Thirteen novel canine dog leukocyte antigen-88 alleles identified by sequence-based typing.

Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.

Canine DLA-79 gene: an improved typing method, identification of new alleles and its role in graft rejection and graft-versus-host disease.

Developing a preclinical canine model that predicts outcomes for hematopoietic cell transplantation in humans requires a model that mimics the degree of matching between human donor and recipient major histocompatibility complex (MHC) genes. The polymorphic class I and class II genes in mammals are typically located in a single chromosome as part of the MHC complex. However, a divergent class I gene in dogs, designated dog leukocyte antigen-79 (DLA-79), is located on chromosome 18 while other MHC genes are on chromosome 12. This gene is not taken into account while DLA matching for transplantation. Though divergent, this gene shares significant similarity in sequence and exon-intron architecture with other class I genes, and is transcribed. Little is known about the polymorphisms of DLA-79 and their potential role in transplantation. This study was aimed at exploring the reason for high rate of rejection seen in DLA-matched dogs given reduced intensity conditioning, in particular, the possibility that DLA-79 allele mismatches may be the cause. We found that about 82% of 407 dogs typed were homozygous for a single, reference allele. Owing to the high prevalence of a single allele, 87 of the 108 dogs (∼80%) transplanted were matched for DLA-79 with their donor. In conclusion, we have developed an efficient method to type alleles of a divergent MHC gene in dogs and identified two new alleles. We did not find any statistical correlation between DLA-79 allele disparity and graft rejection or graft-versus-host disease, among our transplant dogs.

Canine lymphocyte subpopulations.

Ten to 23% of cells in blood, lymph node and bone marrow from normal dogs formed rosettes with human erythrocytes, and 12-27% formed rosettes with erythrocyte-antibody-complement (EAC) complexes. In contrast, only 3% of thymocytes, and 1% of thoracic duct cells formed rosettes with human erythroyctes, and 0 and 15% respectively formed EAC rosettes. When peripheral blood mononuclear cells were separated by rosette sedimentation into populations depleted of, or enriched for, cells forming rosettes with human erythrocytes (H-RFC), the population depleted of H-RFC responded more vigorously to alloantigens in mixed leukocyte culture (MLC) (P < 0.01) and to the mitogens phytohemagglutinin (PHA) (P = 0.01) and concanavalin A (P = 0.01) than did the population enriched for H-RFC. Passage of peripheral blood mononuclear cells over nylon wool columns produced a nonadherent population depleted of H-RFC, EAC rosette-forming cells and cells binding surface immunoglobulin (SIg), while the adherent population was enriched for each of these markers. In 3 dogs 36%, 44% and 64% of adherent cells that formed rosettes with human erythrocytes also possessed SIg, suggesting that canine B cells form rosettes with human red cells. The nonadherent population showed a more vigorous response to alloantigens in MLC (P < 0.01) and to PHA (P < 0.05) than the adherent population, and also stimulated the growth of autologous erythroid colonies better than the adherent population (P = 0.02). A T cell rich population can thus be obtained from canine peripheral blood, but no specific marker for T cells has been identified. Specifically, the capacity to form rosettes with human red cells is not a marker for the canine T cell.

In vitro tests for distinguishing possible immune-mediated aplastic anemia from transfusion-induced sensitization.

Forty-two patients with aplastic anemia (AA) were studied to determine whether or not transfusion-induced sensitization is responsible for the in vitro inhibition by patient lymphocytes of HLA-identical erythroid burst-forming units (BFU-E). The results indicate that lymphocytes from 12 of 34 transfused patients inhibited normal colony growth. In contrast, lymphocytes from none of the 8 untransfused patients demonstrated inhibition. These data were interpreted to mean that coculture studies would not be useful for identifying immune-mediated AA in transfused patients. Therefore, in order to identify possible immune-related AA, we assayed BFU-E from patient blood before and after T-cell depletion. In all 32 patients studied, BFU-E failed to grow from peripheral blood cells before T-cell depletion, but in 8 cases, normal-appearing BFU-E grew after T cells had been removed. Growth of patient BFU-E colonies was inhibited in 6 cases when patient T cells were added back to the culture, indicating that in these 6 patients, an "autoimmune" mechanism may have been present.

Granulocytic colonies on macrophage-coated membranes.

A macrophage cell coating covering a cellulose acetate disk was an effective microenvironment for the production of peroxidase-positive hematopoietic colonies. These developed after intraperitoneal injection of marrow cells with a linear cell relationship of dose to colonies formed. One colony formed for every 2,000 nucleated marrow cells injected. Observation of colony formation daily showed a steady increase in number and size until seven days after cell inoculation. X-irradiation (400 rads) eliminated intrinsic colony formation in BALB/c mice. Irradiation of the donor of the ip marrow cells resulted in a d0 of 95 rads. Treatment of the marrow donor with cytosine arabinoside had a suppressive effect on colony formation as did treatment of the host animal after receipt of the ip marrow. These results indicate that the precursor of the granulocytic colonies seen in the macrophage layers are more similar to committed granulocytic precursors than to the pluripotential stem cell.