Immunohistochemical study of the inflammatory infiltrate associated with feline cutaneous squamous cell carcinomas and precancerous lesions (actinic keratosis).

The distribution of T lymphocytes (CD3+), B lymphocytes (CD79+), immunoglobulin-containing plasma cells (IgG, IgM and IgA), macrophages (Mac387+) and MHC Class II antigen was analysed in the inflammatory infiltrate associated with cutaneous squamous cell carcinomas (SCC) from 23 cats. Peri-tumoural skin (12 cases) and precancerous lesions of actinic keratosis (nine cases) were also evaluated for the expression of MHC Class II. The results revealed that an abundant inflammatory infiltrate was associated with the majority of SCC. This infiltrate was composed mainly of CD3+ T lymphocytes, B cells (CD79+) and IgG-bearing plasma cells, and the intensity of infiltration increased with the degree of invasiveness of the tumour. The number of CD3+ T cells and CD79+ cells was significantly increased in well-differentiated SCC compared with moderately differentiated tumours, whereas the number of IgM+, IgA+ plasma cells and Mac387+ macrophages was low or moderate and did not change significantly with histologic grade or invasiveness. MHC Class II antigen was expressed by infiltrating lymphocytes and macrophages, and by fibroblasts. A variable number of neoplastic cells (10% to 80%) in 10 SCC, and keratinocytes of basal layers in seven of nine cases of actinic keratosis also expressed MHC Class II, whereas keratinocytes of normal skin were always negative for this antigen. These results suggest that CD3+ T lymphocytes, CD79+ B cells and IgG-bearing plasma cells may participate in down-regulation of tumour growth, since these cell types were particularly numerous in well-differentiated and mildly invasive SCC, as well as in actinic keratosis. The expression of MHC Class II by neoplastic cells could enhance this local anti-tumour immune response.     

Immunohistochemical and histopathologic features of 14 malignant fibrous histiocytomas from Flat-Coated Retrievers

Flat-Coated Retrievers seem to be at increased risk of developing soft-tissue sarcomas, and undifferentiated round cell or spindle cell sarcomas account for approximately 59% of sarcomas in the breed. In an attempt to classify these tumors further, formalin-fixed, paraffin-embedded sections from 14 undifferentiated sarcomas from Flat-Coated Retrievers were reviewed and examined with a panel of histologic and immunohistochemical stains. The panel included vimentin, desmin, Myo D1, smooth muscle actin, cytokeratin, S100, von Willebrand factor (factor VIII), Mac 387, CD3, major histocompatibility complex (MHC) class II, and CD79a. The majority of the sarcomas showed greater than 70% staining for MHC class II. We conclude that these undifferentiated sarcomas in Flat-Coated Retrievers belong to a spectrum of tumors with varying proportions of characteristic cell types and morphologic features, some of which fit the diagnostic criteria for malignant fibrous histiocytoma. Many of these sarcomas seem to have a significant myofibroblast component and a mild or moderate T cell infiltrate but the precise cell lineage is still uncertain.     

Feline Hodgkin's-like lymphoma: 20 cases (1992-1999)

We identified 20 cases of feline lymphadenopathy that conform to many clinical and histologic manifestations of human Hodgkin's disease. Histologic subtypes encountered included lymphocyte predominance (nine cases), mixed cellularity (nine cases), and nodular sclerosis (two cases). Two cases were not easily classified; fibrous bands were present, but the absence of nodules supported a subclassification of mixed cellularity Hodgkin's disease. Immunohistochemical staining of the tissues using antibodies against the pan T-cell antigen CD3, the human B-lymphocyte antigen 36 (BLA.36), the pan B-lymphocyte and plasma cell marker CD79a, and a myeloid antigen (MAC387) confirmed the phenotypic heterogeneity of the tumor. Classic Reed-Sternberg (RS) cells and mononuclear, multinucleate, and lacunar cell variants did not stain with any of the antibodies used. In contrast, lymphohistiocytic RS variants (L+H cells) reacted positively to BLA.36 and CD79a B-cell markers. Eighteen of 20 affected cats were > or = 6 years of age (range, 1-14 years). A sex predilection could not be identified. These findings support the existence of Hodgkin's-like lymphoma in the cat. Proper identification of this disease in the cat will enable further characterization of clinical features and biologic behavior to determine whether there are significant differences in the treatment and prognosis of feline Hodgkin's-like lymphoma compared with non-Hodgkin's lymphoma.     

