Fas receptor expression in germinal-center B cells is essential for T and B lymphocyte homeostasis

Abstract

Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.

Figure 1. Fas^fl/flCd19-Cre Mice Die Prematurely Because of Lymphocyte Infiltration in Multiple Organs

(A) Confirmation of Fas deletion. The left side shows a representative Southern blot of naive FAS^FL/fl and Fas^fl/flCd19-Cre B cells showing deletion of the Fas gene in 83% of the latter (87.3% ± 4%, n = 4). The right side shows a representative flow-cytometric analysis of Fas^fl/+Cd19-Cre and Fas^fl/flCd19-Cre B cells (stimulated for 2–4 days with LPS) showing that Fas protein expression was lacking in 81% of the mutant cells (85.4% ± 4.8%, n = 4). (B) Reduced life span. Kaplan-Meier survival curves of Fas^fl/flCd19-Cre mice compared to controls. (C) Organ infiltration. Focal interstitial lymphocyte accumulation is shown in H&E-stained sections of livers, lungs, and kidneys of Fas^fl/fl and Fas^fl/flCd19-Cre mice at the ages of 6 and 14 months. Scale bars apply to all panels in a row.

Figure 2. Hyperproliferation and Activation of T and B Cells and Disrupted Lymphoid-Organ Architecture in Fas^fl/flCd19-Cre Mice

(A) Increased cell numbers. Absolute numbers of total lymphocytes, B cells, and T cells in spleens of Fas^fl/fl and Fas^fl/flCd19-Cre mice at age 6–7 months (n = 3/genotype). For all figures, *p < 0.05 and **p < 0.005. (B) Altered surface marker expression. CD4⁺ T cells, total T cells, and total B cells from Fas^fl/fl and Fas^fl/flCd19-Cre mice at the ages of 2, 6, or 14 months were examined by flow cytometry for expression of CD28, ICOS, ICOSL, and MHCII as indicated. (C) Increased proliferation. BrdU incorporation by B220⁺IgM⁺B cells and CD4⁺ and CD8⁺ T cells was measured by flow cytometry. The percentage of BrdU⁺ cells is indicated. (D and E) Sections of spleen (D) and LN (E) from 14-month-old Fas^fl/fl and Fas^fl/flCd19-Cre mice were stained with H&E (top and middle) or with immunohistochemical staining for detection of T and B cells (bottom). T cells that were stained positively with anti-CD3 were dark blue in color, whereas B cells that were stained positively with B220 antibody were brown. Scale bars apply to control and mutant samples in a pair.

Figure 3. Increased Cytokine Production in Fas^fl/flCd19-Cre Mice

(A) Elevated serum cytokines. Serum amounts of IFN-γ, TNF-α, IL-10, and IL-6 were determined in Fas^fl/fl and Fas^fl/flCd19-Cre mice of age ≥ 11 months. Results shown represent the mean cytokine level ± SD (n = 4–6 mice/genotype). (B) Increased numbers of cytokine-producing cells. Cells from LNs of aged Fas^fl/fl and Fas^fl/flCd19-Cre mice were fixed with 1.6% PFA, subjected to intracellular staining with antibodies recognizing the indicated cytokine, and analyzed by flow cytometry. Numbers are the percentage of cells in the gated lymphocyte population that produced the indicated cytokine (n = 3–4).

Figure 4. Ablation of Fas in GC B Cells Reproduces the Lymphoproliferative Disorder in Fas^fl/flCd19-Cre Mice

