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Blood, Vol. 113, Issue 19, 4780-4789, May 7, 2009
Naturally occurring regulatory dendritic cells regulate murine cutaneous chronic graft-versus-host disease
Blood Sato et al.
113: 4780
Supplemental materials for: Sato et al
Generation of mAb
Crlj:WI rats (Charles River Laboratories) were immunized with CD200R1-GFP-RBL2H3 cells, CD200R2-GFP-RBL2H3 cells, CD200R3-GFP-RBL2H3 cells, or CD200R4-GFP-RBL2H3 cells (2 × 107) three times at 7-day intervals. Three days after the final immunization, the splenocytes were fused with P3U1 mouse myeloma cells as described elsewhere. After hypoxanthine-aminopterin-thymidine selection, the Abs that can react with the transfectants were screened by flowcytometry using R-PE–conjugated F(ab′)2 Fragment Donkey anti-Rat Ig (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA), and several hybridomas producing mAbs were identified by their strong binding to each transfectant and cloned by limiting dilution. mAbs to CD200R1 (13H6E3, rat IgG2b), CD200R2 (B2C2F3, rat IgG2b), CD200R3 (6C4H2, rat IgG2a), and CD200R4 (8B3C9, rat IgG2b) were purified from culture supernatants using HiTrap™ Protein G HP (GE Healthcare). The blocking mAb to CD200R3 (6A5A8-F7, rat IgG2b) was established by its binding to CD200R3-GFP-RBL2H3 cells and inhibition of the suppressive effect of CD200R3ext-huIgFc on CD4+T-cell activation. Generation of human IgFc fusion protein
The DNA sequences containing the extracellular domain lacking the signal sequence of Cd200 , Cd200r1, Cd200r2, Cd200r3, and Cd200r4 were amplified by PCR as described above with a pair of specific primers for Cd200 (5′-tc gaa ttc gca agt gga agt ggt gac cca gga tc-3′ and 5′-ctt aga tct tcc ttt gtc cag act ctg ctt gta g-3′), Cd200r1 (5′-tc gaa ttc gac tga taa gaa tca aac aac aca g-3′ and 5′-ctt cca tgg ctg gtc gta atg att ggt tac cac c-3′), Cd200r2 (5′-tc gaa ttc ggt gta cac cat agt gtc tgt aca g-3′ and 5′-ctt cca tgg cct gga aga aag caa atc cca ctt taa g-3′), Cd200r3 (5′-cgc gat atc gag ttg ttc agt gaa agg a-3′ and 5′-cgc gat atc tta ggc agg atg ctg gtt gt-3′), and Cd200r4 (5′-tc gaa ttc gag ttg tac tga tga gaa tca aac aat-3′ and 5′-ctt cca tgg cac ggg ggg tgg tca ttg tac ctt g-3′), and each of the 5'- and 3'-primers were also tagged (indicated in italics) with EcoRI and BglII sites for Cd200 , EcoRI and NcoI sites for Cd200r1, Cd200r2, and Cd200r4, or an EcoRV site for Cd200r3. The PCR product was subcloned into pCR4-TOPO and the nucleotide sequence was confirmed as described above. After restriction enzyme digestion, the DNA sequence was cloned into the sites of pFUSE-hIgG2-Fc2 (InvivoGen, San Diego, CA), and transfected into 293T cells using FuGENE 6 Transfection Reagent (Roche). huIgFc, CD200ext-huIgFc, CD200R1ext-huIgFc, CD200R2ext-huIgFc, CD200R3ext-huIgFc, or CD200R4ext-huIgFc fusion protein purified from the culture supernatant using HiTrap™ Protein G HP.
Files in this Data Supplement:
- Figure S1. Generation of mAbs to CD200Rs (JPG, 59.3 KB)
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Specific reactivity of mAbs to CD200Rs against CD200Rs-GFP-RBL2H3 cells was analyzed by flow cytometry, and data are represented by a dot plot. Data are representative of 4 replicate experiments.
- Figure S2. Suppressive effect of CD200R3ext-huIgFc on the Ag-specific CD4+T-cell response (JPG, 42 KB)
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(A) Binding of CD200ext-huIgFc to CD200-GFP-RBL2H3 cells or CD200Rs-GFP-RBL2H3 cells (upper panel), and CD200ext-huIgFc or CD200Rsext-huIgFc to CD200-GFP-RBL2H3 cells (lower panel) was analyzed by flow cytometry, and data are represented by a histogram. Data are representative of 4 replicate experiments. (B) Rag2−∕− KJ1-26+T cells (5 × 104) were cultured with OVAp/BM-mDCs (5 × 103) in the presence or absence of huIgFc, CD200ext-huIgFc, CD200Rsext-huIgFc or CTLA-4ext-huIgFc (5 µg/ml) for 3 days, and the proliferative response was measured. *P<0.01 compared with huIgFc. Data were expressed as mean ± SEM, and the results are combined from 4 replicate experiments.
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