Immunomodulatory oligonucleotides
a technology of immunomodulatory oligonucleotides and oligonucleotides, which is applied in the field of immunomodulatory oligonucleotides, can solve the problems of poly(i,c), and achieve the effect of increasing the number of igm secreting b cells and similar increases in response to cpg-odn
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example 1
Effects of ODNs on B Cell Total RNA Synthesis and Cell Cycle
[0097] B cells were purified from spleens obtained from 6-12 wk old specific pathogen free DBA / 2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4+ or CD8+ cells. 8×104 B cells were dispensed in triplicate into 96 well microtiter plates in 100 μl RPMI containing 10% FBS (heat inactivated to 65° C. for 30 mL), 50 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 ug / ml streptomycin, and 2 mM L-glutamate. 20 μM ODN were added at the start of culture for 20 h at 37° C., cells pulsed with 1 μCi of 3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ELISA spot assay after culture of whole spleen cells with ODN at 20 μM for 48 hr. ...
example 2
Effects of ODN on Production of IgM from B Cells
[0100] Single cell suspensions from the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting B cells (J. Exp. Med. 155:1523 (1982). These were cultured as described above in 30 μM ODN or 20 μg / ml LPS for 48 hr. The number of B cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D. M. et al. J. Immunol. 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labelled anti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigrna Chemical Co., St. Louis Mo.) which forms an insoluble blue precipitate in the presence of phosphatase. The dilution of cells producing 20-40 spots / wel...
example 3
B Cell Stimulation by Bacterial DNA
[0101] DBA / 2 B cells were cultured with no DNA or 50 μg / ml of a) Micrococcus lysodeikticus; b) NZB / N mouse spleen; and c) NFS / N mouse spleen genomic DNAs for 48 hours, then pulsed with 3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNAse I for 30 minutes at 37 C prior to addition to cell cultures. E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISA-spot assay.
[0102] DBA / 2 B cells were cultured with either no additive, 50 μg / ml LPS or the ODN 1; 1a; 4; or 4a at 20 uM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10,20,40 or 80 μM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.
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