TACI deficiency impairs sustained Blimp-1 expression in B cells decreasing long-lived plasma cells in the bone marrow by Shoichiro Tsuji, Catarina Cortesão, Richard J. Bram, Jeffrey L. Platt, and Marilia Cascalho Blood Volume 118(22):5832-5839 November 24, 2011 ©2011 by American Society of Hematology

TACI deficiency decreased serum IgM, IgG, and IgA and impaired antigen-specific IgM production in response to a protein antigen. TACI deficiency decreased serum IgM, IgG, and IgA and impaired antigen-specific IgM production in response to a protein antigen. (A) Sera IgM, IgG1, IgG3, IgG2b, and IgA quantified by ELISA. Sera were obtained from nonimmunized C57BL/6-TACI-wt (TACI-wt) or C57BL/6-TACI-ko (TACI-ko) mice. Concentrations were calculated by comparison with standard curves obtained with known concentrations of the respective isotypes. (B-C) Concentrations of NP-specific IgM (B) or IgG (C) determined by ELISA in the sera of C57BL/6-TACI-wt (TACI-wt) or C57BL/6-TACI-ko (TACI-ko) mice immunized with 100 μg NP-OVA 21 days earlier. TACI deficiency decreased NP-specific IgM but not NP-specific IgG. Concentrations were calculated by comparison with standard curves obtained with known concentrations of NP-specific monoclonal antibodies. (D-E) Concentrations of NP-specific IgM (D) or IgG1 (E) determined by ELISA in the sera of QM-TACI-wt or QM-TACI-ko mice after immunization with 100 μg NP-OVA. TACI deficiency decreased NP-specific IgM production and accelerated the production of NP-specific IgG. Concentrations were calculated by comparison with standard curves obtained with known concentrations of NP-specific monoclonal antibodies. Each dot represents results obtained from one mouse. Averages were compared by t test. Significant differences are noted: *P < .05; **P < .01. Shoichiro Tsuji et al. Blood 2011;118:5832-5839 ©2011 by American Society of Hematology

TACI deficiency does not impair B-cell activation and germinal center formation. TACI deficiency does not impair B-cell activation and germinal center formation. (A) The proportion of NP-specific B cells (idiotype-positive) in the spleen of QM-TACI-wt or QM-TACI-ko mice was analyzed by flow cytometry in mice immunized 10 days earlier. Splenocytes were stained with an anti–idiotypic antibody10 and with an anti–IgMa antibody that detects the allotype produced by the heavy chain-targeted allele. (B) Average proportions (above) or average number (below) of idiotype-positive cells obtained from 3 QM-TACI-wt or 3 QM-TACI-ko mice. Averages were compared by t test. Significant differences: *P < .05; **P < .01. (C) TACI deficiency does not decrease B-cell activation after immunization. Splenocytes were obtained from QM-TACI-wt or QM-TACI-ko mice immunized with 100 μg NP-OVA 10 days earlier. Splenocytes were stained with anti-CD19 and CD69 antibodies and analyzed by FACS. Activated B cells (CD19+, CD69+) were as frequent in TACI-proficient as in TACI-deficient mice. Average proportions of CD19+, CD69+ cells obtained from 3 QM-TACI-wt, or 3 QM-TACI-ko mice. Averages were compared by t test. Differences between groups were not significant. (D) TACI deficiency caused increases in size and number of germinal centers 10 days after immunization. Frozen sections of Peyer patches were stained with anti–GL7 antibody (brown), which specifically labels germinal center B cells. The figure is representative of 3 independent experiments. Digital images were obtained by Leica DM6000 B microscope (Leica) and with QCapture Pro Version 6.0.0 software (QImaging). Shoichiro Tsuji et al. Blood 2011;118:5832-5839 ©2011 by American Society of Hematology

TACI is required for differentiation of B cells into ASCs TACI is required for differentiation of B cells into ASCs. TACI-ko animals had decreased NP-specific IgM ASCs or NP-specific IgG ASCs compared with wild-type. TACI is required for differentiation of B cells into ASCs. TACI-ko animals had decreased NP-specific IgM ASCs or NP-specific IgG ASCs compared with wild-type. ASCs were enumerated in B cells isolated from the spleen (A-B) or in bone marrow cell suspensions (C-D) of C57BL/6-wt or C57BL/6-TACI-ko 21 days after immunization, by ELISPOT. Below the graphs, the diagrams show the ELISPOT wells from which the ASCs were enumerated and plated with 106, 5 × 105, or 105 cells, as indicated. Values were compared by t test. Significant differences: *P < .05; **P < .01. Data resulted from the analyses of 5 wild-type mice and 4 TACI-ko mice. Shoichiro Tsuji et al. Blood 2011;118:5832-5839 ©2011 by American Society of Hematology

