Developments and translational relevance for the canine haematopoietic cell transplantation preclinical model.

The development of safe and reliable haematopoietic cell transplantation (HCT) protocols to treat human patients with malignant and non-malignant blood disorders was highly influenced by preclinical studies obtained in random-bred canines. The surmounted barriers included recognizing the crucial importance of histocompatibility matching, establishing long-term donor haematopoietic cell engraftment, preventing graft-vs-host disease and advancing effective conditioning and post-grafting immunosuppression protocols, all of which were evaluated in canines. Recent studies have applied the tolerance inducing potential of HCT to solid organ and vascularized composite tissue transplantation. Several advances in HCT and tolerance induction that were first developed in the canine preclinical model and subsequently applied to human patients are now being recruited into veterinary practice for the treatment of malignant and non-malignant disorders in companion dogs. Here, we review recent HCT advancements attained in the canine model during the past 15 years.

Effect of Irradiation on Incidence of Post-Transplant Lymphoproliferative Disorder after Hematopoietic Cell Transplantation in Miniature Swine.

Post-transplant lymphoproliferative disease (PTLD) is a major complication of clinical organ and cell transplantation. Conditioning and immunosuppressive regimens that significantly impair T cell immunity, including depleting antibodies and calcineurin inhibitors, increase the risk of PTLD after transplantation. Swine PTLD has been shown to closely resemble human PTLD in morphology, histology, and viral-driven reactivation of B cells. Previously, we reported high incidences of PTLD after hematopoietic cell transplantation (HCT) in miniature swine recipients conditioned with thymic irradiation (TI) in addition to T cell depletion and cyclosporine A monotherapy after transplantation. Replacement of TI with 100 cGy of total body irradiation resulted in similar numbers of B cells early post-transplantation, greater numbers of T cells at day 0, and markedly decreased incidence of PTLD, suggesting that a threshold number of T cells may be necessary to prevent subsequent B cell proliferation and development of overt PTLD. Results from this large cohort of animals provide insight into the important effect of irradiation and T cell immunity on the incidence of PTLD after HCT and reinforce the pig model as a valuable tool for the study of PTLD and HCT.

Extracorporeal photopheresis combined with pentostatin in the conditioning regimen for canine hematopoietic cell transplantation does not prevent GVHD.

Extracorporeal photopheresis (ECP) and the purine analog pentostatin exert potent immunomodulatory effects. We evaluated the use of these treatment modalities to prevent GVHD in a canine model of unrelated dog leukocyte Ag-mismatched hematopoietic cell transplantation, after conditioning with 920 cGy TBI. We have shown previously in this model that 36/40 dogs given MTX alone as postgrafting immunosuppression engrafted and that 25 of 40 dogs had severe GVHD and median survival of 21 days. In the current study, nine dogs received conditioning with 920 cGy TBI and postgrafting MTX either with ECP on days -2 to -1 alone (n=5) or ECP on days -6 and -5 combined with two doses of pentostatin (days -4 to -3) (n=4). Seven of nine dogs achieved engraftment. Six dogs developed severe acute GVHD (four in the group with ECP alone and two with pentostatin and ECP). We failed to demonstrate a positive impact of ECP and pentostatin for the prevention of GVHD compared with historical control 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.

Transplantation of CD6-depleted peripheral blood stem cells after DLA-haploidentical bone marrow transplantation contributes to engraftment and tolerance in a preclinical model of stem cell transplantation.

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation is an opportunity for nearly all patients lacking an HLA matched stem cell donor. However, graft rejection and graft-versus-host disease (GvHD) as well as infectious complications still result in high treatment-related mortality. Here, we used the dog as a preclinical model for the study of tolerance induction with the aim to optimize and to improve a clinical protocol of haploidentical stem cell transplantation. For this purpose CD6-depleted peripheral blood stem cells (PBSCs) were transfused 6d after transplantation of unmodified bone marrow from dog leukocyte antigen (DLA)-haploidentical littermate donors in order to induce immune tolerance. Besides hematopoietic stem cells CD6-depleted PBSC contain, NK cells and a minority of suppressive CD8-positive cells that may suppress activated T lymphocytes. Recipients were conditioned with, cyclophosphamide and antithymocyte globulin (ATG) preceded by a transfusion of donor buffy coat and either 1, 2 or 3 × 3.3 Gy total body irradiation (TBI). Postgrafting immunosuppression was limited to 30 d of cyclosporine and methotrexate. The additional administration of CD6-depleted PBSCs after unmodified marrow could not prevent GvHD, but it may improve engraftment and chimerism after conditioning with 2 × 3.3 Gy TBI. Reasons for incomplete suppression and possible improvements for clinical applications are discussed.

