(a–d) Mice were treated with 0.5 µg DT and immunized with 50 µg papain and 5 µg OVA in the footpad. At the time of immunization, different DC subsets were either intact (+) or depleted (∆) depending on the host genotype as indicated in b. All mice received an i.p. injection of 10 µg OVA in PBS without adjuvant on day 14. Serum OVA-specific IgG1 on day 21 was detected by ELISA. Bars indicate mean ± S.E.M. calculated from 3–6 individual mice in each group. Representative data from 2–3 independent experiments are shown. (e) Mgl2-DTR and CD11c-DTR;Mgl2-DTR mice were immunized and boosted as in a, except that the mice were treated with a single dose of 125 ng DT on day −1. Sera were harvested on day 21 from three independent experiments and OVA-specific IgG1 was detected by ELISA. Bars indicate mean ± S.E.M. calculated from five (Mgl2-DTR) and three (Mgl2-DTR;CD11c-DTR) individual mice. n.s., not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Student’s t-test. All statistics indicate comparison to the undepleted (WT or no DT) control shown in each panel except in b and are shown by color-matched asterisks where applicable. (f) WT mice and mice that carry Mgl2-DTR, huLang-DTR and/or msLang-DTR in their genetic construct were treated with DT and immunized as in a, then LNs were harvested on day seven. Tfh and GC B cell percentages in dLNs. Data were pooled from 2–3 independent experiments per group. (g) Mgl2-DTR mice were treated with DT and immunized as in a. Mice received i.p. injections of 50 µg anti-Gr-1 (α-Gr1) or Rat IgG2a isotype control antibodies every other day starting on day two. Left non-draining (L) or right draining (R) popliteal LNs were harvested on day seven and analyzed for GC B and Tfh cells. Data were pooled from two individual experiments per group. Each dot indicates an individual mouse. n.s., not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-tailed Student’s t-test. Bars indicate means ± S.E.M.