Figure 2. Batf contributes to pro-allergic function of IL-4-expressing Tfh cells.

(a) Male WT and Batf KO mice (6–8 weeks old, n=5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum at 2 weeks interval. On day 14, mice were intranasally challenged with Ova followed by three more challenges at days 26, 27 and 28. Twenty-four hours after the last challenge mice were killed. Percentage of IL-4-expressing CD4⁺CD44^hiCXCR5^hiPD1^hi (Tfh) cells and ratio of percentage of IL-4-expressing Tfh cells to percentage of total Tfh cells from spleen and lung lymph nodes of WT and Batf KO asthmatic mice were analysed by flow cytometry. (b–f) Bone marrow chimera mice were generated and subjected to asthma as described in the Methods section. WT (CD45.1⁺) and Batf KO (CD45.2⁺) Tfh cells from the asthmatic mice were sorted and transferred into male C57BL/6 (CD45.2⁺) or B6.SJL (CD45.1⁺; 6–8 weeks old, n=4) mice, respectively. Twenty-four hours later the mice were challenged with Ova intranasally for 5 days and analysed. (b) BALF was analysed to measure airway infiltrating cells. (c) Expression of indicated cytokines in the lung was analysed by qRT–PCR analysis. Data were normalized to beta-actin gene. (d) Levels of Ova-specific IgGs in the serum were analysed by ELISA. (e) Spleen cells from the recipient mice were restimulated with Ova for 72 h and effector Th2 cytokines were analysed by ELISA. (f) Lung and lung lymph node cells were stained with Pacific blue-labelled CD45.1 mAb, PerCP-labelled anti-CD4 mAb, FITC-labelled anti-CD44 mAb and biotinylated anti-CXCR5 mAb, followed by APC-labelled streptavidin (BD Biosciences). Donor WT (CD45.1⁺) and Batf KO (CD45.2⁺) CD4⁺CD44^hi cells were assessed for CXRCR5 and IL-4 expression after restimulation with Ova for 24 h. Results shown are mean±s.e.m. and representative of at least two independent experiments. P values: *<0.05, **<0.01 and ***<0.001. Student's t-test was performed to detect between-group differences.