Fig. S3.

Noncognate immunizations decrease T_FR cells and up-regulate early expression of PD-L1 and PD-L2. Splenocytes harvested from C57BL/6 mice immunized i.v. with 0.5 μg NPαGalCer (NPαGC), 100 μg NP-KLH plus 0.5 μg αGalCer (αGC), or vehicle [PBS/0.1% (wt/vol) BSA] were labeled for FACS analysis to measure (A) T_FR (B220⁻TcRβ⁺CD4⁺CXCR5⁺PD-1⁺FoxP3⁺) and Treg (B220⁻TcRβ⁺CD4⁺CXCR5⁻PD-1⁻FoxP3⁺) cells and (B) iNKT_FR (B220⁻TcRβ⁺CD1dtet⁺CXCR5⁺PD-1⁺FoxP3⁺) and iNKTreg (B220⁻TcRβ⁺CD1dtet⁺CXCR5⁻PD-1⁻FoxP3⁺) cells (representative of two to three experiments; n = 4 mice per group). Splenic B cells were monitored for expression of (C) PD-L1 and (D) PD-L2 by mean fluorescence intensity (MFI). FACS analysis revealed numbers of B220⁺ naïve B cells that are not specific for NP (○) and B220⁺NP⁺Fas⁺GL7⁺ NP-specific germinal center B cells (■ and ▼) at days 4, 6, 8, and 12 after immunization [data represent two to four experiments (n = 8–16 mice) for days 4–8 and one experiment (n = 4 mice) for day 12]. *P ≤ 0.05.