1. Differential Requirement for Malt1 in T and B Cell Antigen Receptor Signaling 
Jürgen Ruland, Gordon S Duncan, Andrew Wakeham, Tak W Mak 
Immunity 
Volume 19, Issue 5, Pages (November 2003)
DOI: /S (03)

2. Figure 1 Generation of Malt1-Deficient Mice
(A) Gene targeting strategy. Top: A portion of the WT murine Malt1 locus showing relevant restriction sites. RV, EcoRV; X, XbaI; HIII, HindIII. Exons 8 (E8) and 9 (E9) are shown as open boxes. The position of the 3′ flanking probe (FP) is indicated. The structure of the targeting vector (middle) and the mutant locus (bottom) are also shown.
(B) Southern blot analysis of tail DNA isolated from WT (+/+), heterozygous (+/−), and Malt1-deficient (−/−) mice. Genomic DNA was digested with EcoRV and hybridized with the flanking probe indicated in (A).
(C) Lack of Malt1 expression in Malt1−/− mice. Thymocyte RNA isolated from Malt1+/+ and −/− mice was subjected to RT-PCR analysis using primer pairs specific for Malt1 or β-actin.
(D) Western Blot analysis of splenic protein lysates from of Malt1+/+, +/−, and −/− mice using antibodies against Malt1.
(E) Total number and relative distribution (%) of Malt1+/+, +/−, and −/− offspring derived from heterozygous intercrosses.
Immunity  , DOI: ( /S (03) )

3. Figure 2
Flow Cytometric Analyses of Lymphocyte Populations in Malt1−/− Mice
(A) Surface IgD and IgM expression on splenic B cells from Malt1+/+ and −/− mice.
(B) Surface CD21 and CD23 expression on B220-gated splenic B cells from Malt1+/+ and −/− mice. The percentage of marginal zone B cells is indicated.
(C) Surface B220 and CD5 expression on peritoneal cavity B cells. The percentage of B1 cells is indicated.
(D) Frequency of CD3−IL-2Rβ+ NK cells and CD3+IL-2Rβ+ NKT cells in the lymphoid compartment of the liver.
(E) Expression of CD4 and CD8 on thymocytes.
(F) CD4 and CD8 expression on splenic T cells.
Immunity  , DOI: ( /S (03) )

4. Figure 3 Immunodeficiency in Malt1−/− Mice
(A) Reduced basal immunoglobulin titers. Concentrations of Ig isotypes were determined in the sera of nonimmunized 8-week-old WT (+/+, open circles) or Malt1-deficient (−/−, filled circles) mice by ELISA.
(B) Impaired T cell-dependent antibody responses. WT (open symbols) and Malt1−/− (closed symbols) mice were immunized with KLH in CFA. KLH-specific IgM and IgG1 production was determined by ELISA at 7, 14, 21, and 28 days after immunization.
(C) Impaired T cell-independent type II antibody responses. WT (open symbols) and Malt1−/− (closed symbols) mice were immunized with TNP-Ficoll, and TNP-specific IgM and IgG3 production was determined at 7, 14, and 21 days after immunization. For (B) and (C), results from two (of six) representative mice are shown per genotype.
Immunity  , DOI: ( /S (03) )

5. Figure 4 Defective T Cell Activation in Malt1−/− Mice
(A) Impaired proliferative responses. T cells were purified from WT, Malt1−/−, and Bcl10−/− mice and stimulated in vitro with medium alone, soluble anti (α)-CD3 (1 μg/ml), anti-CD3 plus anti-CD28 (1 μg/ml), anti-CD3 plus anti-CD28 plus IL-2 (50 U/ml), or PMA (10 ng/ml) for 48 hr. Proliferation was determined by [3H]thymidine incorporation.
(B) Impaired IL-2 production. IL-2 levels were evaluated in the supernatants of Malt1+/+ or −/− T cell cultures stimulated with medium alone (med) or anti-CD3 plus anti-CD28 (100 ng/ml each). n.d., not detectable.
(C) Activation markers. Flow cytometric analysis of CD25, CD44, and CD69 expression 24 hr after stimulation of Malt1+/+ or −/− T cells with medium alone (control) or anti-CD3 plus anti-CD28 (100 ng/ml each) in vitro.
Immunity  , DOI: ( /S (03) )

