Constitutive CD40L expression on B cells prematurely terminates germinal center response and leads to augmented plasma cell production in T cell areas

Abstract

CD40/CD40L engagement is essential to T cell-dependent B cell proliferation and differentiation. However, the precise role of CD40 signaling through cognate T-B interaction in the generation of germinal center and memory B cells is still incompletely understood. To address this issue, a B cell-specific CD40L transgene (CD40LBTg) was introduced into mice with B cell-restricted MHC class II deficiency. Using this mouse model, we show that constitutive CD40L expression on B cells alone could not induce germinal center differentiation of MHC class II-deficient B cells after immunization with T cell-dependent Ag. Thus, some other MHC class II-dependent T cell-derived signals are essential for the generation of germinal center B cells in response to T cell-dependent Ag. In fact, CD40LBTg mice generated a complex Ag-specific IgG1 response, which was greatly enhanced in early, but reduced in late, primary response compared with control mice. We also found that the frequency of Ag-specific germinal center B cells in CD40LBTg mice was abruptly reduced 1 wk after immunization. As a result, the numbers of Ag-specific IgG1 long-lived plasma cells and memory B cells were reduced. By histology, large numbers of Ag-specific plasma cells were found in T cell areas adjacent to Ag-specific germinal centers of CD40LBTg mice, temporarily during the second week of primary response. These results indicate that CD40L expression on B cells prematurely terminated their ongoing germinal center response and produced plasma cells. Our results support the notion that CD40 signaling is an active termination signal for germinal center reaction.

FIGURE 1

Enhanced NF-κB activation in B cells of CD40LBTg mice. MZ and FO B cells from WT control or CD40LBTg mice (CD40L) were stimulated with anti-CD40 Ab (10 μg/ml) for the indicated time periods, and cell lysates were analyzed by Western blotting with specific Abs against pIκBα, IκBα, p100, and p52. A, Representative blot images with β-actin control staining. B, Fold change expression level of pIκBα, IκBα, p100, and p52 in MZ and FO B cells before (0 h) and after stimulation with anti-CD40 Ab at each time point, compared between WT control (●) and CD40LBTg (□) mice. Each protein expression level was normalized based on β-actin staining and compared with the level before stimulation (0 h: 1).

FIGURE 2

QRT-PCR analysis of genes responsible for B cell differentiation. Purified MZ B cells and FO B cells from control WT mice or CD40LBTg mice were cultured in vitro with a combination of IL-4, anti-IgM [F(ab′)₂], and anti-CD40 Ab or with CpGODN for 40 h and lysed for preparation of cDNA. QRT-PCR analysis was performed with an iQ5 cycler using primers for indicated genes. Statistical analysis of gene-expression profiles from at least three experiments using β-actin expression as internal control is shown.

FIGURE 3

B cell-specific MHC-II–deficient CD40L Tg mice as a model to study the effect of autonomous CD40/CD40L signaling in the absence of cognate T–B interaction. A, Possible T–B interaction of B cells from WT, CD40LBTg, IA-B, and IA-B/CD40LBTg mice. B, Representative FACS profiles of MHC-II (IA-b) expression on B220⁺ B cells from WT, CD40LBTg, IA-B, and IA-B/CD40LBTg mice at 8 wk of age. Mean fluorescent intensities of IAb expression for WT and CD40LBTg mice are shown. C, CD40L expression on MZ and FO B cells from IA-B mice (IAB) and IA-B/CD40LBTg mice (IAB/CD40L) was analyzed after stimulation with anti-CD40 Ab (10 μg/ml) for 18 h. Representative graphs of CD40L expression level with (shaded graph) or without (solid line) stimulation compared with isotype control (dotted line) are shown. D, Representative FACS profiles of B220⁺ B cells from WT, CD40LBTg, IA-B, and IA-B/CD40LBTg mice at 8 wk of age. The frequencies of T1 (CD21⁻CD23⁻), MZ (CD21^hiCD23⁻), and FO (CD21^loCD23⁺) cells among B220⁺ cells are shown.

FIGURE 4

Enhanced early, but impaired late, humoral response in CD40LBTg mice. A, Anti-NP IgM and IgG3 serum Ab end point titers in IA-B and IA-B/CD40LBTg mice were determined by ELISA at the indicated time points after immunization with NP-Ficoll. Averages of four mice per group are shown. B, Anti-NP IgM serum Ab end point titers in IA-B and IA-B/CD40LBTg mice were determined by ELISA at the indicated time points after immunization with NP-CGG plus alum or alum alone as control. Averages of at least three mice per group are shown. C, Anti-NP IgG1 serum Ab end point titers in control WT, CD40LBTg, IA-B, and IA-B/CD40LBTg mice were determined by ELISA at the indicated time points after immunization with NP-CGG plus alum. Averages of at least three mice per group are shown. D, Averages of anti-NP serum Ab affinity maturation of each group at 20 wk postimmunization. *p < 0.05.

