Experimental design (A) showing tamoxifen (TAM) treatment of day 90 PR8
flu memory
Tbx21fl/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
Tbx21fl/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+IgDneg 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
Tbx21fl/fl.hCD20-TAM-cre
flu memory mice.
(J-L) Enumeration of mdLN NP+ memory
(NP+CD38+IgDneg) B cells (J) 8 days
post-TAM treatment of
Tbx21fl/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 Tbx21fl/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.