Immunocytochemical demonstration of lymphocyte subsets and MHC class II antigen expression in synovial membranes from dogs with rheumatoid arthritis and degenerative joint disease

The study describes the distribution of canine leucocyte antigens in synovial membrane biopsies from six dogs with canine rheumatoid arthritis (CRA) and from eight dogs with osteoarthritis (OA) secondary to spontaneous rupture of the cranial cruciate ligament (CCL) (n = 5) or patellar luxation (n = 3). Synovial membranes from five dogs without evidence of joint lesions were used as control tissues. In the subsynovium of dogs with normal joints CD5+, CD4+, CD8+ and alpha beta TCR+ lymphocytes were present only in low numbers. With monoclonal antibody (mAb) to MHC class II antigen, either none or up to 20-30% of synovial lining cells were immunoreactive. Furthermore, scattered MHCII+ stromal cells were seen in the deeper subsynovial layer. In synovial membrane biopsies from dogs with CRA numerous diffusely and perivascularly distributed CD5+ lymphocytes were found in the subsynovium. CD4+ cells outnumbered CD8+ cells and were more numerous in the perivascular areas. In all the CRA cases examined, there were markedly higher numbers of alpha beta TCR+ cells compared with gamma delta TCR+ cells. With mAb to CD21, low numbers of immunoreactive lymphocytes were demonstrated. In all the CRA cases, a marked increase of MHC class II antigen expression was noted. In the majority of samples, 50% or more than 90% of the synovial lining cells were strongly MHC class II+. Throughout the subsynovial layer there were numerous MHC class II+ cells and included those with dendritic morphology and inflammatory mononuclear cells. Furthermore, marked perivascular immunoreactivity for MHC class II antigen was found. In biopsies from dogs with OA, there were markedly lower numbers of subsynovial CD5+, CD4+ and CD8+ lymphocytes. T-cells were mainly diffusely distributed. In three of the eight OA dogs examined, there was an increased percentage of synovial lining cells expressing MHC class II. The majority of OA cases had subsynovial major histocompatibility complex (MHC) class II+ cells with a dendritic morphology.     

Immunohistological study of the immune system cells in paraffin-embedded tissues of conventional pigs

The distribution of different cells of the immune system has been studied in formalin-fixed paraffin-embedded tissues from conventionally reared healthy pigs, using immunohistological techniques. The samples collected were: lungs, tonsils, lymph nodes (mediastinal, mesenteric, inguinal and submandibular), pancreas, spleen, liver, kidney, adrenal gland, ileum and stomach. A total of six primary antibodies anti-CD3, anti-CD79alpha, Mac 387, anti-lysozyme, anti-CD45RA (3C3/9) and anti-SLA-II-DQ (BL2H5) were used with a standard avidin-biotin peroxidase (ABC) method. Anti-CD3 and anti-CD79alpha mAb-reacted, respectively with cells located in T cell areas and B cell areas. Mac 387 recognised circulating polymorphonuclear leukocytes, while anti-lysozyme-stained resident macrophages in all tissues. 3C3/9 and BL2H5, were assessed in formalin-fixed paraffin-embedded tissues for the first time. 3C3/9 identified B lymphocytes, in primary follicles and mantle zones, a subpopulation of T cells, especially located in the marginal zone of the spleen and a variable number of immunoblasts, in the germinal centres. BL2H5 reacted with B cells in the mantle zones of the follicles of lymphoid tissues, with dendritic and interdigitating cells in all studied lymphoid tissues and with a variable number of resting and activated T cells in the periarteriolar lymphoid sheath (PALs), marginal zone and red pulp of the spleen. Furthermore, it stained Kupffer and perivascular macrophages in the liver. This study represents a detailed histological study of the distribution of the most important subpopulations of immune system cells in conventional, healthy pigs. In our view, these tools should be useful for future comparative studies in disease conditions.     

B-cell immunoblastic lymphoma with multinucleated giant cells in a cat

A 10-year-old male mixed breed cat died after six months history of intermittent vomiting and weight loss. At necropsy, large white-colored foci were found in both kidneys, and whitish thickening of the gastric wall was present at the pyloric part of the stomach. Histopathological examination revealed that both lesions consisted of proliferation of large-sized neoplastic lymphocytes intermingled with multinucleated giant cells. Immunohistochemically, the neoplastic cells were positive for both B-cell antigen receptor complex (CD 79 alpha cy) and MHC class II, although multinucleated giant cells were negative. The present case was diagnosed as B-cell immunoblastic lymphoma with multinucleated giant cells.     

