Influence of Fas on the regulation of the response of an anti-nuclear antigen B cell clonotype to foreign antigen

Abstract

A peripheral B cell tolerance checkpoint appears to be operative during the germinal center (GC) reaction. We previously showed that a transgenic BCR clonotype that is 'dual reactive' for the hapten arsonate (Ars) and nuclear auto-antigens is stimulated to enter the GC response via Ars immunization. However, the participation of this clonotype in this response wanes with time and it gives rise to few memory B cells capable of mounting a secondary anti-Ars IgG response. Enforced expression of Bcl-2 partially rescues the GC and memory B cell responses of this clonotype, suggesting that apoptotic pathways are involved in the action of the GC tolerance checkpoint. Since GC B cells substantially up-regulate levels of expression of the Fas apoptotic death receptor, we determined whether an intrinsic Fas deficient could rescue the participation of this clonotype in the GC response. It could not, strongly indicating that Fas expression by autoreactive GC B cells is not necessary for their elimination. In addition, experiments in which Fas-sufficient dual reactive clonotype B cells were transferred to Fas-deficient hosts revealed an absence of participation of these B cells in the GC and IgG anti-Ars responses. We present data consistent with the idea that T cells in Fas-deficient hosts are primed to express elevated levels of FasL and eliminate antigen-activated B cells that up-regulate Fas.

Fig. 1.

Primary B cell development in Fas-deficient HKI65 and HKIR mice. (A) Splenocytes were isolated from Fas-sufficient and Fas-deficient HKI65 and HKIR hemizygous mice as well as B6 mice and were stained with anti-B220 and anti-IgM^a and anti-IgM^b allotypic mAbs and analyzed by flow cytometry. HKI knock-in loci encode the IgM^a allotype. The data shown are from pooled cells from two mice of each genotype. (B) Splenocytes from mice of the indicated genotypes were stained with anti-B220, anti-CD21 and anti-CD23 and analyzed by flow cytometry. Follicular (CD23^high CD21^low) and marginal zone (CD23^low CD21^high) sub-populations are gated. The data shown are representative of at least three independent experiments. (C) Splenocytes from mice of the indicated genotypes were stained with anti-B220, anti-IgD and anti-clonotypic E4 mAbs and analyzed by flow cytometry. All data shown in this figure are from cells in the B220⁺ gate. The results are representative of at least three independent experiments.

Fig. 2.

Increased numbers of splenic transitional B cells in Fas-deficient mice. Spleen cells from mice of the indicated genotypes were stained with anti-B220, anti-clonotypic E4 mAb and AA4.1 and analyzed by flow cytometry. The upper panel shows the percentage of immature AA4.1⁺ and mature AA4.1⁻ B cells out of total cells in the lymphocyte gate. The lower panel shows the percentage of immature AA4.1⁺ cells out of gated B220⁺ and E4⁺ cells. The data shown are representative of two independent experiments.

Fig. 3.

An intrinsic Fas deficiency does not influence the primary AFC and GC responses of canonical HKIR B cells. The indicated types of chimeric mice were created as described in Methods using 2 × 10⁶ B cell-enriched splenic donor cells per recipient and immunized 12 h later with 100 μg of Ars–KLH in alum. Mice were sacrificed on day 6 after immunization and the E4⁺ clonotype-specific immune response was assessed using ELISpot and flow cytometry. (A) The ELISpot data shown represent the number of E4⁺ IgG-and IgM-producing AFCs per million splenocytes. (B) Splenocytes were stained with anti-B220, E4 and PNA and the number of PNA⁺, E4⁺ cells out of total B220⁺ cells evaluated by flow cytometry and the values plotted. Black filled circles and diamonds represent data obtained from Fas-sufficient HKI65 B cells in individual mice and open circles and diamonds data represent from Fas-deficient HKI65 B cells in individual mice. Horizontal bars show the mean value for each genotype. P values for differences between data from different types of chimeric mice, calculated using the Student’s t-test, are shown.

Fig. 4.

