Functional characterization of canine lymphocyte subsets

Abstract

Functional characterization of subsets of T lymphocytes is essential for transplantation studies in dogs, as it is in other species. We studied the function of T cells separated by two mouse monoclonal antibodies recognizing complementary subsets--an antibody directed to canine T cells (MdT-P1) with an up-regulating function, and an antibody directed to human CD 8 (MT811) that cross-reacts with down-regulating canine T cells. Immunorosetting with sheep red blood cells and Percoll gradient allowed us to study depleted and enriched fractions. Their function was tested in mixed lymphocyte culture (MLC), cell-mediated cytotoxicity (CML), and coculture with B cells in a hemolytic plaque assay (PFC). In MLC, MdT-P1-positive cells showed a high proliferative response, and MT811-positive cells responded poorly to allogeneic cells. Vice versa, MT811- negative cells responded strongly, and MdT-P1-negative cells were poor responders but strong stimulators. Effector cells of CML were separated following 8 days of culture and prior to mixing with target cells. Enriched and depleted fractions with either antibody showed low cytotoxic activity as compared with unseparated cells. When added to unseparated effector cells MT 811-positive cells suppressed cytotoxicity. B cells were obtained by rosetting with staphylococcal protein A (SPA). Their immunoglobulin production was studied following 6 days of culture stimulated by pokeweed mitogen in a reverse hemolytic plaque assay. Again, MT 811-positive cells added to the culture suppressed, and MT 811-negative cells enhanced immunoglobulin production. In conclusion, immunorosetting with two monoclonal antibodies allowed us to distinguish subpopulations of canine T cells with up-regulating (helper/inducer) from those with down-regulating (suppressor) activity.

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining

“…Therefore, the search for monoclonal antibodies generated against antigens derived from other species for their cross-reactivity and systematic studies have been performed [ 18 , 20 , 25 – 28 ]. Some cross-reacting antibodies have been employed to study canine hematopoietic cells [ 29 , 30 ], canine lymphomas [ 31 ], canine lymphocyte subsets [ 32 ] or the canine leukocyte adhesion deficiency syndrome (CLAD) [ 33 ].…”