1. Volume 45, Issue 3, Pages 513-526 (September 2016)
The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis 
Silvia Alvarez-Diaz, Christopher P. Dillon, Najoua Lalaoui, Maria C. Tanzer, Diego A. Rodriguez, Ann Lin, Marion Lebois, Razq Hakem, Emma C. Josefsson, Lorraine A. O’Reilly, John Silke, Warren S. Alexander, Douglas R. Green, Andreas Strasser 
Immunity 
Volume 45, Issue 3, Pages (September 2016)
DOI: /j.immuni
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2. Figure 1 Casp8−/−Mlkl−/− Mice Are Viable
(A) Expected and observed frequency of mice of the indicated genotypes in offspring from crosses of mice with the indicated genotypes.
(B) Photograph of a 14-week-old Casp8−/−Mlkl−/− mouse bred from a double-deficient cross alongside a WT mouse.
(C) Immunoblotting for Caspase-8, MLKL, and HSP70 (loading control) from solid organs (left) and lymphoid tissues (right) of mice of the indicated genotypes.
(D) Expected and observed frequency of mice of the indicated genotypes in offspring from crosses of mice with the indicated genotypes.
(E) Photograph of an 8-week-old Fadd−/−Mlkl−/− mouse alongside a littermate control mouse.
(F) Immunoblotting for FADD, MLKL, Actin, and HSP70 (last two used as loading controls) from solid organs and lymphoid tissues of mice of the indicated genotypes.
See also Figure S1.
Immunity  , DOI: ( /j.immuni )
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3. Figure 2
Cells from Casp8−/−Mlkl−/− Mice Are Resistant to Diverse Necroptotic Cell-Death Stimuli
(A and B) Primary MDFs (A) and BMDMs (B) from the indicated genotypes were treated with TNF (100 ng/mL), FASL (10 ng/mL), poly(I:C) (25 μg/mL), or LPS (25 μg/mL) in the presence or absence of the caspase inhibitor Q-VD-OPh (25 μM), z-VAD-FMK (20 μM), or SMAC mimetic (CpdA: 500 nM) or were left untreated for 24 hr. Abbreviations are as follows: T, TNF; S, Smac-mimetic; Q, Q-VD-Oph; F, FASL; Z, z-VAD-FMK; P, poly(I:C); L, LPS. Cell viability was determined by staining with propidium iodide (PI) and flow cytometric analysis. Data are presented as mean ± SEM (n = 3 for each genotype). ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
See also Figure S2.
Immunity  , DOI: ( /j.immuni )
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4. Figure 3
Casp8−/−Mlkl−/− Mice Develop Progressive Severe Lymphadenopathy
(A) Cross-section of immune cells in bone marrow, spleen, and lymph nodes from mice of the indicated genotypes at 20 days and 70 days of age (n = 3). Different cell subsets were determined by flow cytometry using the following markers: B cells (B220+ or CD19+), T cells (CD3+), Monocytes and Macrophages (MAC-1+), Granulocytes (GR-1+), Erythroid cells (TER119+), and NK cells (NK1.1+ and NKp46+). Data represent mean ± SD (n = 3).
(B and C) Weight and total cell numbers of the lymph nodes (axillary, brachial, inguinal, and mesenteric) and spleen from mice of the indicated genotypes at (A) 50 days and (B) 100 days. ∗p < 0.05, ∗∗∗p < compared to WT mice; #p < 0.05, ##p < 0.01, ###p < compared to Casp8−/−Ripk3−/− mice.
(D) Kaplan-Meyer survival curves for WT (n = 15), Mlkl−/− (n = 19), Casp8+/−Mlkl−/− (n = 39), Casp8−/−Mlkl−/− (n = 22), and Casp8−/−Ripk3−/− (n = 31) mice.
(E) Kaplan-Meyer survival curves for control (MLKL) (n = 32, FaddwtMlkl−/− or Fadd+/−Mlkl−/−), Fadd−/−Mlkl−/− (n = 34), control (RIP3) (n = 44, FaddwtRipk3−/− or Fadd+/−Ripk3−/−), and Fadd−/− Ripk3−/− (n = 28) mice. Survival of mice of different genotypes was compared by log-rank test. ∗∗p < 0.001, ∗∗∗p <
See also Figure S3.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
Casp8−/−Mlkl−/− Mice Accumulate “Unusual” B220+CD3+CD4−CD8− T Cells
(A) Numbers of B and T cells recovered from spleens from mice of the indicated genotypes. Values represent mean ± SD (n = 3–5 mice/genotype), ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(B) Representative flow cytometric images of the unusual DN T cells (numbers indicate percentage of cells in each quadrant) (n = 4).
