Human Anti-CD200R1 Recombinant Antibody (clone OX108); Fab Fragment (CAT#: PFBC-020)

Figure 1 sCD200 protein and OX108 mAb binding to mutants of sCD200R analyzed by surface plasmon resonance.

OX68 mAb was immobilized on the BIAcore™ chip and mutants bound from spent tissue culture from 293T cells. sCD200 was passed over each protein in the four flow cells sequentially, followed after washing, with OX108 mAb. (A) sCD200 gave no binding to sCD4d3+4 (the signal is bulk effect due to high protein concentration) but bound well to wild-type (CD200R/CD4) and mutant A123D. sCD200 gave no binding to mutant T73R. OX108 mAb binds both mutants to the same extent as the wild type. (B) Similar experiment showing sCD200 bound with reduced activity to mutant P62F that still retains OX108 mAb binding activity. (C) Mutant D127R showed reduced binding for both sCD200 and OX108 mAb. (D) R67D shows normal sCD200 binding but no OX108 mAb binding.

Hatherley, D., & Barclay, A. N. (2004). The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains. European journal of immunology, 34(6), 1688-1694.

Figure 2 CD200R engagement inhibits activation of U937 cells.

A, Wild-type (black bars) and truncated (gray bars) CD200R were engaged on U937 cells using different reagents as indicated. Plate-bound OX108 or isotype control were immobilized at 40 μg/ml, soluble OX108 was used at 10 μg/ml, and cross-linking anti-mouse IgG was used at 20 μg/ml. Cells were then stimulated overnight with 20 ng/ml LPS and culture supernatants assayed for IL-8 by ELISA. B, Wild-type or truncated CD200R was engaged on U937 using 100 μg/ml plate-bound OX108 mAb and cells were stimulated overnight using different reagents as indicated. Neisseria indicates ethanol-killed N. meningitides diluted in culture medium. , p < 0.05 according to two-tailed Student's t test; ** , p < 0.005, N.S., nonsignificant. Results are expressed as means of duplicate or triplicate wells ± SD and are representative of two or more independent experiments.

Mihrshahi, R., Barclay, A. N., & Brown, M. H. (2009). Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells. The Journal of Immunology, jimmunol-0901531.

Figure 3 Flow cytometry

Cell surface CD200R and CD206 were detected by flow cytometry in U937 cells stimulated with IL-4 for 24 h. Data are representative of two-three separate experiments. Statistical significance was determined by Student's t test; *, indicates p < 0.05.

Warren, K. J., Fang, X., Gowda, N. M., Thompson, J. J., & Heller, N. M. (2016). The TORC1-activated proteins, p70S6K and GRB10, regulate IL-4 signaling and M2 macrophage polarization by modulating phosphorylation of insulin receptor substrate-2. Journal of Biological Chemistry, 291(48), 24922-24930.

Figure 4 Macrophages express CD200R and are able to bind CD200- and K14-coated beads.

(A) Flow cytometry shows expression of CD200R on human macrophages stained with MAb OX108 (thick line) compared to an isotype-matched control MAb, OX1 (shaded histogram). Macrophages did not express CD200 when stained with MAb OX104 (dotted line [but indistinguishable from the shaded histogram]). (B) CD200-CD4d3 + 4-coated fluorescent beads bind macrophages (thick line) compared to beads coated with negative control CD4d3 + 4 (shaded histogram). (C) K14-CD4d3 + 4-coated fluorescent beads bind macrophages (thick line) compared to beads coated with negative control CD4d3 + 4 (shaded histogram).

Foster-Cuevas, M., Wright, G. J., Puklavec, M. J., Brown, M. H., & Barclay, A. N. (2004). Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor. Journal of virology, 78(14), 7667-7676.

Figure 5 Immunohistochemistry

(A-C) CD200R immunoreactivity is seen on perivascular cells in cryosections of the cerebellum (A) and spinal cord (B). (C, D) Adjacent sections of white matter show CD200R staining of perivascular cells (C) and mannose receptor expression in the same cells (D). Scale bars = (A-D) 10 μm.

Koning, N., Swaab, D. F., Hoek, R. M., & Huitinga, I. (2009). Distribution of the immune inhibitory molecules CD200 and CD200R in the normal central nervous system and multiple sclerosis lesions suggests neuron-glia and glia-glia interactions. Journal of Neuropathology & Experimental Neurology, 68(2), 159-167.