Figure 8.

ICOS-L on B cells is required for Tfh-cell development and GC responses when Ag-presenting B cells or Ag are limited. (A) CD19^−/− mice received 1 × 10⁶, 0.5 × 10⁶, 0.25 × 10⁶ or 0.12 × 10⁶ NP-specific or NP-specific ICOS-L^−/− B cells co-transferred with 0.5 × 10⁶ CD4⁺ Thy1.1⁺ OT-II T cells, with spleens of recipients harvested 7 days after immunization with NP-OVA. Percentages of CD4⁺ Thy1.1⁺ CD44⁺ CXCR5^hi PD-1^hi T and B220⁺ IgD⁻ GL-7^hi CD95^hi B cells from animals receiving various numbers of NP-specific B cells are shown. (C-E) Representative flow cytometry plots of splenic CD4⁺ Thy1.1⁺ OT-II CD44⁺CXCR5^hiPD-1^hi or CD4⁺Thy1.1⁺CD44⁺CXCR5^hiBcl6^hi T cells, or B220⁺IgD^loGL-7^hiCD95^hi B cells. Experiments were performed 3 times with n ≥ 5 per group. (F) CD4⁺ Thy1.1⁺ OT-II cells were transferred into CD19-deficient mice, followed by immunization with 100 μg NP-OVA. Two days after priming, 0.05 × 10⁶ OT-II T cells were re-transferred, along with 1 × 10⁶ ICOS-L-intact or –deficient NP-specific B cells, into CD19^−/− animals primed 2 days earlier with 25 or 50 ug of NP-OVA, with spleens of recipients harvested 7 days after immunization. Representative flow cytometry plots of splenic CD4⁺ Thy1.1⁺ OT-II CD44⁺CXCR5^hiPD-1^hi Tfh cells with the percentages of such cells among CD4⁺ Thy1.1⁺ cells shown on the graph on the right. n = 3 mice per group. **p < 0.003; *p < 0.02 by Sidak's multiple comparison test comparing the means of Tfh (A and F) and GC B (B) cell percentages (of transferred cells) in recipient mice. Error bars represent standard deviation.