MALT1 Proteolytic Activity Suppresses Autoimmunity in a T Cell Intrinsic Manner

Abstract

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.

Keywords: Breg; CTLA-4; Treg; autoimmunity; inflammation; lymphocyte; paracaspase; protease.

Figure 1

Malt1-PD and Malt1-PDT mice display ataxia and weight retardation. (A,B) Representative pictures of Malt1-PD and -PDT mice suffering from ataxia, characterized by leg clutching. (C) Ataxia onset in male and female control, Malt1-PD and -PDT mice monitored from week 8 to 15 (male mice: control n = 41, Malt1-PD n = 16, and Malt1-PDT n = 8; female mice: control n = 37, Malt1-PD n = 26 and Malt1-PDT n = 7). (D) Weight retardation in male and female Malt1-PD and -PDT mice monitored from week 8-12 until termination. Values after week 12 are excluded due to a too low number of surviving Malt1-PDT mice, causing very broad 95% confidence intervals. Male mice: week 8: control n = 41, Malt1-PD n = 9 and Malt1-PDT n = 6; week 9: control n = 49, Malt1-PD n = 8 and Malt1-PDT n = 8; week 10: control n = 50, Malt1-PD n = 11 and Malt1-PDT n = 7; week 11: control n = 61, Malt1-PD n = 16 and Malt1-PDT n = 8; week 12: control n = 59, Malt1-PD n = 16 and Malt1-PDT n = 7 and female mice: week 8: control n = 37, Malt1-PD n = 10 and Malt1-PDT n = 3; week 9: control n = 49, Malt1-PD n = 18 and Malt1-PDT n = 6; week 10: control n = 50, Malt1-PD n = 23 and Malt1-PDT n = 5; week 11: control n = 56, Malt1-PD n = 27 and Malt1-PDT n = 6; week 12: control n = 54, Malt1-PD n = 26 and Malt1-PDT n = 6.

Figure 2

Multi-organ inflammation in Malt1-PD and Malt1-PDT mice. (A,B) Representative pictures of stomach of Malt1-PD and -PDT mice with increased vascularization compared to their corresponding controls. (C,D) H&E staining showing immune infiltration in the submucosa of the stomach of Malt1-PD (C) and Malt1-PDT (D) mice. Scale bar 100 μm. (E,F) H&E staining showing immune infiltration in the salivary gland of Malt1-PD (E) and Malt1-PDT (F) mice. Scale bar 100 μm. (G,H) H&E staining showing immune infiltration in the lacrimal gland of Malt1-PD (G) and Malt1-PDT (H) mice. Scale bar 100 μm.

Figure 3

Inflammation in Malt1-PD and Malt1-PDT mice is local and not systemic. (A) CD4⁺ T cells in Malt1-PD (top panel) and Malt1-PDT (bottom panel) mice. (B) CD44⁺CD4⁺ T cells in Malt1-PD (top panel) and Malt1-PDT (bottom panel) mice. (C) CD44⁺CD4⁺ T cells producing IFN-γ in Malt1-PD (top panel) and Malt1-PDT (bottom panel) mice. For (A–C) lymphocytes from cLN were stimulated for 4 h with PMA/ionomycin and brefeldin A to determine the different cell populations by flow cytometry. Malt1-PD mice: n = 11, corresponding control mice: n = 12; Malt1-PDT mice: n = 6, corresponding control mice: n = 9. (D) Serum levels of IL-2, IL-4, IL-6, IL-17, IFN-γ, and TNF in Malt1-PD (top panels) and Malt1-PDT (bottom panels) mice. Data represent three separate serum collections: collection 1 = filled squares, collection 2 = open squares and collection 3 = open circles. Malt1-PD mice: n = 10, corresponding control mice: n = 11 and Malt1-PDT mice: n = 11, corresponding control mice: n = 10. The mean ± SEM is indicated on the graphs. The statistical significance between groups was calculated with an unpaired 2 tailed Student's t-test: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Figure 4

Reduced Treg frequency and reduced surface CTLA-4 expression on Tregs and effector CD4⁺ T cells in Malt1-PD and Malt1-PDT mice. (A,B) Treg frequency (Foxp3⁺CD25⁺CD4⁺CD8⁻ T cells) in thymus of young Malt1-PD (n = 6) (A) and Malt-PDT (n = 3) (B) mice and their corresponding controls (n = 5 and n = 3, respectively). (C,D) Treg frequency in cLN of young Malt1-PD (n = 6) (C) and Malt1-PDT (n = 3) (D) mice and their corresponding controls (n = 5 and n = 3, respectively). (E,F) Treg frequency in Malt1-PD (n = 11) (E) and Malt1-PDT (n = 6) (F) mice suffering from ataxia and their corresponding controls (n = 12 and n = 9, respectively). Lymphocytes were stimulated for 4 h with PMA/ionomycin and the data represent three individual experiments: experiment 1 = filled squares, experiment 2 = open squares and experiment 3 = open circles. (G,H) Normalized CTLA-4 expression on the surface of Tregs (G) and CD44⁺CD4⁺ T cells (H) from young disease free Malt1-PDT mice (n = 15) and their corresponding controls (n = 15). The individual percentages of Foxp3⁺CD4⁺ T cells or CD44⁺CD4⁺ T cells that express CTLA-4 on their surface is normalized against the average percentage of the corresponding control mice of each individual experiment. Lymphocytes were stimulated for 4 h with PMA/ionomycin and data represent two individual experiments: experiment 1 = filled squares, experiment 2 = open squares. For (A–H): all data were obtained via flow cytometry. The mean ± SEM is indicated on the graphs. The statistical significance between groups was calculated with an unpaired 2 tailed Student's t-test: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Figure 5

Bregs are almost absent in Malt1-PD mice, but are present in Malt1-PDT mice. IL-10 secretion by control (n = 4), Malt1-PD (n = 3), and Malt1-PDT (n = 3) splenic B cells after overnight stimulation with TLR9 ligand CpG. The mean ± SEM is indicated on the graphs. The statistical significance between groups was calculated with an unpaired 2 tailed Student's t-test: ^*p < 0.05 and ^**p < 0.01.

Figure 6

Expression of hMALT1 in T cells of Malt1-PD mice rescues the disease phenotype. (A,B) H&E staining of stomach (A) and salivary gland (B) of Malt1-PD (PD) and Malt1-PD mice expressing wild type human MALT1 in T cells (Malt1^PD/- Rosa26^LSL-MALT1-WT CD4-Cre = PD hWT-T) mice. (C) Percentage of CD44⁺CD4⁺ T cells producing IFN-γ in PD and PD hWT-T mice. (D) Treg frequency (Foxp3⁺CD25⁺CD4⁺CD8⁻ T cells) in thymus of PD and PD hWT-T mice. (E) Treg frequency in cLN of PD and PD hWT-T mice. For (C–E): all data were obtained via flow cytometry and for (E), lymphocytes were stimulated for 4 h with PMA/ionomycin. Data represent two individual experiments: experiment 1 = open squares and experiment 2 = filled squares. Malt1-PD mice: total n = 7 and Malt1-PD hWT-T mice: total n = 5, age 13–18 weeks. The mean ± SEM is indicated on the graphs. The statistical significance between groups was calculated with an unpaired 2 tailed Student's t-test: ^*p < 0.05 and ^****p < 0.0001.