Immunohistochemical characterisation of PCV2 associate lesions in lymphoid and non-lymphoid tissues of pigs with natural postweaning multisystemic wasting syndrome (PMWS)

The lymphoid, renal, pulmonary, and hepatic lesions of naturally occurring postweaning multisystemic wasting syndrome (PMWS) affected pigs have been studied by means of immunohistology. Ten conventionally reared pigs showing acute clinical signs of PMWS were selected from a farm on which animal were seronegative to porcine reproductive and respiratory virus and to Aujeszky's disease virus. All pigs were positive in tests for porcine circovirus type 2 by ISH and IHC. Monoclonal and polyclonal antibodies to CD3, CD79alpha, CD45RA (3C3/9), lysozyme, SLA-II-DQ (BL2H5), and MAC387 were used to characterise cells in PMWS lesions. The most relevant changes were reduction or loss of B and T lymphocytes, increased numbers of macrophages, and partial loss and redistribution of antigen presenting cells throughout lymphoid tissues compared to uninfected controls. The characteristics of lymphoid lesions in the present study strongly suggest an immunosuppressive effect of PMWS in affected pigs.     

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.     

Malignant histiocytosis in a domestic cat: cytomorphologic and immunohistochemical features

Malignant histiocytosis (MH) was diagnosed in a 13-year-old neutered male Domestic Shorthair cat on the basis of light microscopic and immunohistochemical findings. Thoracic fluid analysis showed a modified transudate which contained a very few atypical discrete cells. Cytologic and histologic evaluation of mediastinal and splenic masses revealed a pleomorphic population of large, discrete, round cells 10 to 30 micrometers in diameter with marked cellular atypia. Nuclei were oval to reniform, often with prominent, bizarre nucleoli. Multinucleated cells and mitotic figures were commonly seen. Erythro- and leucocytophagia were noted. Immunohistochemistry indicated a scattered positive staining pattern with the histiocytic antigenic marker Mac387 and a minor population of cells showing positive reactivity for lysozyme. This report describes the characterization of MH in a cat and emphasizes that MH should be considered as a differential diagnosis in proliferative disorders of discrete-cells in this species.     

Influence of age and parity on the distribution of cells expressing major histocompatibility complex class II, CD4, or CD8 molecules in the endometrium of mares during estrus

OBJECTIVE: To evaluate effect of age and parity on distribution and number of cells expressing major histocompatibility complex (MHC) class II, CD4, or CD8 molecules in the endometrium of mares during estrus. ANIMALS: 32 gynecologically healthy mares, categorized as young (3 to 8 years; n = 17) or old (9 to 16 years; 15) and nulliparous (n = 6), nulliparous embryo donors (16), or parous (10). PROCEDURES: Endometrial specimens collected from the uterine body and horns during estrus were stained by use of the avidin-biotin-peroxidase method, using monoclonal antibodies against equine MHC class II, CD4, and CD8 molecules. Labeled cells in the stratum compactum within 5 randomly selected fields at 400x magnification (total area = 0.31 mm2) were counted, and numbers were compared among groups and between locations. RESULTS: Age did not affect cell numbers within the 3 cell subsets examined. Numbers in each subset were higher in the uterine body than in the horns, although the difference was not significant for cells expressing MHC class II. Significantly more cells expressing MHC class II molecules were detected in the uterine body of nulliparous and parous mares than in embryo donors, whereas in the horns, these cells were significantly higher in number only in parous mares. Parity did not affect number of CD4+ or CD8+ cells. CONCLUSIONS AND CLINICAL RELEVANCE: The increased likelihood for endometritis to develop in mares as they age cannot be explained by a decrease in number of cells expressing MHC class II, CD4, or CD8 molecules within the endometrium. However, greater number of cells within these 3 subsets detected in the uterine body, compared with the horns, during estrus suggests a local readiness to act against microorganisms or semen introduced during mating or insemination.     

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.     

Cross-reactivity of human and bovine antibodies in striped dolphin paraffin wax-embedded tissues

This study evaluates the cross-reactivity of seven anti-human and one anti-bovine antibodies in formalin-fixed, paraffin-embedded tissue samples of liver and mesenteric lymph nodes of 13 striped dolphins (Stenella coeruleoalba). Four antibodies (CD3, IgG, lysozyme and S100 protein) reacted with striped dolphin lymph nodes in a similar pattern to that observed in the species of origin. The anti-human MHC class II mAb reacted strongly with macrophages and dendritic-like cells of striped dolphins, whereas a small number of lymphocytes were labelled with this antibody. These antibodies were used to study the immunophenotype of the inflammatory infiltrated in non-specific chronic reactive hepatitis (eight cases) and chronic parasite cholangitis (two cases) and normal liver (three cases) of striped dolphins. Non-specific chronic reactive hepatitis was composed of inflammatory infiltration of CD3+ T lymphocytes and IgG+ plasma cells in portal spaces and hepatic sinusoids. Lymphonodular aggregates observed in chronic parasitic cholangitis showed a cellular distribution similar to that found in lymph node cortex, including the presence of S100+ and MHC class II+ dendritic-like cells in lymphoid follicles and interfollicular areas. This result suggests that those inflammatory infiltrates are highly organised to enhance antigen presentation to B and T cells.     

