Fig 2: TACI is required for T-independent antibody responses. (A) Mixed bone marrow chimera generation for studies of T-independent antibody responses. Bone marrow from Tnfrsf13b+/+ or Tnfrsf13b−/− littermates on the C57BL/6 (B6) background (Ly9.1−) was mixed 50:50 with bone marrow from WT 129S7 (129) mice (Ly9.1+), then injected into sublethally irradiated Rag1−/− mice, to generate WT:wt and KO:wt chimeras, respectively. 9 wk later, mice were injected i.p. with TNP-Ficoll or PBS as a control. 7 days after immunization, spleens and blood were harvested for analysis. Antibodies produced by C57BL/6 or 129S7 cells are of the Ighb or Igha allotype, respectively. (B) Stacked bar chart showing the percentages of B6 and 129 cells within the total splenic MZ B cell populations (B220+CD19+CD93−CD1dhiIgMhi) in WT:wt and KO:wt chimeras. Cells were analyzed by flow cytometry using the gating strategy shown in Fig. S2 G. Bars show the mean ±95% CI. (C) Proportion of MZ B cells (CD1dhiIgMb hi) within the B6 mature splenic B cell populations (Ly9.1−CD93−B220+CD19+CD138−) and cell numbers in WT:wt and KO:wt chimeras. Bars show the mean. Each point represents one mouse. Black: WT B6; red: TACI KO B6. (D) Stacked bar chart showing the percentages of B6 and 129 cells within the total splenic FO B cell populations (B220+CD19+CD93−CD1dmedIgM+) in WT:wt and KO:wt chimeras. Cells were analyzed by flow cytometry using the gating strategy shown in Fig. S2 G. Bars show the mean +95% CI. (E) Proportion of FO B cells (CD1dmedIgMb+) within the B6 mature splenic B cell populations (Ly9.1−CD93−B220+CD19+CD138−) and cell numbers in WT:wt and KO:wt chimeras. Bars show the mean. Each point represents one mouse. Black: WT B6; red: TACI KO B6. (F) TNP-specific IgMb and IgMa antibody responses in WT:wt and KO:wt chimeras 7 days after immunization with TNP-Ficoll or PBS control. IgMb originates from B6 B cells (black: WT; red: TACI KO), whereas IgMa originates from WT 129 B cells (gray). Antibody levels in serum were determined by ELISA and measured as absorbance at 450 nm (A450). Bars show the mean. Each point represents one mouse. (G and H) Percentage and numbers of B6 cells (Ly9.1−) within the splenic PCs (TCRβ−CD138+B220loCD19int-lo) (G) or PBs (TCRβ−CD138+B220hiCD19int) (H) in WT:wt and KO:wt chimeras 7 days after immunization with TNP-Ficoll or PBS control. Bars show the mean. Each point represents one mouse. Black: WT B6; red: TACI KO B6. (I) In vitro plasmablast differentiation assay. WT:WT and KO:WT mixed bone marrow chimeras were generated as in Fig. 1 B. MZ B cells (CD21hiCD23loCD93−CD138−CD19+) were purified from spleens by FACS 10 wk later, using the gating strategy shown in Fig. S3 A, then cultured with LPS for 3 days in vitro before analysis of generated plasmablasts. (J) Ratio of CD45.2+ to CD45.1+ PBs (CD138+B220lo) generated after 3 days of LPS stimulation of FACS-purified MZ B cells from WT:WT or KO:WT chimeras, normalized to the ratio of CD45.2+/CD45.1+ MZ B cells at day 0 (input). Cells were analyzed by flow cytometry as shown in Fig. S3 B. Bars show the mean. Each point represents one biological replicate. Statistical tests: unpaired t test (C, E, and J), two-way ANOVA with Fisher’s LSD test (B, D, and F–H). Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. Sample numbers: 5 (B–E, WT:wt; F–H, WT:wt +PBS; F, KO:wt +PBS IgMb only), 6 (B–E, KO:wt; F–H, WT:wt +TNP-Ficoll, KO:wt +PBS except for IgMb in F), 7 (F–H, KO:wt +TNP-Ficoll), 23 (J, WT:WT), 24 (J, KO:KO). Data are representative of two (B–H) or pooled from three (J) independent experiments. PCs, plasma cells; PBs, plasmablasts.

