Fig. 5. Elevated CD200 expression on DFT cells.

(A) GOIs for this study are plotted as a log₂-transformed transcripts per million (TPM) heat map with dark blue indicating the most highly expressed genes. Technical replicates (N = 2) from separate flasks were used for the cell lines (C5065, RV) and biological replicates (N = 2) were used for primary tissues, except peripheral nerve (PN) (N = 1). (B) Wild-type DFT1.C5065, DFT2.JV, DFT2.SN, and DFT1.C5065 transfected to overexpress CD200 or CD200R1 were stained with either CD200R1-mOrange or CD200-mOrange FAST protein. Histograms filled with blue or red highlight expected strong binding interactions. The percentage of events that falls within the marker is shown. Results are representative of N = 2 replicates per treatment. (C) Mice were immunized with 41BB or CD200 FAST proteins. Black, preimmune; gray, immune sera from a mouse immunized with 41BB; red, preimmune; blue, immune sera from a mouse immunized with CD200. CHO cells transfected with either full-length 41BB or CD200 were stained with sera and then anti-mouse AF647. Results are representative of N = 2 per treatment. (D) Sera were used to screen two strains each of DFT1 and DFT2 cells for 41BB and CD200 expression. Results are representative of N = 3 per treatment. (E) DFT1 C5065 transfected with either vector control, CD200, or CD200R1 was stained with purified polyclonal anti-CD200 and anti-mouse IgG AF647 (black, no antibodies; red, secondary antibody only; blue, primary and secondary antibody). Results are representative of N = 2 per treatment.