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Toll-Like Receptor 4-Dependent Activation of Dendritic Cells by Greek Letter Beta-Defensin 2
Arya Biragyn, Pier Adelchi Ruffini, Cynthia A. Leifer, Elena Klyushnenkova, Alexander Shakhov, Oleg Chertov, Aiko K. Shirakawa, Joshua M. Farber, David M. Segal, Joost J. Oppenheim, and Larry W. Kwak

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Fig. S1. Schema of protein constructs used. All recombinant proteins contained a short c-Myc tag and six His residues (Tag) fused at the carboxyl-terminus. Several different variants of mDF2Greek Letter Beta were tested; mDF2Greek Letter Beta alone (N21mDF2Greek Letter Beta), or fusion proteins N2mDF2Greek Letter Beta or N24mDF2Greek Letter Beta of mDF2Greek Letter Beta with murine single-chain antibodies (sFv38 or sFv315, nonimmunogenic tumor idiotypes cloned from murine 38C13 or MOPC315 B cell tumors, respectively). Control proteins consisted of sFv alone (sFv315), or sFv fused with functionally active murine Greek Letter Beta-defensin 3 (mDF3Greek Letter Beta), or MCP-3 (MCP3), or with a naturally inactive murine pro�Greek Letter Beta-defensin 2 (mproDF2Greek Letter Beta). The chemoattractant moiety was separated from sFv with an 11�amino acid. spacer peptide (SP).

Figure S1

Fig. S2. A representative experiment of dot plot of expression of CD40 and B7.2 in CD11c+ cells. Proportion of triple-positive cells for CD11+/CD40+/B7.2+ is shown (%). Cells were stained after 18 hours of incubation in culture medium alone (no treatment), with 5 Greek Letter Mug/ml N2mDF2Greek Letter Beta or mproDF2Greek Letter Beta, or with 1 ng/ml or 10 ng/ml LPS, respectively.

Figure S2

Fig. S3. (A) DCs treated with 5 Greek Letter Mug/ml of various mDF2Greek Letter Beta containing recombinant proteins (N2mDF2Greek Letter Beta, N24mDF2Greek Letter Beta and N21mDF2Greek Letter Beta, see Fig. S1) induced comparable activation of iDC, as judged by increase in proportion of CD11+/CD40+/B7.2+ cells. The control DCs were left untreated (CM) or incubated with 5 Greek Letter Mug/ml sFv alone or fused with pro�Greek Letter Beta-defensin (mproDF2Greek Letter Beta), murine Greek Letter Beta-defensin 3 (mDF3Greek Letter Beta), MCP-3 (MCP3), or 10 ng/ml LPS. To confirm specificity, iDCs were incubated with supernatants from the N2mDF2Greek Letter Beta, N24mDF2Greek Letter Beta, or mproDF2Greek Letter Beta samples pretreated with 9E10 mAb, specific for Myc tag, coupled with protein A-Sepharose beads (N2mDF2Greek Letter Beta*, N24mDF2Greek Letter Beta* or mproDF2Greek Letter Beta*, pretreated with mAb, repeated twice). ***P < 0.001 is for comparison of pooled data between treated with mAb and untreated mDF2Greek Letter Beta. Pooled data are from five independent experiments. mDF2Greek Letter Beta activated iDCs were isolated from both BALB/c (B) and C57/BL6 (C) strains of mice, which cannot be inhibited by treatment with 5-20 Greek Letter Mug/ml RsDPLa (mDF2Greek Letter Beta+RsDPLa). In contrast, the RsDPLa treatment completely abrogated LPS-induced DC maturation (LPS+RsDPLa). The experiment was repeated three times. (D) mDF2Greek Letter Beta-matured DCs produce proinflammatory cytokine IL-6. Conditioned media from DCs incubated for 18 hours with N24mDF2Greek Letter Beta, or mproDF2Greek Letter Beta with or without proteinase K (PK), or boiled mDF2Greek Letter Beta (mDF2name+boil) were measured by ELISA. Control groups were treated with 10 ng/ml LPS (boiled or not boiled) with or without PK pretreatment. Representative data from three independent experiments.

Figure S3