FIGURE 5

Early termination of MHC-II–dependent germinal center response in IA-B/CD40LBTg mice. A, Percentage of NP-specific B cells with germinal center phenotype (lineage⁻B220⁺NP-binding CD38⁻ cells) in control WT, CD40LBTg, IA-B, and IA-B/CD40LBTg mice were determined by FACS at the indicated time points after immunization with NP-CGG plus alum. Data represent averages with standard deviations from at least three mice per group. B, Representative FACS profiles of early germinal center B cell and memory B cell differentiation in IA-B and IA-B/CD40LBTg mice after immunization with NP-CGG plus alum. Shaded graphs represent the MHC-II (IA-b) expression of NP-binding germinal center B cells, and open graphs represent the MHC-II (IA-b) expression of total lineage⁺B220⁺ naive B cells in each mouse. WT and CD40LBTg mice with a CD19 heterozygous background were used for this experiment.

FIGURE 6

Impaired generation of Ag-specific IgG1⁺ memory B cells and long-lived plasma cells in mice expressing B cell-specific CD40LTg. A, Numbers of NP-binding IgG1⁺B220⁺ cells and total IgG1⁺B220⁺ cells in spleens of control WT, CD40LBTg, IA-B, and IA-B/CD40LBTg mice at 20 wk after immunization with NP-CGG plus alum. At least 2 × 10⁶ events were collected for each analysis by FACS. Data represent averages with standard deviations from at least five mice per group. B, Numbers of NP-binding IgG1-secreting cells and IgG1-secreting cells in bone marrow of WT and CD40LBTg mice were measured by ELISPOT 20 wk after immunization with NP-CGG plus alum. Data represent averages with standard deviations from at least four mice per group. C, Ratio of NP-specific total (captured by NP15-BSA) and high-affinity (captured by NP2-BSA) IgG1-secreting cells in bone marrow of WT and CD40LBTg mice at 20 wk after immunization with NP-CGG plus alum. Data represent averages with standard deviations from at least five mice per group. WT and CD40LBTg mice with a CD19 heterozygous background were used for this experiment.

FIGURE 7

Effect of CD40L expression on germinal center B cell generation by adoptive-transfer experiment. A, MZ B cells or FO B cells from control WT (cd19 cre/⁺, MHC-II sufficient), IA-B (>95% MHC-II deficient), or IA-B/CD40LBTg mice (>95% MHC-II deficient with CD40L expression) were transferred into iab neo/neo cd19 cre/cre mice and immunized with NP-CGG plus alum 1 d after transfer. Among lineage⁻B220⁺ cells, NP-binding cells were analyzed for differentiation into PNA⁺CD38⁻ germinal center B cells. Representative FACS profiles of recipient mice at 7 d after immunization with NP-CGG plus alum are shown, with event counts in the germinal center PNA⁺CD38⁻ gate. A total of 10⁶ events were collected for each analysis. A FACS profile of an immunized recipient without B cell transfer is shown as a control. B, Frequencies of NP-binding lineage⁻PNA⁺CD38⁻ germinal center B cells in iab neo/neo cd19 cre/cre mice that received MZ B cells or FO B cells from control WT, IA-B, or IA-B/CD40LBTg mice are shown after day 7 immunization with NP-CGG plus alum. Each dot represents individual recipient mice. C, MZ B cells or FO B cells from QM mice on a WT and CD40LBTg background were transferred into CD45.1 B6 mice, which were subsequently immunized with NP-CGG plus alum 1 d after transfer. Data show representative FACS profiles of recipient mice at 7 d after immunization with NP-CGG plus alum from one of three similar experiments. The top panels show donor-derived CD45.2⁺ (R1 gate) NP-binding cells among live lymphocytes with the frequencies of each gated population. CD95⁺CD38⁻ germinal center B cell population in R1 was analyzed in the bottom panels. A total of 10⁶ events were collected for each analysis. D, Frequencies of CD45.2⁺ NP-binding CD95⁺CD38⁻ germinal center B cells in CD45.1 B6 mice that received MZ B cells or FO B cells from control WT or CD40LBTg mice are shown after day 7 immunization with NP-CGG plus alum. Each dot represents individual recipient mice. E, Frequencies of CD45.2⁺ NP-binding CD95⁺CD38⁻ germinal center B cells in CD45.1 B6 mice that received B cells from control WT (○) or CD40LBTg mice (●), or no B cells (■) are shown after day 5 and 7 immunization with NP-CGG plus alum. Each dot represents individual recipient mice. F, In the same experiment as in E, end point titers of donor-derived IgMa (white bars) or IgG1a (black bars) serum titers in CD45.1 B6 mice that received B cells from control WT or CD40LBTg mice or no B cells are shown after day 5 and 7 immunization with NP-CGG plus alum. Data represent averages with standard deviations.

FIGURE 8

Ag-specific germinal center and plasma cell differentiation in CD40LBTg and IA-B/CD40LBTg mice. A, Three serial spleen sections from WT mice and CD40LBTg mice at days 7, 14, and 21 postimmunization with NP-CGG plus alum were stained with biotinylated anti-IgD Ab plus streptavidin-Alexa 488 plus NP-PE, CD138-PE, or anti–TCRβ-PE. Serial sections are arranged in a row. Arrowheads indicate NP-specific germinal centers. B, Three serial spleen sections from IA-B mice and IA-B/CD40LBTg mice at day 14 postimmunization with NP-CGG plus alum were stained as in A. Arrowheads indicate NP-specific germinal centers. Original magnification ×100.