Fas-sufficient canonical HKI65 B cells do not participate in the Ars-induced IgG AFC response in Fas-deficient hosts. Splenocytes from HKI65 mice were adoptively transferred into B6 or B6.lpr mice and the resulting chimeric mice immunized 12 h later with 100 μg of Ars–KLH in alum. Mice were sacrificed on day 6 after immunization and the E4 clonotype-specific immune response was assessed using ELISpot analysis of splenocytes. ELISpot membranes were coated with either anti-mouse IgG (gamma chain specific) or anti-mouse IgM (mu chain specific) and ELISpots developed with the E4 mAb. Data from individual mice are indicated by open and filled circles. P values for comparisons of data from chimeras generated using Fas-sufficient and -deficient hosts, calculated using the Student’s t-test, are shown.

Fig. 5.

Effects of a host Fas deficiency on Fas-sufficient donor B cell homing to and stability in follicles in unimmunized chimeric mice. MACS-enriched B cells from B6.CD45.1 mice were adoptively transferred into either Fas-sufficient (B6.CD45.2) or Fas-deficient (B6.lpr) recipients of the CD45.2 allotype (5 × 10⁶ cells per mouse). Mice were sacrificed either on day 1 or day 7 after transfer. Left panels: splenocytes were isolated, stained with anti-B220, anti-IgD and anti-allotypic CD45.1 mAbs and analyzed by flow cytometry. Gated donor CD45.1⁺ cells are shown. The numbers shown above the gates indicate the mean percentage of CD45.1⁺ cells out of total B220⁺ or IgD⁺ cells and the standard deviation from three separate experiments. Right panels: immunofluorescent staining of spleen sections of mice described above on day 1 after transfer. Red staining indicates donor CD45.1 cells, while green staining with MOMA-1 delineates the border of the follicle and the marginal zone. Original magnification of images was ×140.

Fig. 6.

Fas expression and GC responses of B cells in Fas-sufficient -> Fas-deficient chimeric mice. (A) Chimeric mice created by transfer of 2 × 10⁶ HKI65/Vκ10A double transgenic B cells into B6.lpr hosts were either immunized the day after donor cell transfer with 100 μg of Ars–KLH in alum or left naive. On day 7 after transfer, mice were sacrificed, spleen cells isolated, stained with anti-B220 and anti-CD95 (Fas receptor) and analyzed by flow cytometry. Colored lines in the histograms represent B220-gated cells from individual mice. (B) Splenic B cells from HKI65/Vκ10A double transgenic mice were transferred into B6 or B6.lpr hosts and chimeric mice immunized as described above. Six days after immunization, chimeric mice were sacrificed, spleens cells isolated and stained with E4 and PNA and analyzed by flow cytometry. The E4⁺, PNA⁺ canonical GC B cell and E4⁻, PNA⁺ host GC B cell gates are shown.

Fig. 7.

CD8 T cells in Fas-deficient mice are primed to express elevated levels of FasL upon activation. Enriched splenic T cells from B6 and B6.lpr mice were activated for 4 h by plate-bound anti-CD3e antibody as described in Methods. Cells were then stained with antibodies for the indicated markers and levels of expression of surface FasL on various sub-populations evaluated by flow cytometry. FasL levels on cells gated for expression of CD3 only, CD3 and CD4 or CD3 and CD8 are shown.

Fig. 8.

A Fas deficiency in recipient mice results in an enhanced IgM response and compromised IgG AFC and GC responses from Fas-deficient donor B cells. Splenocytes from HKI65.lpr mice were adoptively transferred into B6 or B6.lpr mice (5 × 10⁶ per mouse) and chimeric mice immunized 12 h later with 100 μg of Ars–KLH in alum. Mice were sacrificed on day 6 after immunization and the clonotype-specific E4⁺ immune response was assessed using ELISpot and flow cytometry as described in the legends to Figs 3 and 4. (A) The number of E4⁺ IgM- and IgG-producing AFCs per million splenocytes is illustrated. (B) The percentages of PNA⁺, E4⁺ B cells out of the total number of B220⁺ lymphocytes are shown. Filled and open circles represent transfer into Fas⁺-deficient or Fas-deficient recipients, respectively. Each circle represents an individual mouse. Horizontal bars show the mean values for each data set. Statistical significance was assessed by the Student’s t-test.