Figure 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. Refer to the image caption for details. Panel A: A schematic diagram illustrates the experimental setup for generating mixed bone marrow chimeras, where bone marrow from different donor genotypes is combined and transferred into recipient mice to assess B cell development and responses. Panel B: A stacked bar graph shows the percentage distribution of marginal zone (MZ) and follicular (FO) mature splenic B cells in WT:WT and KO:WT chimeras. The y-axis represents the percentage of B cells, and the x-axis shows the chimera types. Panel C: Bar graphs show ICOSL (Inducible T cell Co-Stimulator Ligand) surface expression on splenic FO B cells from WT:WT and KO:WT chimeras. The y-axis represents normalized geometric mean fluorescence intensity (MFI), and the x-axis shows chimera types. Panel D: A stacked bar graph shows the percentage of follicular (FO) B cells in WT:WT and KO:WT chimeras. The y-axis represents percent FO, and the x-axis shows chimera types. Panel E: Bar graphs show the number of splenic plasma cells in WT:WT and KO:WT chimeras. The y-axis represents cell numbers, and the x-axis shows chimera types. Panel F: Scatter plots (with bars) show antigen-specific antibody responses, including anti-TNP IgM and anti-TNP IgG1 levels, following immunization. The y-axis represents absorbance (A450), and the x-axis shows experimental conditions. Panel G: Bar graphs show the frequency and number of B6 plasma cells (PC) in WT:WT and KO:WT chimeras after immunization. The y-axis represents percentage or cell number, and the x-axis shows chimera types. Panel H: Bar graphs show the frequency and number of B6 plasmablasts (PB) in WT:WT and KO:WT chimeras. The y-axis represents percentage or cell number, and the x-axis shows chimera types. Panel I: A schematic diagram illustrates the adoptive transfer or chimera-based experimental design involving different donor cell populations and recipients, including gating strategy setup for downstream analysis. Panel J: A bar graph shows CD45.2 to CD45.1 ratios in plasmablasts (PB) after stimulation (e.g., LPS treatment). The y-axis represents normalized ratios, and the x-axis shows chimera groups.

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.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal