How to Build a Dog: Genetics of Complex Traits

2 Veterinary Student Platform Presentation Canine PBMC NSG mice: A model of canine specific immunity to allogeneic canine mammary tumor-DC fusion vaccines Jeremy Foote Ph.D., Patricia Deinnocentes, Farruk M.L. Kabir, Bruce Smith Ph.D., VMD and Richard Bird Ph.D. Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL Introduction. Invasive breast cancer is the most frequently occurring malignancy in women and canine populations. Both incidence and prevalence of breast cancer are increasing, making development of new strategies for breast cancer treatment, in particular those designed to treat individuals suffering from early stage disease. Recently our lab has developed a cellular vaccine fusion incorporating patient dendritic cells with an immunogenic cell line of canine mammary tumors (CMT28), which has been shown in preliminary trials to generate both humoral and cell mediated tumor specific immunity. However, our ability to assess patient specific immune function in response is limited for client convenience and patient well-being. To evaluate patient immunity to allogeneic DC-CMT28 vaccines we generated a mouse model of canine tumor immune responses. Our initial emphasis was developing this model using canine peripheral blood mononuclear cells (PBMCs) from healthy donors to determine the feasibility of this project using guide lines established from human PBMC NOD SCID IL2R common gamma chain -/mouse (PBMC NSG mice) studies. Methods. Peripheral blood was collected from 2 healthy Labrador Retrievers and PBMC’s were purified using Ficoll density centrifugation. 10 PBMC’s were injected by the intraperitoneal route into NSG mice 48 hours after radiation treatment with a 100cGy dose of γ radiation. On 1, 7, 14, 21, and 42 days post reconstitution, engraftment of canine WBCs was determined by flow cytometry using antibodies specific to helper T cells (CD45+ CD4+), cytotoxic lymphocytes (CD45+ CD8+), regulatory T cells (CD45+ CD4+ Foxp-3+), naïve (CD45+ IgM+) or class switched B cells (CD45+ IgG+) and IgM (CD45 low IgM intracellular+) or IgG (CD45 low IgG intracellular+) secreting plasma cells. Onset of xenogenic graft versus host disease (GVHD), which has been documented to occur with transfer of human lymphocytes into NSG mice, was assessed by biweekly measurements of weight where a 15% reduction in body weight, or anemia, and thrombocytopenia determined on the day of FACS analysis is indicative of xenogenic GVHD. Results. Engraftment of NSG with canine PBMC’s resulted in detection of CD45+ lymphocytes in the blood, bone marrow, spleen, and peritoneal cavity. The proportions of lymphocytes in the spleen steadily increased over time to represent 70% of the total splenic cells on 42 days post engraftment. In comparison the bone marrow and peritoneal cavity (PEC) canine lymphocytes represented less than 20% of total cells indicating that the spleen was the primary tissue of engraftment. Evaluation of lymphocyte subsets revealed an early predominance of helper and regulatory T cells which decreased over time in comparison to increasing proportions of CTL’s and IgM+ secreting plasma cells, which correlated with clinical symptoms (weight loss, anemia, thrombocytopenia) consistent with the onset of GVHD 42 days post engraftment. Conclusions. NSG mice successfully engraft with canine lymphocytes from healthy donors. The inverse relationship between decreasing proportions of helper and regulatory T cells in comparison CTLs and plasma cells may suggest a potential role in the development of humoral and cell-mediated immunity to mouse tissue auto-antigens signifying an onset of GVHD 42 days post engraftment. These findings suggest that NSG mice have the potential to be a model with which we can model patient specific immunity to allogeneic DC-CMT28 vaccines. Future vaccination studies will be conducted early post reconstitution to precede the activation of canine lymphocytes by mouse autoantigens and the development of GVHD. Acknowledgment: NIH, CVM Interdepartmental & Animal Health Research Grants, Allison Church Bird for expert flow cytometry analysis and cell sorting.