Graft-versus-host reaction (GVHR) in clonal amago salmon, Oncorhynchus rhodurus.

The graft-versus-host reaction (GVHR) was demonstrated in a salmonid model system of clonal diploid and triploid amago salmon. Triploid operculum grafts on clonal diploid evoked an acute rejection within 12 days. Grafts exchanged among triploid amago salmon exhibited prolonged survival for 18 days. In contrast, diploid grafts on triploid, and allografts among clonal diploid amago salmon were accepted. A typical GVHR was induced in triploid recipients by intraperitonal injection of head kidney cells from sensitised diploid donors. The clinical signs of graft-versus-host disease (GVHD) were observed in the recipients after 1 week of cell injection as a loss of appetite and appearance of solid faeces, followed by haemorrhage, local swelling of ventral skin and an enlarged spleen. Three of six fish died within 1 month. Water temperature and frequency of sensitisation are critical to induce GVHR. Diploid donors had to be sensitised three times at 20 degrees C to induce the typical GVHR. GVHR was most effectively induced by head kidney cells, followed by peripheral blood leucocytes (PBL) and spleen cells. Ploidy analysis by flow cytometry revealed that the donor head kidney cells greatly increased in the recipient liver, head kidney and spleen, and reached the peak after 9 days of donor cell injection. The results in the present study are quite similar to the findings in ginbuna and ginbuna-gold fish hybrid system, suggesting the presence of T cells in salmonid as well as cyprinid fish.

Cytotoxic T cell function in fish.

Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

Immune reactivity following CD40L blockade: role in autoimmune glomerulonephritis in susceptible recipients.

To investigate the role of costimulation in autoimmune glomerulonephritis that develops in the setting of murine chronic graft-vs-host disease (cGVHD), we examined the effects of blocking CD40L, a costimulatory marker expressed on activated CD4+ T cells, in recipient mice. These studies addressed the potential role of CD40L blockade in preventing disease and in downregulating its expression in animals with evidence of autoreactivity. Animals treated acutely with anti-CD40L antibody at disease induction do not develop circulating anti-DNA antibodies, proteinuria, or histologic evidence of renal disease. If treatment is delayed for two weeks, after circulating anti-DNA antibodies are apparent, all animals develop massive proteinuria by 14 weeks after disease induction. Renal histology of kidneys from the delayed treatment and control groups reveal similar glomerular immune deposits, and intense staining for CD4, ICAM-1, and I-A(b) in areas of mononuclear cell infiltration. Long-term treatment studies begun two weeks after disease induction is not disease-protective, as all animals develop massive proteinuria and renal disease by 14 weeks. These studies suggest that early CD40L signaling events are critical to induction of allogeneic interactions and autoreactivity in cGVHD, but that short-term or chronic CD40L blockade, once autoreactivity is evident, does not abrogate systemic autoreactivity and renal involvement.

Chronic tubulointerstitial nephritis and wasting disease in marmosets (Callithrix jacchus).

Six marmosets (Callithrix jacchus), 20 months to 16 years old, died from a disease characterized by weakness and paralysis of the hind legs, weight lose, anemia and transient diarrhea. The lesions most prominent at necropsy were subacute to chronic tubulointerstitial nephritis, subacute to chronic pancreatitis, and generalized hemosiderosis. Chronic protein deficiency is believed to be the underlying pathogenic mechanism, since the diet contained no more than 15% animal protein during the last two years, and increasing the amount of protein was accompanied by disappearance of the syndrome.

[Wasting disease produced in hamsters after the inoculation of a bovine leukemic material]. / Production d'un syndrome débilitant chez le hamster après l'inoculation de matériel leucémique bovin.

Wasting disease was observed in hamsters inoculated at birth with a cell-free bovine leukemic extract. The autopsy and histological examination of the runted hamsters revealed visible lesions in the lymphoid tissues. These lesions consisted mostly in depletion of lympocytes in the spleen and in the cortex of the lymph nodes and in atrophy of the Peper's patches. Thymic lesions were also observed. Anemia and a decrease of circulating lymphocytes were observed in the peripheral blood.