(C) The percentages and numbers of the unusual DN T cells in the lymph nodes of mice of the indicated genotypes were measured by flow cytometric analysis. Values represent mean ± SD (n = 3–5 mice/genotype), ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(D) The percentages and numbers of the unusual DN T cells in the peripheral blood of mice of the indicated genotypes were measured by flow cytometric analysis. Values represent mean ± SD (n = 3–5 mice/genotype).
(E) Proliferation of CD3+CD8+ T cells from mice of the indicated genotypes was determined by staining for Ki67 after fixation and permeabilization. Percentages of Ki67+ cells in 20- and 70-day-old mice of the indicated genotypes (n = 3) (left) and representative flow cytometric images of the CD3+CD8+ T cells (numbers indicate percentage of cells in each quadrant) (n = 3) (right) are shown. Data are presented as mean ± SD for each genotype; ∗∗p < 0.001, ∗∗∗p <
(F) Immunoblotting for RIPK3, MLKL, and HSP70 (loading control) in extracts from purified DN T cells (Casp8−/−Mlkl−/− and Casp8−/−Ripk3−/− mice) or total T cells (WT mice). Two mice per genotype were used.
See also Figure S4.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5 Casp8−/−Mlkl−/− Mice Develop Autoimmune Glomerulonephritis
(A) Representative H&E-stained sections of the kidneys from mice of the indicated genotypes were examined for pathological changes, such as hyper-cellularity, glomerular enlargement, or leukocyte infiltration. Kidneys from 6 mice of each genotype were analyzed. Magnification × 40.
(B) Representative confocal photomicrographs of frozen sections (kidneys from three mice of each genotype analyzed) stained for the presence of IgA-, IgG-, or IgM-containing immune complexes (green) in glomeruli. Nuclei are revealed by staining with DAPI (blue). Scale bars represent 25 μm.
(C and D) Sera of mice of the indicated genotypes were collected, and the concentrations of (C) ANA or (D) total antibodies of the different Ig isotypes were quantified by ELISA (age 100 days). Values in graphs represent mean ± SEM (n = 6–13 mice/genotype); ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(E) RBC and reticulocyte numbers were measured in blood samples of mice of the indicated genotypes. Data represent mean ± SD (n = 9–53 mice/genotype); ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(F) RBC numbers were measured in blood samples of mice of the indicated genotypes at 60 days of age. Data represent mean ± SD (n = 5 mice/genotype).
Immunity  , DOI: ( /j.immuni )
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7. Figure 6 Casp8−/−Mlkl−/− Mice Have Abnormally Low Platelet Counts
(A) Platelet counts in mice of the indicated genotypes at 50 days (left) and 100 days (right) of age. Data represent mean ± SD (n = 6–19 mice/genotype); ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(B) Platelet counts (left) and percentages of reticulated platelets (right) in mice of the indicated genotypes and age. Data represent mean ± SEM (n = 7–10 mice/genotype); ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(C) Megakaryocyte counts in H&E-stained sections of bone marrow (left) and spleen (right). Data represent mean per field of view (FOV; × 200) from 10 fields per individual mouse (n = 3–14 mice/genotype).
Immunity  , DOI: ( /j.immuni )
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8. Figure 7
Casp8−/−Mlkl−/− Mice Have Abnormally Increased Amounts of Certain Pro-inflammatory Cytokines and Chemokines
(A and B) The concentrations of 23 cytokines and chemokines (indicated in Experimental Procedures) were measured in the sera of mice of the indicated genotypes by the multiplex system at (A) 50 days when animals still appeared healthy or (B) 100 days of age when Casp8−/−Mlkl−/− mice were showing clear signs of illness. The concentrations of RANTES, IL-10, and IL-12p40 are shown. Each dot represents a single mouse; the bar indicates the average; the error bars represent SEM; ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p <
(C and D) 23 cytokines and chemokines were measured in the supernatants of purified T cells activated with PMA (2 ng/mL) plus ionomycin (10 ng/mL) or CD3 (10 μg/mL) plus CD28 (10 μg/mL) antibodies (C) or BMDMs stimulated with poly(I:C) (25 μg/mL) or LPS (25 μg/mL) (D) from mice of the indicated genotypes. The concentrations of RANTES, IL-10, and IL-12p40 are shown. Data represent mean ± SD (n = 3 mice/genotype). ∗∗p < Casp8−/−Mlkl−/− mice compared to any of the other genotypes.
See also Figures S5, S6, and S7.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions