Fig 1: 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 (CD1dhiIgMhi) and FO (CD1dmedIgM+) 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 (CD1dhiIgMhiCD93−B220+CD19+) and FO B cells (CD1dmedIgM+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 (CD1dhiIgMhiCD93−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 (CD1dhiIgMhiCD93−B220+CD19+) and FO B cells (CD1dmedIgM+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.
Fig 2: Analysis of the BAFFR interactome and TACI-deficient mice. (A) Left, diagram of the Tnfrsf13cem1Tyb allele showing exons 1–3 (E1–E3); filled boxes indicate coding sequence; open boxes indicate untranslated regions. Two Strep-tag II sequences were inserted after the last coding codon in E3, before the stop codon. Right, diagram of a BAFFR-Twin-Strep-tag trimer showing the location of the affinity tag at the C terminus of the protein. (B) Volcano plots of proteins identified by mass spectrometry in BAFFR-Twin-Strep-tag APs from B cells activated with LPS, either resting or after stimulation with BAFF for 15 min. The plots show the log2FC in abundance of each protein in BAFFR APs compared with control APs from WT cells (log2FC(BAFFR v control)) or in stimulated BAFFR APs compared with resting BAFFR APs (log2FC(BAFFR +BAFF v BAFFR resting)) from three independent experiments, and the –log10p value was determined by two-tailed Student’s t tests. The thresholds used to determine specific BAFFR interactors (upper right quadrant) are drawn at twofold enrichment and P = 0.05. The full list of proteins identified is in Table S1. (C and D) Numbers (C) and proportions (D) of FO (CD1dmedIgM+) and MZ (CD1dhiIgMhi) mature splenic B cells (CD93−B220+CD19+) in Tnfrsf13b+/+ and Tnfrsf13b−/− mice. Bars show the mean of n = 7 mice, from two independent analyses. Each point represents one mouse. (E) ICOSL surface expression, measured by flow cytometry, on splenic FO B cells (CD1dmedIgM+CD93−B220+CD19+) from Tnfrsf13b+/+ and Tnfrsf13b−/− mice, quantified as the geometric MFI and normalized to the mean MFI of Tnfrsf13b+/+ (WT) FO B cells within the same experiment. Bars show the mean of n = 7 mice, from two independent analyses. Each point represents one mouse. Statistical tests: unpaired t test. Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. MFI, mean fluorescence intensity; FC, fold change; AP, affinity purification.
Fig 3: TACI deficiency leads to reduced MZ B cells. (A) Concentration of BAFF in the serum of Tnfrsf13b+/+ and Tnfrsf13b−/− mice. Bars show the mean. Each point represents one mouse. (B) Mixed bone marrow chimera generation. Bone marrow from Tnfrsf13b+/+ or Tnfrsf13b−/− littermates (expressing the CD45.2+ allele) was mixed 50:50 with bone marrow from WT CD45.1+ mice, then injected into sublethally irradiated Rag1−/− mice. B cells were analyzed at least 8 wk later. (C) Concentration of BAFF in the serum of WT:WT and KO:WT chimeras. Bars show the mean. Each point represents one mouse. (D) Stacked bar charts showing the percentages of CD45.2+ and CD45.1+ cells within the splenic MZ (CD93−CD1dhiIgMhi), FO (CD93−CD1dmedIgM+), and T2 (CD93+IgD+IgM+) B cell (B220+CD19+) populations in WT:WT and KO:WT chimeras. Cells were analyzed by flow cytometry using the gating strategy in Fig. S2 A. Bars show the mean ±95% CI. (E) Stacked bar chart showing the percentages of CD45.2+ and CD45.1+ cells within the splenic PC population (TCRβ−CD138+CD19lo) in WT:WT and KO:WT chimeras. Bars show the mean ±95% CI. (F) Representative flow cytometry plots showing the proportions of MZ and FO B cells within the CD45.2+ and CD45.1+ mature splenic B cell populations (CD93−B220+CD19+CD138−) in WT:WT and KO:WT chimeras. MZ and FO cells are gated via two different approaches. Numbers indicate the percentage of cells in that gate. (G and H) Proportions (G) and numbers (H) of MZ (CD1dhiIgMhi) and FO (CD1dmedIgM+) cells within the CD45.2+ and CD45.1+ mature splenic B cell populations (CD93−B220+CD19+CD138−) in WT:WT and KO:WT chimeras. Bars show the mean. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. Statistical tests: Welch’s t test (A and C), two-way ANOVA with Fisher’s LSD test (D and E), repeated-measures two-way ANOVA with Fisher’s LSD test (G and H). Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. Sample numbers: 6 (A and D), 11 (C), 5 (E), 16 (G and H). Data are pooled from two experiments representative of four experiments (D), one out of two experiments (E), and pooled from six (A), five (C) and four (G and H) independent experiments. PC, plasma cell.
Fig 4: AKT-mTORC1 signaling is impaired in TACI KO MZ B cells. (A) Flow cytometry histograms comparing pAKT-T308 levels in TACI KO CD45.2+, WT CD45.2+, and WT CD45.1+ resting MZ B cells at 37°C from WT:WT and KO:WT chimeras. Red: TACI KO CD45.2+ MZ; black: WT CD45.2+ MZ; gray: WT CD45.1+; dashed lines: cells treated with the PI3Kδ inhibitor idelalisib (PI3Ki). (B) pAKT-T308 MFI, measured by flow cytometry, in CD45.2+ and CD45.1+ resting MZ B cells from WT:WT and KO:WT chimeras. WT:WT, n = 4; and KO:WT, n = 5. Lines connect cells from the same sample. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. (C) Flow cytometry histograms comparing pAKT-S473 levels in TACI KO CD45.2+, WT CD45.2+, and WT CD45.1+ resting MZ B cells at 37°C from WT:WT and KO:WT chimeras. Red: TACI KO CD45.2+ MZ; black: WT CD45.2+ MZ; gray: WT CD45.1+; dashed lines: cells treated with the PI3Kδ inhibitor idelalisib (PI3Ki). (D) pAKT-S473 MFI, measured by flow cytometry, in CD45.2+ and CD45.1+ resting MZ B cells from WT:WT and KO:WT chimeras; n = 4. Lines connect cells from the same sample. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. (E) Flow cytometry plots and histograms comparing pS6-S235/236 levels in WT and TACI KO CD45.2+ resting MZ B cells at 37°C from WT:WT and KO:WT chimeras. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT cells stained with isotype control antibody (Isotype). (F) Percentage of pS6+ cells (left) and pS6 MFI within pS6+ cells (right), measured by flow cytometry, in CD45.2+ and CD45.1+ resting MZ B cells from WT:WT and KO:WT chimeras; n = 5. Lines connect cells from the same sample. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. (G) Immunoblot anti-TACI immunoprecipitation (IP) from LPS-activated B cells, alongside a control IP using isotype control antibody (IC) and the input cell lysates. Cells were stimulated with APRIL, BAFF, or media-only control for 15 min at 37°C. The immunoblot was probed with anti-TACI, anti-p110δ, anti-p85α, anti-mTOR, and anti-MyD88. The same membrane sections are displayed at two different image intensities for p110δ, p85α, and mTOR due to higher protein levels in the input lysates. Statistical test: repeated-measures two-way ANOVA with Fisher’s LSD test (B, D, and F). Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. Data are from one of four (B and F) or two (D and G) independent experiments. Source data are available for this figure: SourceData F6.
Supplier Page from Abcam for Mouse BAFF ELISA Kit (TNFSF13B)