Progression of an orbital T-cell rich B-cell lymphoma to a B-cell lymphoma in a dog

An 11-year-old Shetland Sheepdog was presented for exophthalmos caused by a locally extensive, poorly defined mass located behind the right eye. The primary orbital mass was identified by light microscopy and immunohistochemistry as a T-cell rich B-cell lymphoma (TCRBCL) composed predominantly of BLA.36-positive large neoplastic lymphoid cells admixed with fewer CD3- and CD79a-positive small lymphocytes. The dog was treated for lymphoma, but 6 months after presentation it was euthanatized for suspected hepatic and gastrointestinal metastasis. Gross findings revealed an enlarged liver with multiple well-demarcated, randomly distributed 0.1-1.5-cm white nodules, five firm white submucosal jejunal nodules, and ileocecal, mediastinal, and hilar lymphadenopathy. Metastatic liver lesions consisted of sheets of monomorphic large neoplastic lymphoid cells that effaced and expanded portal and centrilobular zones. These cells were morphologically similar to the large neoplastic cells of the original orbital tumor and were CD3-negative and variably BLA.36-positive, consistent with B-cell lineage. Similar cells comprised the jejunal nodules and effaced the lymph nodes. The progression of TCRBCL to a diffuse B-cell lymphoma in this case is consistent with reported human cases and has not been previously reported in the dog.     

Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.     

Sows intramammarily inoculated with Escherichia coli at parturition. II. Effects on the densities of MHC class II(+), CD4(+) and CD8(+) cells in the mammary gland

The aim of this study was to determine the density of MHC class II, CD4 and CD8 positive cells in mammary glands of sows around parturition, and whether the densities were altered following intramammary inoculation with Escherichia coli prior to parturition. Also, animals developing clinical disease after inoculation were compared with animals not developing clinical disease. Fourteen cross-bred primiparous sows were subject to intramammary inoculation with E. coli bacteria 24h before estimated parturition. Mammary gland biopsies were collected and clinical observations were made. Four sows were categorised as clinically ill based on general condition, body temperature and gross mammary affection. There were no changes in density of MHC class II, CD4 and CD8 positive cells in non-inoculated glands around parturition, while significant changes in densities were shown in inoculated glands. Here, the density of MHC class II, CD4 and CD8 positive cells reached a peak at 72 h post-inoculation (p<0.01). In sows developing clinical disease, there was a tendency to an over all lower density (p=0.07) of MHC class II positive cells in inoculated glands compared with sows not developing clinical disease. When comparing the categories with respect to the density of CD4 and CD8 positive cells, the sows developing clinical disease showed a higher density (p=0.03) of CD4 and CD8 positive cells in inoculated glands than sows not developing disease. No differences were shown between categories in non-inoculated glands. It is concluded that the density of MHC class II, CD4 and CD8 positive cells seems to be unaltered around parturition. However, there is a rapid increase in density of these cells following intramammary inoculation with E. coli. Also, the data suggest that there is a difference between sows developing and sows not developing clinical disease after inoculation with respect to the increase in density of MHC class II, CD4 and CD8 positive cells in the mammary gland.     

Immunohistochemical characterisation of the lesions of canine idiopathic pericarditis

Pericardial tissue was obtained from 14 dogs with idiopathic pericarditis, and from three dogs with pericardial effusion associated with neoplastic disease, for histopathological assessment and characterisation of infiltrating leucocytes by immunohistochemistry. The major pathological change was extensive pericardial fibrosis which was generally accompanied by a mixed inflammatory response that was of greatest intensity at the cardiac surface of the tissue. Perivascular lymphoplasmacytic aggregates were present at the pleural surface and within the fibrosed pericardium. There were no features that clearly distinguished the samples from dogs with neoplastic disease from dogs with idiopathic pericarditis. The pericardial infiltrates were dominated by MAC 387+ monocyte-macrophages and plasma cells expressing immunoglobulin (Ig)A or IgG. CD3+ T lymphocytes and major histocompatibility complex (MHC) class II+ macrophages were less common, although the perivascular aggregates were mixtures of T and B lymphocytes and a proportion of fibroblasts expressed MHC class II. There was no vascular pathology or deposition of immunoglobulin or complement within vessel walls. These findings are consistent with an immune response dominated by humoral effector mechanisms (Th2 immunity) but do not clearly support a primary immune-mediated pathogenesis for idiopathic pericarditis.     

