Volume 29, Issue 6, Pages 922-933 (December 2008) Prevention of Autoimmunity and Control of Recall Response to Exogenous Antigen by Fas Death Receptor Ligand Expression on T Cells  Imed Mabrouk, Stéphanie Buart, Meriem Hasmim, Christelle Michiels, Elizabeth Connault, Paule Opolon, Gilles Chiocchia, Matthieu Lévi-Strauss, Salem Chouaib, Saoussen Karray  Immunity  Volume 29, Issue 6, Pages 922-933 (December 2008) DOI: 10.1016/j.immuni.2008.10.007 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Efficient and Specific Cre-Mediated Fasl−/− Gene Deletion (A) Cell-specific deletion of Fasl gene. Q-RT-PCR and classical PCR were performed on cells from control mice, from Fasl−/− mice, and from mice with FasL loss on T cells, B cells, or myeloid cells. FasL mRNA expression was normalized to control littermates. Numbers correspond to tissues used for both Q-RT-PCR and PCR and are as follows: for T-Fasl−/− mice, 1 = thymocytes, 2 = purified T cells, 3 = LN cells, 4 = T cell-depleted splenocytes, 5 = splenocytes, and 6 = tail tissue; for B-Fasl−/− mice, 8 = splenocytes, 9 = purified B cells, and 10 = tail tissue; for M-Fasl−/− mice 11 = bone marrow cells, 12 = tail tissue, and 13 = CD11b+Gr1+ purified bone marrow cells; and 7 = tissue from FasL-deficient mice. Results shown represent mean ± SEM and are from one representative experiment out of two (n = 2–3 mice per genotype). ∗∗∗p < 0.0001 for thymocytes versus splenocytes in TFasl−/− mice; p = 0.078 and p = 0.08 are respectively for B-Fasl−/− and M-Fasl−/− tissues as compared to Fasl−/− tissues. (B and C) FACS analysis of surface and intracellular FasL expression on control activated T cells, B cells, and respective gene-targeted mice. Purified T cells (B) and B cells (C) were respectively activated with CD3 antibody (5 μg/ml) and 10 μg/ml of LPS for 24 hr. Plots are from one representative experiment out of three (n = 2–3 mice per genotype). Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 FasL Is Required for the Abnormal T Cell Development and the Homeostasis of LN-Resident Cells (A–E) Increased cell number in T-Fasl−/− mice. (A) LN cell counts at indicated age from littermates (n = 5), T-Fasl−/− mice (n = 5), and Fasl−/− mice (n = 4) were 3.38 ± 1.17 × 106, 8.9 ± 2.33 × 106, and 294.5 ± 34.26 × 106, respectively. ∗p = 0.02 and ∗∗p = 0.002 as compared to littermates. ∗∗∗p < 0.0001 as compared to Fasl−/− mice. (B) Characteristic CD3+B220+CD8−CD4− cells from TFasl−/− mice (n = 5), littermates (n = 5) and Fasl−/− mice (n = 4) were quantified by flow cytometry after staining with CD3 and B220 antibodies. Results shown represent cellularity ± SEM. ∗p = 0.014 as compared to littermates, ∗∗p = 0.003 as compared to Fasl−/− mice. (C–E) Conventional B cell (C), CD8+ T cell (D), and CD4+ T cell (E) numbers were determined by staining with CD19 and a mixture of CD4 and CD8 antibodies, respectively. Results shown represent cellularity ± SEM. For CD19+ B cells, ∗p ≤ 0.03 and ∗∗∗p = 0.0003 as compared to littermates; ∗∗p = 0.0022 as compared to Fasl−/− mice. For CD8+ T cells, ∗p = 0.013 for 5-month-old T-Fasl−/− mice versus 9 month-old T-Fasl−/− mice, and ∗∗p = 0.003 as compared to Fasl−/− mice. For CD4+ T cells, ∗∗p = 0.0086 for 5-month-old T-Fasl−/− mice versus age-matched Fasl−/− mice; ∗∗p = 0.0028 for 9-month-old T-Fasl−/− mice versus age matched littermates, and ∗∗∗p = 0.0005 for 5-month-old T-Fasl−/− mice versus 9-month-old T-Fasl−/− mice. (F and G) Normal cell distribution in B-Fasl−/− mice and M-Fasl−/− mice. Absolute number of total cells (four to six mice per genotype) from axillary LNs isolated from B-Fasl−/− mice, M-Fasl−/− mice, and littermates at 5 months of age were 3.160 ± 0.33 × 106; 4.30 ± 0.86 × 106 and 3.38 ± 1.14 × 106, respectively (F); conventional T cells and B cells as well as abnormal CD3+B220+CD4−CD8− in B-Fasl−/− mice, M-Fasl−/− mice, and littermates at 7 months of age (G). Results shown represent cellularity ± SEM. (H). Histological analysis of LNs from mice at 5 months of age. Fixed sections were stained with hematoxylin + eosin + saffranin or Ki67 antibody for proliferating cells. Scale bars represent 100 μm. (I) Quantification of Ki67+ cells was determined from three mice per group and control littermates were considered to be the reference point. Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 DC Accumulation in T-Fasl−/− Mice (A and B) Spleen cellularity in T-Fasl−/− mice and Fasl−/− mice at indicated ages (A) and in 5- to 7-month-old B-Fasl−/− mice and M-Fasl−/− mice (B). Number of total spleen cells of T-Fasl−/− mice (n = 6–7), Fasl−/− mice (n = 4), B-Fasl−/− mice (n = 5), M-Fasl−/− mice (n = 5), and littermates (n = 6–7) were 87.33 ± 8.38 × 106, 242 ± 14.27 × 106, 87.75 ± 6.39 × 106, 81.25 ± 8.53 × 106, and 78.43 ± 5.12 × 106, respectively. ∗∗∗p < 0.0001 as compared to Fasl−/− mice. (C and D) Increased CD11c cell numbers. In (C), splenocytes were stained for different markers and numbers in the quadrant indicate percentage of high CD11c+I-Ab+ cells; plots are from a representative experiment out of three independent experiments (n = 2 per genotype for each experiment). As shown in (D), numbers of CD11chiand CD11clo were determined from (C). Results shown represent mean ± SEM. ∗∗p ≤ 0.0074 for T-Fasl−/− mice versus Fasl−/− mice or littermates and ∗∗∗p ≤ 0.0009 for T-Fasl−/− mice versus Fasl−/− mice. Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Reduced Number of CD8+ T Cells in the Spleen of T-Fasl−/− Mice (A and B) Number of CD8+ T cells and abnormal CD3+B220+CD4−CD8− cells were determined by flow-cytometry analysis. Results shown represent cellularity ± SEM (n = 4–7 mice for each genotype). ∗p = 0.002 as compared to littermates. ∗∗p < 0.005, and ∗∗∗p < 0.0001 as compared to Fasl−/− mice. (C–E) Conventional CD4+, CD19+, and CD8+ cells from spleens were numbered at the indicated ages in T-Fasl−/− mice and Fasl−/− mice (C and D), and at 7 months of age in B-Fasl−/− mice and M-Fasl−/− mice (E). Results shown represent cellularity ± SEM (n = 4–5 mice per genotype). ∗p = 0.025 for B-Fasl−/− mice versus littermates, ∗∗p ≤ 0.001 for T-Fasl−/− mice versus Fasl−/− mice or littermates, and ∗∗∗p = 0.0004 for T-Fasl−/− mice versus Fasl−/− mice. (F) Disorganized spleen architecture in the absence of splenomegaly. Spleens from 5-month-old littermates, T-Fasl−/− mice, B-Fasl−/− mice, M-Fasl−/− mice, and Fasl−/− mice were fixed and sections were stained with hematoxylin + eosin + saffranin. Scale bars represent 100 μm. Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Activated Phenotype of FasL-Deficient T Cells (A) Analysis of CD62L and CD44 expression gated on splenic CD4+ T and CD8+ T cells so that the proportion of naive versus activated and memory cells could be determined. Plots are from a representative experiment; mice were 5 months old (n = 3 per group). (B) Fas and DR5 expression on cell-sorted splenic CD4+ T and CD8+ T cells from 5-month-old mice (n = 4 per group). All plots are from a representative experiment. (C) Analysis of KLF2 mRNA expression in thymocytes and purified splenic