TACI-deficient MZ B cells do not have a survival, homing, or localization defect. (A) Schematic of in vivo continuous EdU labeling. WT:WT and KO:WT mixed bone marrow chimeras were generated as in Fig. 1 B. 9 wk later, mice were injected i.p. once with EdU, and then, EdU was maintained in drinking water for 42 days. Spleens were harvested at 4 h, and 14, 28, and 42 days following EdU injection. (B) Proportion of EdU⁺ cells within MZ (CD1d^hiIgM^hi) and FO (CD1d^medIgM⁺) CD45.2⁺ mature splenic B cell populations (CD93⁻B220⁺CD19⁺) from WT:WT and KO:WT chimeras at 4 h, 14, 28, and 42 days after start of EdU treatment. Cells were analyzed by flow cytometry using the gating strategy shown in Fig. S3 E. Each point represents one mouse (4 h WT, n = 4; KO, n = 5; 14 days, n = 4; 28 and 42 days, n = 5). Lines connect the mean at each time point. Black: WT CD45.2⁺; red: TACI KO CD45.2⁺. (C) Schematic of adoptive transfer. WT:WT and KO:WT mixed bone marrow chimeras were generated as in Fig. 1 B. At least 10 wk after reconstitution, splenic B2 cells were harvested from chimeras and transferred by i.p. injection into CD45.1⁺CD45.2⁺ recipients. Spleens of recipient mice were harvested either 7 days or 1 h later. The ratio of CD45.2⁺/CD45.1⁺ cells recovered was normalized to the ratio of CD45.2⁺/CD45.1⁺ cells in the injected mix (input). (D) Ratio of CD45.2⁺ to CD45.1⁺ MZ B cells (CD1d^hiIgM^hiCD93⁻B220⁺CD19⁺) and FO B cells (CD1d^medIgM⁺CD93⁻B220⁺CD19⁺) recovered from the spleens of recipient mice 7 days after AT of B cells from WT:WT or KO:WT chimeras, normalized to the ratio of CD45.2⁺ to CD45.1⁺ MZ or FO B cells in the B cells that were transferred (input). Cells were analyzed by flow cytometry using the gating strategy shown in Fig. S3 G. Bars show the mean of n = 7 (WT:WT) and n = 8 (KO:WT) recipient mice. Each point represents one recipient mouse. (E) Viability of CD45.2⁺ and CD45.1⁺ MZ B cells (CD1d^hiIgM^hiCD93⁻CD138⁻B220⁺CD19⁺) from WT:WT or KO:WT chimeras after 24 or 48 h in vitro culture with or without 200 ng/ml BAFF, normalized to the mean viability in the absence of BAFF at 24 h within the same genotype and experiment. Bars show the mean of n = 8; each point represents a biological replicate. (F) Ratio of CD45.2⁺ to CD45.1⁺ MZ B cells (CD1d^hiIgM^hiCD93⁻B220⁺CD19⁺) and FO B cells (CD1d^medIgM⁺CD93⁻B220⁺CD19⁺) recovered from the spleens of recipient mice 1 h after adoptive transfer of B cells from WT:WT or KO:WT chimeras, normalized to the ratio of CD45.2⁺ to CD45.1⁺ MZ or FO B cells in the B cells that were transferred (input). Cells were analyzed by flow cytometry using the gating strategy shown in Fig. S3 H. Bars show the mean of n = 5 (WT:WT) and n = 6 (KO:WT) recipient mice. Each point represents one recipient mouse. (G) Flow cytometry histograms comparing in vivo labeling of splenic TACI KO and WT MZ and FO CD45.2⁺ B cells from WT:WT and KO:WT chimeras 5 min after i.v. injection of anti-CD19-PE antibody. Red: TACI KO CD45.2⁺ MZ; black: WT CD45.2⁺ MZ; blue: WT CD45.2⁺ FO; orange: KO CD45.2⁺ FO; filled light and dark gray: background staining of MZ and FO CD45.2⁺CD45.1⁺ B cells, respectively, spiked in during ex vivo homogenization of spleens. (H) Percentage of splenic TACI KO and WT MZ and FO CD45.2⁺ B cells labeled in vivo with anti-CD19-PE 5 min after i.v. injection of the antibody into WT:WT and KO:WT chimeras. Bars show the mean of n = 9 (WT:WT) and n = 7 (KO:WT) mice. Each point represents one mouse. Statistical tests: two-way ANOVA with Šídák’s multiple comparisons test (B and E), unpaired t test (D, F, and H). Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. Data are pooled from four (B) or two independent experiments (D–F and H). AT, adoptive transfer.