(A) Confirmation of Fas deletion in GC B cells. The top portion shows flow-cytometric analysis of GC B cells (PNA^hiCD38^lo) among gated CD19⁺ B cells of Fas^fl/lpr and Fas^fl/lprIghg1-Cre mice. Numbers are the percentage of GC B cells among total B cells. The bottom-right portion shows the absence of Fas expression in GC B cells from mLN of Fas^fl/lprIghg1-Cre mice. The bottom-left portion shows a Southern blot of T cells (T), follicular B cells (FB), and marginal zone B cells (MZ) from Fas^fl/+Ighg1-Cre mice confirming the absence of Cre-mediated recombination in cells other than GC B cells. (B) Increased weight of spleens from 3.5- and 8-month-old Fas^fl/lpr and Fas^fl/lprIghg1-Cre mice. Each symbol represents an individual mouse. (C) Increased absolute numbers of total lymphocytes and B and T cells in spleens of 8-month-old Fas^fl/lpr and Fas^fl/lprIghg1-Cre mice. Results shown represent the mean ± SD (n = 3/genotype). (D) Disrupted splenic architecture. We stained spleen sections from 8-month-old Fas^fl/lpr (left) and Fas^fl/lprIghg1-Cre (right) mice with H&E to reveal the splenic architecture. Scale bars apply to control and mutant samples in a pair. (E) Accumulation of GC B cells. Representative flow-cytometric analyses of GC B cells (PNA^hiCD38^lo) within gated B cells in spleen (SP) and mLN of 3- to 3.5-month-old Fas^fl/lpr and Fas^fl/lpr Ighg1-Cre mice before (no imm) and 30 days after immunization with SRBC. (F) Accumulation of GC B cells in Fas^fl/flCd19-Cre mice. Representative flow cytometric analyses of GC B cells among B220⁺ B cells in spleen (SP) and mLN of unimmunized Fas^fl/fl and Fas^fl/flCd19-Cre mice. Numbers in (F) and (G) are the percentage of GC B cells (PNA^hiCD38^lo) among gated B cells.

Figure 5. Hyperproliferation and Activation of Fas-Deficient B Cells Is T Cell Dependent

Fas^fl/flCd19-CreTcrb^−/− mice lack T cells and have Fas-deficient B cells. B cells from Fas^fl/fl, Fas^fl/flCd19-Cre, and Fas^fl/flCd19-CreTcrb^−/− mice were examined for (A) MHC class II expression as in Figure 2B; (B) BrdU incorporation as in Figure 2C; and (C) B cell cellularity in the LN as in Figure 2A. The mean cellularity ± SD is shown (n = 3–9 mice/genotype). (D) H&E stained sections of spleen and liver from mice of the indicated genotypes. Scale bars apply to an entire row.

Figure 6. The Lymphoproliferative Disorder in Fas^fl/flCd19-Cre Mice Is Caused by Memory B Cells and Requires CD28-CD80 Signaling

Fas^fl/flCd19-CreCd28^−/− mice lack CD28 in all cells and Fas in B cells only. Fas^fl/fl, Fas^fl/flCd19-Cre, and Fas^fl/flCd19-CreCd28^−/− mice were examined for (A) percentage of B220⁺IgM⁻IgD⁻ B cells. Mean numbers ± SD of B220⁺IgM⁻IgD⁻ B cells in spleens of Fas^fl/fl, Fas^fl/flCd19-Cre, and Fas^fl/flCd19-CreCd28^−/− mice (n = 3–4/genotype) were 7.6 ± 1.78 × 10⁵, 37.8 ± 5.1 × 10⁵ and 4.8 ± 0.96 × 10⁵, respectively. Fas^fl/fl, Fas^fl/flCd19-Cre, and Fas^fl/flCd19-CreCd28^−/− mice were examined for (B) percentage of B220⁺IgG1⁺ memory B cells (top) and CD80 and CD86 expression (bottom). Mean numbers ± SD of B220⁺IgG1⁺ memory B cells in spleens of Fas^fl/fl, Fas^fl/flCd19-Cre and Fas^fl/flCd19-CreCd28^−/− mice (n = 3–4/genotype) were 2.9 ± 1.3 × 10⁴, 85.2 ± 58.6 × 10⁴ and 1.73 ± 1.17 × 10⁴, respectively. (C) shows the expression of the indicated markers on T and B cells as in Figure 2B; (D) shows STAT1 phosphorylation in IFN-γ-stimulated T cells as in Figure S4B; (E) shows BrdU incorporation as in Figure 2C; and (F) shows LN cellularity (right) as in Figure 2A; the mean cellularity ± SD is shown (n = 3–7 mice/genotype). *(*)p < 0.05 for Fas^fl/fl versus Fas^fl/flCd19-Cre mice but p < 0.005 for Fas^fl/flCd19-Cre versus Fas^fl/flCd19-CreCd28^−/− mice. Mice were ≥11 months old (A, B, C, E, and F) or 5 to 6 months old (D).