate Thoroughbred lymphocytes and titration of equine anti-CD4 and anti-CD8 antibodies were performed. Connemara pony and Thoroughbred BM-MSCs were isolated, expanded and characterised by tri-lineage differentiation. Finally, BM-MSCs from both breeds were set up in co-culture at different ratios with stimulated Thoroughbred lymphocytes. Proliferation of CD4(+) and CD8(+) cells was determined by flow cytometry. RESULTS A high proportion of CD4/CD8 double-positive lymphocytes were found in freshly isolated PBMCs, although this percentage decreased after 4 days of culture. Mismatched BM-MSCs inhibited proliferation of stimulated lymphocytes in a dose-dependent manner, with the greatest suppression occurring at a 1:10 ratio of BM-MSCs to PBMCs. Proliferation of CD4(+) and CD8(+) subpopulations decreased in 1:10 co-culture, with statistical significance in the case of CD8(+) cells, while that of the CD4/CD8 double-positive population was similar to the phytohaemagglutinin control. CONCLUSIONS The results demonstrate dose-dependent immunosuppression of stimulated lymphocytes by mismatched equine BM-MSCs, supporting their future application in allo-MSC clinical treatments.

Down-regulation followed by re-expression of equine CD4 molecules in response to phorbol myristate acetate.

The regulatory effects of phorbol myristate acetate (PMA) on the expression of the CD4 molecule on horse T cells were investigated. On both peripheral blood lymphocytes and thymocytes, PMA resulted in a rapid and transient down-regulation of equine CD4 expression, but had no such effect on the surface expression of equine CD5, CD8 or major histocompatibility complex (MHC) class I and class II molecules. Over 75% of the surface CD4 molecules per cell were lost after a 4 h exposure to PMA at 37 degrees C. The regulation of equine CD4 expression induced by PMA was temperature dependent and reversible. The PMA-mediated loss of CD4 expression was inhibited at 4 degrees C. After 24 h of exposure to PMA, CD4 molecules were re-expressed on the cell surface, even in the continued presence of PMA. These findings demonstrate that equine CD4+ T cells undergo alterations in CD4 expression in response to PMA, and suggest that the equine homolog of the CD4 molecule is regulated by PMA in a similar manner to the human CD4 molecule.