CD4+ T cells. Mice were 4 months old T-Fasl−/− (n = 5) and littermates (n = 3). Data represent mean ± SEM from three independent experiments. (D) T cell proliferation response. Purified T cells from 5-month-old T-Fasl−/− mice (n = 4), littermates (n = 4), and Fasl−/− mice (n = 2) were stimulated with coated CD3 antibody for 3 days. Results shown represent mean ± SEM. ∗p = 0.034 as compared to Fasl−/− mice. (E) CD44, CD62L, Fas, and DR5 expression was determined on gated splenic CD11b+ cells and CD19+ cells from M-Fasl−/− mice and B-Fasl−/− mice, respectively. Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 FasL-Dependent Autoimmunity (A) Sera from mice that lack FasL on T cells, B cells, or myeloid cells and from age-matched control mice were analyzed for anti-dsDNA IgG. T-Fasl−/− mice were aged from 5 to more than 9 months (5-month-old mice, n = 3; 7-month-old mice, n = 5; 9-month-old mice, n = 5; and >9-month-old mice, n = 4); B-Fasl−/− mice were 7 to 9 months old (n = 8); M-Fasl−/− mice were more than 7 months of age (n = 6) and Fasl−/− mice were from 7 to 12 months of age (n = 6). ∗p ≤ 0.05 for littermates versus T-Fasl−/− mice or B-Fasl−/− mice, and ∗∗p ≤ 0.0099 as compared to Fasl−/− mice. (B and C) IgG subclasses and IgM concentrations in the sera from 7- to 9-month-old T-Fasl−/−mice (n = 8–10), Fasl−/− mice (n = 5), and littermates (n = 8–10) (B), or 7-month-old B-Fasl−/− mice and M-Fasl−/− mice (n = 7–10) (C). ∗p < 0.05, ∗∗p < 0.005, and ∗∗∗p ≤ 0.0002. (D) Kidney histological analysis. Kidney from 7- to 9-month-old littermates, T-Fasl−/− mice, B-Fasl−/− mice, M-Fasl−/− mice, and FasL-deficient mice were fixed, and sections were stained with hematoxylin + eosin + saffranin. Scale bars represent 100 μm. Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Impaired Secondary Response to Exogenous Antigen in T-Fasl−/− Mice T-Fasl−/− mice, B-Fasl−/− mice, M-Fasl−/− mice, and littermates were immunized with OVA-CFA (50 μg) and challenged with OVA-IFA (50 μg) at day 7. Mice were analyzed 1 week and 2 weeks after secondary immunization for OVA-IgM and OVA-IgG1, respectively. Data are from three independent experiments for T-Fasl−/− mice and two independent experiments for both B-Fasl−/− mice and M-Fasl−/− mice (six mice per genotype for each experiment). (A) Sera level of OVA-specific IgG1 antibodies. ∗p = 0.005 for T-Fasl−/− mice as compared to littermates. (B) Purified splenic CD4+ T cells from TFasl−/− mice were restimulated with OVA (100 μg/ml) for 90 hr, and IFN-γ secretion was detected in the supernatants by ELISA. Results shown represent the amount of cytokine ± SEM. ∗p = 0.019 as compared to controls. (C) Sera titer of OVA-specific IgM was determined at day 14 after primary immunization. (D) Immunohistochemistry of spleen follicles from immunized T-Fasl−/− and littermates for GC identification in cryostat sections stained with PNA. (E) Cryostat sections from littermates, T-Fasl−/− mice, B-Fasl−/− mice, and M-Fasl−/− mice were stained with CD35 and CD45R antibodies to show FDC and B cells, respectively. For (D) and (E), each image is representative of spleen sections from two mice in two independent experiments. Scale bars represent respectively 30 μm in (D) and in the top panel of (E) and 100 μm in the lower panel of (E). Results represent mean ± SEM. (F) Ratio of surface follicles to total surface was quantified as described in the Experimental Procedures. Immunity 2008 29, 922-933DOI: (10.1016/j.immuni.2008.10.007) Copyright © 2008 Elsevier Inc. Terms and Conditions