Contrasting genetic approaches find a role for CARD11

CARD11 (also known as CARMA1) — a member of the membrane-associated guanylate kinase (MAGUK) family of proteins — is essential for lymphocyte activation, as now shown by gene targeting and genome-wide mouse mutagenesis screens.

MAGUK proteins are involved in the assembly and clustering of receptors and intracellular signalling molecules at the neuronal synapse. Structural similarities between the synaptic junctions in neurons and lymphocytes, as well as findings from studies using cell lines, led Hara et al. to generate Card11^−/− mice to investigate the role of this protein in lymphocyte signalling.

What effect did the absence of Card11 have on lymphocyte signalling? Card11^−/− T cells showed impaired proliferation and reduced production of interleukin-2 after antigen-receptor stimulation, and failed to proliferate in response to activation of protein kinase C (PKC). Card11^−/− B cells also showed defective proliferation and impaired cell-cycle progression after antigen-receptor stimulation. In addition, the proliferation of Card11^−/− B cells in response to lipopolysaccharide (LPS) — the ligand for Toll-like receptor 4 (TLR4) — was reduced. So, Card11 seems to be required for the transduction of signals that are downstream of antigen receptors and TLRs.

Further examination of the signalling events in Card11^−/− lymphocytes showed that the proliferation defects result from impairments in the activation of JUN N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) signalling pathways, and confirmed that Card11 functions downstream of PKC activation. Finally, the authors tested the response of Card11^−/− mice to T-cell-dependent and -independent antigens, and showed that Card11 is required for the activation of mature T and B cells in vivo.

Using the chemical mutagen ethylnitrosourea (ENU), Jun et al. generated a library of random, genome-wide point mutations to try to identify regulators of immune responses. Amongst others, they derived a mutant mouse strain, termed unmodulated, in which high levels of cell-surface IgM antigen receptors are expressed by circulating IgD⁺ B cells. Mapping and sequencing experiments showed that the phenotype resulted from a point mutation in the coiled-coil (CC) domain of Card11, which is thought to destroy the structure of the CC domain, but the mutated Card11 protein is expressed at normal levels.

B cells from unmodulated mice showed defective proliferation in response to antigen-receptor stimulation, but the response of these cells to LPS was normal. T-cell receptor (TCR)-mediated activation was also normal, but co-stimulation through CD28 was impaired. In agreement with the findings in the Card11^−/− mice, the authors of this study found that the defective B-cell proliferative responses in the unmodulated mice resulted from impaired activation of JNK and NF-κB signalling pathways.

The levels of IgM and IgG3 in the sera of unmodulated mice were markedly reduced, which led Jun et al. to investigate B-cell antibody responses in these mice. Responses to both T-cell-dependent and -independent antigens were defective in these mice, with T helper 1 (T_H1) responses being more severely affected than T_H2 responses. In addition, high levels of serum IgE developed with increasing age, resulting in spontaneous atopy in these mice. The authors concluded that Card11 has a crucial role in regulating humoral immunity and atopy.