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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: J Immunol. 2012 Jun 25;189(3):1406–1417. doi: 10.4049/jimmunol.1200120

FIGURE 4.

FIGURE 4

Hyperresponsive and anergy phenotypes are not necessary for decreased diabetogenic activity of NR4 B cells. (A–D) To test hyperresponsiveness, B cells were purified from pooled spleens of three 6–8 week-old female NOD, NR4, NR4S1, NR4S2, NR4S3, NR4S4 and NR4S5 mice. Triplicate aliquots of 1×105 B cells were stimulated in culture with (A) 10µg/mL anti-IgM-F(ab’)2; (B) 10µg/mL anti-IgM-F(ab’)2 and 5µg/mL anti-CD40; (C) 5µg/mL anti-CD40 or; (D) 10µg/mL LPS. Proliferation over the final 24h of a 72h incubation period was measured by [3H]thymidine incorporation and is shown as a mean fold-change compared to NOD B cells (±SEM) in 2 to 4 independent experiments for each congenic strain. *p<0.05, **p<0.01, ***p<0.001 compared to NOD B cells (one-way ANOVA, Tukey post-test). (E–H) To test anergy induction, B cells were purified from pooled spleens of three 6–8 week-old female IgHEL single-tg or IgHEL/sHEL double-tg mice with NOD, NR4, (NOD×NR4)F1, (NR4S2×NR4)F1, (NR4S3× NR4)F1, (NR4S4×NR4)F1 or (NR4S5×NR4)F1 genetic backgrounds. Triplicate aliquots of 1×105 B cells were stimulated in culture with: (E) 10µg/mL anti-IgM-F(ab’)2; (F) 10µg/mL anti-IgM-F(ab’)2 and 5µg/mL anti-CD40; (G) 5µg/mL anti-CD40; or (H) 10µg/mL LPS. Proliferation over the final 24h of a 72h incubation period was measured by [3H]thymidine incorporation. Bars represent the mean fold-change in proliferation of B cells from IgHEL/sHEL double-tg mice compared to single IgHEL counterparts (±SEM) in 2 to 4 independent experiments for each subcongenic background, while the NOD, NR4 and (NOD × NR4)F1 background controls were performed 5, 4 and 8 times, respectively. *p<0.05 compared to NOD and (NOD × NR4)F1 background group (one-way ANOVA, Tukey post-test).