Immunohistochemical characterization of inflammatory cell populations and adhesion molecule expression in synovial membranes from dogs with spontaneous cranial cruciate ligament rupture

This study describes the distribution of CD4+ and CD8alpha+ T lymphocytes, B lymphocytes, macrophages, MHC class II antigens, immunoglobulin (IgG, IgM, IgA)-containing cells and of adhesion molecules belonging to the CD11/CD18 family in synovial membrane biopsies from 28 dogs with spontaneous rupture of the cranial cruciate ligament (CCL). Synovial membranes from 11 dogs without evidence of joint lesions were used as control tissues. The main cell types in synovial membranes from dogs with CCL rupture were B lymphocytes and plasma cells belonging to the IgG isotype. The severity of inflammatory cell infiltration in CCL cases was positively correlated with the expression of adhesion molecules. Double immunofluorescence labelling of frozen sections revealed that in the inflamed synovium of dogs with CCL rupture numerous dendritic cells expressing MHC class II antigen and canine CD1c were present. The findings further support the view that in the synovium of dogs with CCL rupture an immunologic response is going on in which dendritic cells are possibly involved by presenting hitherto unknown antigens to T lymphocytes.     

Primary ureteral giant cell sarcoma in a Pomeranian

An 8-year-old male neutered Pomeranian dog was presented to the Veterinary Teaching Hospital at Oregon State University for surgical treatment of hydronephrosis of the left kidney and a left cranial abdominal mass. A primary ureteral mass was found during exploratory surgery, and the mass was resected and ureteral anastomosis was performed. Cytologic evaluation of the mass revealed 3 distinct cell populations, including a large number of multinucleated giant cells, a moderate number of thin spindle-shaped cells, and cohesive clusters of transitional epithelial cells. The cytologic diagnosis was giant cell sarcoma. The diagnosis was confirmed by histologic examination, and immunohistochemical staining was performed. The spindle-shaped cells and multinucleated giant cells were both immunoreactive for vimentin and spindle-shaped cells for S-100. Tumor cells did not express wide-spectrum cytokeratin, broad-spectrum muscle actin, smooth muscle actin, sarcomeric actin, desmin, BLA36, Mac 387, synaptophysin, neuron-specific enolase, glial fibrillary acid protein, or von Willebrand factor. These findings are most consistent with an anaplastic sarcoma with giant cells. This is the first case report of a primary ureteral giant cell sarcoma in a dog.     

Monoclonal antibodies to sheep MHC class I and class II molecules: biochemical characterization of three class I gene products and four distinct subpopulations of class II molecules

The availability of a panel of monoclonal antibodies to sheep MHC class I and class II gene products has allowed for the first time an assessment of the relative complexity of the sheep MHC. By using four monoclonal antibodies to MHC class I, and seven monoclonal antibodies to MHC class II molecules together with one-dimensional SDS-PAGE, sequential immunoprecipitation and 2-dimensional gel analysis, three class I gene products and four distinct subsets of class II molecules have been identified. Sheep class I molecules showed heterogeneity on 2-dimensional gels and as in mouse and man, represented the products of at least three different non-allelic class I genes. Interestingly, the sheep beta 2 microglobulin molecule also displayed heterogeneity, consistent with either two primary gene products or allelic variation. Four sheep class II monoclonal antibodies identified distinct, non-overlapping subsets of sheep class II molecules of Mr 32-36 K (alpha chain) and 25-28 K (beta chain). These class II molecules were co-expressed on sheep B lymphocytes and represented the primary products of different sheep MHC class II genes. The class II molecules within three of these subsets displayed allelic polymorphism essentially restricted to their beta polypeptides, while the fourth subset of class II molecules showed allelic variation in both their alpha and beta polypeptides. The results of this study represent the first evidence for gene duplication and heterogeneity within the sheep MHC. The identification of three primary class I gene products and four distinct subsets of class II molecules suggests three class I loci and up to four distinct class II subregions within the sheep MHC. Potentially large numbers of allelic variants of these different gene products may be expressed in normal sheep.