Figure 7. ASC development after flu challenge infection requires T-bet⁺ memory B cells.

Experimental design (A) showing tamoxifen (TAM) treatment of day 90 PR8 flu memory Tbx21^fl/fl.hCD20-TAM-cre (B-M), hCD20-TAM-cre (J-M) and B6 (B-I) mice in order to inducibly delete T-bet from B cells in Tbx21^fl/fl.hCD20-TAM-cre mice. Mice analyzed (resting memory, B-F, J-L) 8 days following last TAM treatment or challenged with X31 influenza and analyzed 5 days later (memory recall, G-I, M).

(B-E) Enumeration of CXCR3⁺ mdLN cells following TAM treatment, showing flow plots (B, E) and frequency of CXCR3⁺ T cells (C), B cells (D) and NP⁺CD38⁺IgD^neg memory B cells (Bmem).

(F) Enumeration of NP-specific Bmem cells following TAM-treatment.

(G-I) Enumeration of mdLN CXCR3⁺ NP⁺ GCB and ASCs (G) and NP⁺ GCB (H) and ASCs (I) in day 5 primary X31 infected B6 mice and X31-challenged, TAM-treated B6 and Tbx21^fl/fl.hCD20-TAM-cre flu memory mice.

(J-L) Enumeration of mdLN NP⁺ memory (NP⁺CD38⁺IgD^neg) B cells (J) 8 days post-TAM treatment of Tbx21^fl/fl.hCD20-TAM-cre and hCD20-TAM-cre mice. Identification (K) and enumeration (L) of IgM, IgG 1, IgG2c and IgG2b expressing NP⁺ Bmem cells.

(M) Enumeration of total and IgG2c⁺ NP⁺ mdLN ASCs in TAM-treated memory Tbx21^fl/fl .hCD20-TAM-cre and hCD20-TAM-cre mice 5 days after X31 challenge.

Representative data from one of 2 (K-M) or 3 (B-J) independent experiments shown as the mean + SD of 3-6 mice/group. p values determined using one-way ANOVA (G (for NP⁺ ASCs), I) or Student’s t test (all others). *p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001 or “ns” not significant. “TFTC” too few to count (<10 cells /sample). See also Figure S7.