T cells from Cbl-b-deficient mice can be activated in the absence of co-stimulation, leading to spontaneous autoimmunity, but the mechanism responsible for this has been unclear until now. In an ionomycin-induced model of T-cell anergy, wild-type T cells showed a 90% reduction in proliferation in response to CD3- and CD28-specific antibodies after ionomycin treatment, and this was associated with an increase in Cbl-b expression. By contrast, Cbl-b-deficient T cells showed a 40% reduction in proliferation, showing that lack of Cbl-b inhibits anergy induction. For wild-type T cells, the induction of anergy by ionomycin can be attributed partly to reduced calcium mobilization, and the authors showed that calcium responses are maintained in the absence of Cbl-b. They explain this by showing that Cbl-b activity during anergy induction results in a reduced level of phosphorylated (active) phospholipase C-γ1 (PLC-γ1), which in turn prevents the release of calcium from intracellular stores. In the absence of Cbl-b, phosphorylated PLC-γ1 accumulates, and calcium can be mobilized. Interestingly, although Cbl-b — which is best known for its role in ubiquitylation-dependent proteasomal degradation of proteins — did induce ubiquitylation of PLC-γ1 in wild-type T cells after ionomycin treatment, this was not associated with increased degradation, in contrast to previous suggestions (see Further reading). The authors suggest that rapid ubiquitylation-dependent protein modifications (such as dephosphorylation) are more important than degradation during the early phases of anergy induction.
To show the relevance of these results in vivo, mice that express a transgenic T-cell receptor (TCR) specific for an MHC-class-I-restricted non-self-peptide were crossed with Cbl-b-deficient mice. Repeated injection of TCR-transgenic mice with the non-self-peptide resulted in peptide-specific CD8+ T-cell anergy, but on the Cbl-b-deficient background, rechallenge with the peptide resulted in increased T-cell proliferation, which was associated with increased mortality. Whereas all Cbl-b-sufficient TCR-transgenic mice survived a second challenge with peptide, 50% of those lacking Cbl-b died, as a result of high levels of T-cell cytokine production. Similarly, the authors showed that Cbl-b controls in vivo tolerance of antigen-specific CD4+ T cells. This demonstration of the importance of the Cbl-b pathway of anergy induction in vivo leads the way for further studies of the physiological role of the E3 ubiquitin ligases GRAIL and ITCH in T-cell tolerance, both of which have been associated with anergy induction in vitro.
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