Figure 7. Cell types affected in Bovine Neonatal Pancytopenia are characterized by high MHC class I expression.

(a–c) Bone marrow and endothelial cells were isolated from late gestation bovine foetuses (n = 3). MHC class I expression and BNP Ab binding was assessed using flow cytometry (a,b) and confocal imaging (c). Data are representative for three different animals. (a) Endothelial cells were mixed with peripheral blood leukocytes from a healthy donor to compare MHC class I expression and BNP Ab binding between tissues. Endothelial cells and PBMC/Granulocytes were selected on forward and sideward scatter (FSC and SSC) and on CD31^high and CD31^low expression, respectively. Expression index = Δ Ab/Δ Isotype. (b) MHC class I expression and BNP Ab binding of bone marrow cells. Bone marrow cells divided into MHC class I^high and MHC class I^low expression (upper panel) were compered for BNP alloantibody binding (lower panel). Cells were gated on live cells based on FSC and SSC (confirmed with DAPI) and on autofluorescent-negative cells. (c) Confocal images of bone marrow cells stained with isotype, anti-MHC class I or anti-CD41 (Green). DAPI (blue) was used as a nuclear stain. Megakaryocytes are cells with large nuclei and anti-CD41 was used as a positive control for megakaryocytes. (d) Cryopreserved bone marrow cells isolated from a neonatal calf were measured using flow cytometry to evaluate MHC class I and Integrin beta-1 expression of cells with low and high BNP Ab binding. Intact cells were gated based on FSC and SSC and dead cells were excluded using DAPI staining. Data are representative for Abs isolated from two different BNP dams.