Isolation and characterisation of equine dendritic cells.

This research examines the characteristics and isolation methods of dendritic cells in horses and elucidates their importance in influencing T-cell responses.

Methodology

• The researchers developed a process from existing techniques to isolate equine blood dendritic cells by exploiting adherence and metrizamide gradients.
• They utilized numerous monoclonal antibodies (mAbs), including some known to label dendritic cells in other species, to test for immunochemical reactions with these isolated equine dendritic cells, as well as sections of lymph node and spleen.

Findings

• From the blood cells isolated, about 62% were Major Histocompatibility Complex (MHC) Class II positive, exhibiting standard dendritic cell morphology. Only around 4% contained non-specific esterase, an indicator of mature macrophages.
• These dendritic cells also showed expression of MHC Class I, LFA-1, EqWC1, and EqWC2.
• Among the possibly cross-reactive antibodies, a mAb against bovine CD1b was particularly notable as it stained lymph node dendritic cells, but not those in the blood.
• The researchers used monoclonal antibodies targeting equine CD5 (T-cells), surface immunoglobulin (B-cells), and macrophages (CZ2.2) to count the contaminating cells in blood preparations via flow cytometry. Approximately 39% of the cells were large, MHC Class II positive, but did not express T and B cell markers or CZ2.2.

Conclusions and Interpretations

• Comparison of immunochemistry and flow data, combined with examination of alveolar macrophages and adhered blood cells, led to the conclusion that CZ2.2 detects a myeloid marker not seen on mature macrophages and possibly shared with dendritic cell precursors.
• The isolated dendritic cells were tested on their functional capacity using their stimulation ability in the mixed leukocyte reaction (MLR). Isolates with enriched dendritic cells were found to stimulate MLRs more strongly than peripheral blood mononuclear cells or adherent cells.
• In conclusion, the equine dendritic cells isolated from blood expressed high levels of MHC Class I and II, and LFA-1 and stimulated robust MLRs. These cells didn’t express markers characterizing T and B cells, but through the expression of equine macrophage marker CZ2.2, they appear closely related to mononuclear phagocytes.