MALT1 is a central signaling component in innate and adaptive immunity by regulating NF-κB and other key signaling pathways in different cell types. Activities of MALT1 are mediated by its scaffold and protease functions. Because of its role in lymphocyte activation and proliferation, inhibition of MALT1 proteolytic activity is of high interest for therapeutic targeting in autoimmunity and certain lymphomas. However, recent studies showing that Malt1 protease-dead knock-in ( Malt1 -PD) mice suffer from autoimmune disease have somewhat tempered the initial enthusiasm. Although it has been proposed that an imbalance between immune suppressive regulatory T cells (Tregs) and activated effector CD4 + T cells plays a key role in the autoimmune phenotype of Malt1 -PD mice, the specific contribution of MALT1 proteolytic activity in T cells remains unclear. Using T cell-conditional Malt1 protease-dead knock-in ( Malt1 -PDT) mice, we here demonstrate that MALT1 has a T cell-intrinsic role in regulating the homeostasis and function of thymic and peripheral T cells. T cell-specific ablation of MALT1 proteolytic activity phenocopies mice in which MALT1 proteolytic activity has been genetically inactivated in all cell types. The Malt1 -PDT mice have a reduced number of Tregs in the thymus and periphery, although the effect in the periphery is less pronounced compared to full-body Malt1 -PD mice, indicating that also other cell types may promote Treg induction in a MALT1 protease-dependent manner. Despite the difference in peripheral Treg number, both T cell-specific and full-body Malt1 -PD mice develop ataxia and multi-organ inflammation to a similar extent. Furthermore, reconstitution of the full-body Malt1 -PD mice with T cell-specific expression of wild-type human MALT1 eliminated all signs of autoimmunity. Together, these findings establish an important T cell-intrinsic role of MALT1 proteolytic activity in the suppression of autoimmune responses.

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Summary The protease MALT1 is a key regulator of NF-κB signaling and a novel therapeutic target in autoimmunity and cancer. Initial enthusiasm supported by preclinical results with MALT1 inhibitors was tempered by studies showing that germline MALT1 protease inactivation in mice results in reduced regulatory T cells and lethal multi-organ inflammation due to expansion of IFN-γ-producing T cells. However, we show that long-term MALT1 inactivation, starting in adulthood, is not associated with severe systemic inflammation, despite reduced regulatory T cells. In contrast, IL-2-, TNF-, and IFN-γ-producing CD4 + T cells were strongly reduced. Limited formation of tertiary lymphoid structures was detectable in lungs and stomach, which did not affect overall health. Our data illustrate that MALT1 inhibition in prenatal or adult life has a different outcome and that long-term MALT1 inhibition in adulthood is not associated with severe side effects.

Summary T cells play a key role in adaptive immunity. Defects in specific T cell receptors or signaling proteins can alter their frequency and activation status, which may be associated with immune disease or cancer. Monitoring of T cell frequency and function in genetically modified mice or murine models of disease is therefore of high interest. Here, we provide a detailed protocol to analyze regulatory T cells, T cell activation, and cytokine production in thymus, spleen, or blood via flow cytometry. For complete details on the use and execution of this protocol, please refer to Demeyer et al. (2020) .