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Blood, Vol. 109, Issue 7, 2961-2967, April 1, 2007
TACI regulates IgA production by APRIL in collaboration with HSPG
Blood Sakurai et al.
109: 2961
Supplemental materials for: Sakurai et al, Vol 109, Issue 7, 2961-2967
Files in this Data Supplement:
- Table S1. APRIL- and BAFF-induced IgM production and viable cell number (PDF, 48.8 KB) -
B cells treated with control siRNA, TACI siRNA, or heparatinase (10 U/mL) were cultured in a 96-well plate (1.0 × 105/well) with anti-BCR antibodies (anti-Ig and anti-Igλ; 0.5 µg/mL each), CD40L (2 µg/mL), BAFF (4 µg/mL), APRIL (8 µg/mL), anti-TACI mAb (11H3) (5 µg/mL), or control mouse IgG2a (5 µg/mL), in the presence or absence of IL-4 (20 U/mL) and TGF- (1 ng/mL) for 10 days. IgM concentration was measured by ELISA. Viable cell number was determined by trypan blue exclusion method. Data are presented as mean ± SD and are representative of 3 independent experiments with similar results.
- Figure S1. Characterization of mouse anti–human TACI mAb (11H3) (JPG, 40.4 KB)
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To construct a TACI expression vector, a cDNA encoding full-length TACI (GenBank no. AF023614), subcloned from human peripheral blood mononuclear cells using RT-PCR with linker primers, was inserted into pBluescript SK(+) at the EcoRI and BamHI sites and finally into the pBCMGSneo expression vector1 at the XhoI and NotI sites (pBCMGS-TACI). Murine pre-B cells (cell line 300-19) were electroporated with pBCMGS-TACI, and stable transfectants were selected by G418 treatment. Cells with high-density TACI were cloned by flow cytometry. Methods used to generate the hybridoma that produced TACI-specific antibody and 3× FLAG-soluble BAFF fusion protein have been described previously.2 Anti–human TACI mAb (clone 11H3, mouse IgG2a, IgG2) was established and purified from cultured supernatant with the use of a mAbTrap kit (Amersham Biosciences, Piscataway, NJ). (A) Flow cytometric analysis of TACI expression using 3× FLAG-soluble BAFF and 11H3 mAb. TACI and mock transfectants were stained with FLAG-tagged BAFF- and FITC-labeled anti–FLAG M2 antibody or 11H3 mAb and FITC-labeled anti–mouse immunoglobulins with the reference of isotype control IgG2a. (B) Mock or TACI transfectants (2 × 106 per well) were cultured with recombinant BAFF (5 µg/mL), 11H3 mAb (10 µg/mL), or isotype-matched IgG2a (10 µg/mL) for 30 minutes, and nuclear extracts were prepared to evaluate nuclear translocation of NF-B by immunoblot analysis, as described in “Materials and methods.”
- Figure S2. Expression levels of BCMA, BAFF-R, and CD40 on B cells are not altered by TACI siRNA or heparatinase (JPG, 37.4 KB)
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Control or TACI siRNA was transfected into human B cells. After 16-hour incubation, cells were stained with anti-BCMA, anti–BAFF-R, or anti-CD40 mAb in the presence or absence of heparatinase (10 U/mL). Stained cells were analyzed by flow cytometry. Data are representative of 3 independent experiments with similar results.
REFERENCES
1. Karasuyama H, Kudo A, Melchers F. The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain. J Exp Med. 1990;172:969-972.
2. Hase H, Kanno Y, Kojima M, et al. BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex. Blood. 2004;103:2257-2265.
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