TACI deficiency decreases production of ASCs in a B cell–autonomous manner. TACI deficiency decreases production of ASCs in a B cell–autonomous manner. (A-B) The number of ASCs was restored by adoptive transfer of TACI-wt B cells but not by transfer of TACI-wt T cells into TACI-deficient recipients. NP-specific IgM (A) or NP-specific IgG (B) ASCs were enumerated by ELISPOT of splenocytes 14 days after immunization, and after reconstitution by adoptive transfer of QM-TACI-wt or QM-TACI-ko B cells, and C57BL/6-wt or C57BL/6-TACI-ko T cells. Representative pictures of the ELISPOT wells are shown below each graph. Transfer efficiency was equivalent in all experiments and determined by enumerating IgMa-positive donor B cells by FACS analysis in the spleen of recipients at the time of analysis. The number of IgM-secreting cells was significantly decreased whenever reconstitution was done with TACI-ko B cells: *P = .016 (2-way ANOVA). The number of IgG-secreting cells in animals reconstituted with TACI-ko B cells and TACI-wt T cells was significantly decreased compared with animals reconstituted with TACI-wt B and T cells: *P = .041 (t test). Data resulted from analysis of 3 recipient mice for each type of transfer. Shoichiro Tsuji et al. Blood 2011;118:5832-5839 ©2011 by American Society of Hematology

TACI is necessary and sufficient to induce Blimp-1 in B cells. TACI is necessary and sufficient to induce Blimp-1 in B cells. (A-B) TACI deficiency abrogates Blimp-1 expression by B cells 14 days after immunization. Blimp-1 mRNA expression was detected by RT-PCR (A) and quantified by real-time PCR (B). Relative expression of Blimp-1 was calculated by determining the ratio of Blimp-1/β-actin in immunized B cells compared with the Blimp-1/β-actin ratio in B cells from nonimmunized mice. Results represent the mean ± SEM of 3 measurements/mouse of each of 4 mice/genotype. Values were compared by t test and found to be significantly different: ***P < .0001. (C-D) Expression of TACI is sufficient to induce Blimp-1. The 18.81 B cells were transfected with a vector encoding TACI and EGFP from a single bicistronic mRNA or with a control vector encoding only EGFP. RNA was isolated from sorted GFP-positive transfected cells, 24 hours after transfection and converted to cDNA. Blimp-1 mRNA expression was studied by RT-PCR, after 30 PCR cycles (C) or quantitative RT-PCR (D). Blimp-1 expression by 18.81 B cells expressing TACI was calculated relative to the Blimp-1 expression by 18.81 cells transfected with a GFP control vector. Results represent mean ± SEM of 3 separate measurements. Values were compared by t test and found to be significantly different: *P = .0103. (E) Blimp-1 and TACI expression decreased and BCL-6 expression increased with time in 18.81 B cells transfected with a TACI expression vector. The 18.81 B cells transfected with TACI and EGFP or with EGFP control vectors were cultured up to 3 days with or without 100 ng/mL APRIL, as indicated. TACI, BCL-6, and Blimp-1 mRNA expression was studied by RT-PCR, after 30 PCR cycles. Cells were collected at 0, 24, 48, and 72 hours in culture. Results are representative of 3 independent experiments. Shoichiro Tsuji et al. Blood 2011;118:5832-5839 ©2011 by American Society of Hematology

Ionizing radiation induces Blimp-1 expression independently of TACI Ionizing radiation induces Blimp-1 expression independently of TACI. (A-B) X-ray irradiation of 18.81 cells (A) or C57BL/6-TACI-ko cells (B) induces γH2AX foci, indicating accumulation of DNA double-strand breaks. Ionizing radiation induces Blimp-1 expression independently of TACI. (A-B) X-ray irradiation of 18.81 cells (A) or C57BL/6-TACI-ko cells (B) induces γH2AX foci, indicating accumulation of DNA double-strand breaks. Cytospin slides of nonirradiated (0 Gy) or irradiated (4 Gy) cells were stained with an anti-γH2AX antibody (top diagrams) and counterstained with 4,6-diamidino-2-phenylindole (bottom diagrams). (C) Irradiation of 18.81 B cells with 1 Gy or 4 Gy induces Blimp-1 expression transiently, detected by quantitative RT-PCR. Relative expression of Blimp-1 was calculated by determining the ratio Blimp-1/β-actin for each sample. Values reflect 3 independent measurements. (D) Irradiation induces Blimp-1 expression independently of TACI. TACI-ko B cells, isolated from C57BL/6-TACI-ko mice, were irradiated with 4 Gy and cultured with 15 μg/mL of LPS for 24 hours. Blimp-1 expression increased after irradiation of TACI-deficient B cells. RNA was obtained after 24 hours in culture and expression of Blimp-1 or β-actin analyzed by quantitative RT-PCR. Relative expression of Blimp-1 was calculated by determining the ratio Blimp-1/β-actin for each sample. Values reflect 3 independent measurements. Averages were compared by t test. Significant differences were noted: *P < .05, **P < .01, and ***P < .001. Shoichiro Tsuji et al. Blood 2011;118:5832-5839 ©2011 by American Society of Hematology