Fig 3: Analysis of plasmablast differentiation, NP-CGG immunization, EdU labeling, and adoptive transfer studies. (A) Gating strategy used to purify MZ B cells from the spleens of WT:WT and KO:WT chimeras by FACS for in vitro plasmablast differentiation assays. (B) Gating strategy used to identify CD45.2+ and CD45.1+ plasmablasts (CD138+B220lo) after 3 days of in vitro LPS activation of MZ B cells. (C) Gating strategy used to identify NP-specific CD45.2+ and CD45.1+ splenic GCBs (CD19+CD138−FAS+GL7hiIgD−CD38loNP+) 7 days after immunization of WT:WT and KO:WT chimeras with PBS or NP-CGG with alum. (D) Numbers of NP-specific CD45.2+ and CD45.1+ splenic GCBs in WT:WT and KO:WT chimeras, 7 days after immunization with PBS or NP-CGG with alum. Bars show the mean of n = 8 (WT:WT +PBS), n = 7 (KO:WT +PBS), or n = 10 (WT:WT and KO:WT +NP-CGG) mice, pooled from two independent experiments. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. Statistical test: two-way ANOVA with Šídák’s multiple comparisons test. Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. (E) Gating strategy used to identify EdU+ CD45.2+ FO and MZ B cells in splenocytes from WT:WT and KO:WT chimeras after continuous EdU labeling in vivo. (F) Proportion of EdU+ cells within T1 (IgDlo), T2 (IgD+IgM+), and T3 (IgDhiIgMlo) CD45.2+ immature splenic B cell populations (CD93+B220+CD19+) from WT:WT and KO:WT chimeras at 4 h, and 2, 3, and 5 days after EdU injection (performed at 12 wk after reconstitution) and EdU continuously administered via drinking water. Each point represents one mouse (4 h WT, n = 4; KO n = 5; 2, 3, and 5 days, n = 4). Lines connect the mean at each time point. Black: WT CD45.2+; red: TACI KO CD45.2+. No difference reported between WT and KO means at any time point as determined by two-way ANOVA with Šídák’s multiple comparisons test. (G) Gating strategy used to identify transferred CD45.2+ and CD45.1+ FO and MZ B cells in the spleens of CD45.2+CD45.1+ recipients, 7 days after adoptive transfer. (H) Gating strategy used to identify transferred CMFDA-labeled CD45.2+ and CD45.1+ FO and MZ B cells in the spleens of CD45.2+CD45.1+ recipients, 1 h after adoptive transfer. GCBs, germinal center B cells.

Fig 4: Analysis of mixed chimeras. (A) Gating strategy used to identify FO, MZ, T1, T2, and T3 B cells and PCs from the spleens of WT:WT and KO:WT chimeras. (B) Total number of MZ (CD1dhiIgMhi) and FO (CD1dmedIgM+) mature splenic B cells (CD93−B220+CD19+CD138−; CD45.2+ and CD45.1+ combined) in WT:WT and KO:WT chimeras. Bars show the mean of n = 16 mice, from four independent analyses. Each point represents one mouse. (C) ICOSL surface expression, measured by flow cytometry, on CD45.2+ and CD45.1+ splenic FO B cells (CD1dmedIgM+CD93−B220+CD19+) from WT:WT and KO:WT chimeras, quantified as the geometric MFI and normalized to the mean MFI of WT CD45.2+ cells within the same experiment. Bars show the mean of n = 6 mice, from two independent analyses. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+; gray: WT CD45.1+. (D) TACI surface expression, measured by flow cytometry, on TACI KO and WT CD45.2+ splenic PCs (TCRβ−CD138+CD19lo), MZ (CD93−CD1dhiIgMhi), FO (CD93−CD1dmedIgM+), T2 (CD93+IgD+IgM+) and T1 (CD93+IgDlo) B cells (B220+CD19+), and T cells (TCRβ+CD19−) from WT:WT and KO:WT chimeras. Left: Representative flow cytometry histograms showing TACI expression on each cell type. Filled: WT; unfilled: TACI KO. Right: TACI expression quantified as the geometric MFI. Bars show the mean of n = 5 mice, representative of two independent analyses. Each point represents one mouse. Black: WT CD45.2+; red: TACI KO CD45.2+. (E) Numbers of CD45.2+ and CD45.1+ splenic PCs (TCRβ−CD138+CD19lo) in WT:WT and KO:WT chimeras. Bars show the mean of n = 10 mice, from two independent analyses. Each point represents one mouse. Colors are as in C. (F) Numbers of CD45.2+ and CD45.1+ splenic T2 (CD93+IgD+IgM+) B cells (B220+CD19+CD138−) in WT:WT and KO:WT chimeras. Bars show the mean of n = 16 mice, from four independent analyses. Each point represents one mouse. Colors are as in C. (G) Gating strategy used to identify B6 (Ly9.1−) and 129 (Ly9.1+) FO B cells, MZ B cells, PCs (CD138+B220loCD19int-lo), and PBs (CD138+B220hiCD19int) from the spleens of WT:wt and KO:wt chimeras, for the data shown in Fig. 2. Statistical tests: unpaired t test (B), repeated-measures two-way ANOVA with Fisher’s LSD test (C, E, and F), unpaired Welch’s t test corrected for multiple comparisons using the Holm–Šídák method (D). Numbers above graphs indicate P values where P ≤ 0.05, otherwise no value is shown. PCs, plasma cells; PBs, plasmablasts; MFI, mean fluorescence intensity.

Fig 5: 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.