6. Figure 5 Signal Transduction from the TCR to NF-κB in Malt1−/− Mice
(A) Normal overall tyrosine phosphorylation pattern in Malt−/− T cells. Malt1+/+ and −/− lymph node T cells were treated with hamster anti-CD3ϵ antibody (10 μg/ml) followed by crosslinking with anti-hamster antibody (1 μg/ml) for the indicated times in minutes. Protein lysates were subjected to Western blotting using antibodies against phospho-tyrosine.
(B) Normal Erk activation. T cells were stimulated as in (A), and protein lysates were subjected to Western blotting using antibodies specific for phospho-Erk.
(C) Impaired TCR/CD28- and PMA-induced NF-κB activation. Purified WT, Malt1−/−, and Bcl10−/− T cells were incubated with medium alone (control), anti-CD3 (10 μg/ml) plus anti-CD28 (1 μg/ml), or PMA (50 ng/ml) plus ionomycin (10 ng/ml). After 8 hr, nuclear extracts were prepared and subjected to gel mobility shift assays with radiolabeled probes containing NF-κB or AP-1 binding site sequences.
(D) Normal TNF-α-induced NF-κB activation. WT or Malt1−/− T cells were stimulated for 30 min with 10 ng/ml TNF-α. Nuclear extracts were subjected to gel mobility shift analysis with probes containing NF-κB binding site sequences.
(E) Defective IKK activation. WT, Malt1−/−, and Bcl10−/− T cells were stimulated with PMA plus ionomycin for the indicated time in minutes. The IKK complex was immunoprecipitated and IKK activity was determined in vitro using GST-IκBα as a substrate.
(F) Defective IκBα phosphorylation and degradation. After stimulation of WT, Malt1−/−, and Bcl10−/− T cells with PMA plus ionomycin for the indicated time in minutes, phosphorylation and degradation of IκBα was investigated by Western blotting using anti-phospho-IκBα (P-IκBα), anti-IκBα, and anti-actin (loading control) antibodies.
(G) CARMA3- and IAP2-MALT1-induced NF-κB activation in Bcl10−/− cells. Bcl10+/− or −/− MEFs were cotransfected with a NF-κB reporter plasmid plus either the empty pcDNA3 vector (control), pcDNA3-Flag-IAP2-MALT1, or pcDNA3-CARMA3-HA. After 48 hr, NF-κB activation was determined as described in the Experimental Procedures.
(H) Defective Jnk and p38 MAP kinase signaling in Malt1−/− T cells. Purified T cells were stimulated with PMA plus ionomycin for the indicated time in minutes. Protein lysates were subjected to Western blotting using antibodies against phospho-Jnk, Jnk, phospho-p38, p38, or actin (loading control).
Immunity  , DOI: ( /S (03) )

7. Figure 6
B Cell Activation and BCR-Mediated NF-κB and AP-1 Activation in Malt1−/− B Cells
(A) Proliferative response. Purified WT, Malt1−/−, and Bcl10−/− splenic B cells were stimulated for 48 hr with medium alone, anti-IgM (10 μg/ml), anti-IgM (10 μg/ml) plus anti-CD40 (5 μg/ml), or LPS (20 μg/ml), and B cell proliferation was assessed by [3H]thymidine incorporation.
(B) NF-κB and AP-1 activation. WT, Malt1−/−, and Bcl10−/− B cells were incubated for 8 hr with medium alone (control) or anti-IgM (5 μg/ml). Nuclear extracts were prepared and subjected to gel mobility shift assays using probes containing NF-κB or AP-1 binding site sequences.
(C) IκBα degradation induced by BCR signaling. Purified WT, Malt1−/−, and Bcl10−/− B cells were stimulated with anti-IgM (5 μg/ml). Degradation of IκBα was determined by Western blotting using anti-IκBα and anti-actin (loading control) antibodies.
Immunity  , DOI: ( /S (03) )

8. Figure 7
A Model for the Differential Requirement of MALT1 in TCR versus BCR Signal Transduction Leading to NF-κB Activation
See text for details.
Immunity  , DOI: ( /S (03) )