Figure 4. Blimp1 Deletion in T_FR Cells after Immunization Impairs T_FR Suppressive Activity.

(A) Schematic diagram of experimental protocol. Donor CD45.2⁺ Prdm1^fl/fliCre⁻ (WT), Prdm1^fl/fliCre⁺ (Blimp1-deleted; del), and CD45.1 ⁺ mice were immunized with NP-OVA in CFA for 7 days; tamoxifen was administered to WT and Del mice on day 6. On day 7, donors were euthanized and sorted CD45.2⁺ T_FR (CD4⁺CD3⁺PD-1⁺CXCR5⁺GITR⁺) along with CD45.1⁺ T_FH cells (CD4⁺CD3⁺PD-1⁺CXCR5⁺GITR⁻) were transferred into Tcra^−/− hosts followed by immunization with NP-OVA in CFA and tamoxifen administration daily from days 7 to 9. Spleens from euthanized hosts were analyzed on day 18.

(B) Histogram (upper) and MFI (bottom) of Blimp1 and Bcl6 expression in donor CD45.2⁺ T_FR cells after tamoxifen injection. Ctrl, T_FR cells from Prdm1^fl/flFoxP3^Cre (left), or Bcl6^fl/flFoxP3^Cre mice (right).

(C) Frequency of CD45.2⁺ T_FR (CD45.2⁺CD4⁺PD-1⁺CXCR5⁺FoxP3⁺), CD45.1⁺ T_FH (CD45.2⁻CD4⁺Bcl6⁺CXCR5⁺), and GC B cells (B220⁺CD19⁺GL7⁺Fas⁺).

(D and E) Expression (D) and quantification (E) of IL-17A, IFNγ, and IL-10 by donor CD45.2⁺ T_FR and ex-T_FR cells.

(F) WT and KO mice were immunized with NP-OVA in CFA. Seven days later, T_FR (WT versus KO: CD4⁺CD3⁺PD-1⁺CXCR5⁺YFP⁺) and non-T_FR (WT versus KO: CD4⁺CD3⁺CXCR5⁺YFP⁺) were sorted and transferred (10⁵/mouse) into Tcra^−/− hosts followed by immunization with NP-OVA in CFA; hosts were further challenged with NP-OVA in IFA at day 13. Spleens or mLNs were analyzed on day 20 (n = 3/group). Splenic (upper) or mLN (center) T_FR, T_FH, intracellular expression of IL-17A by donor T_FR and non-T_FR cells. At right, serum anti-NP₃₀ IgG, and anti-NP₄ IgG levels.

In (A)–(E), the data are representative of two independent experiments (B–E: n = 3–4/group). N.S., no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 (unpaired two-tailed Student’s t test). Error bars indicate means ± SEMs.

See also Figure S3.