Unrestrained cleavage of Roquin-1 by MALT1 induces spontaneous T cell activation and the development of autoimmunity

Abstract

Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1^TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1^Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1^Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1^TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1^Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκB_NS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1^Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity.

Keywords: RNA-binding protein; T cell differentiation; antigen receptor signaling; autoimmunity; paracaspase.

Fig. 1.

Continuous Roquin cleavage leads to autoimmunity. (A) Western blot analysis of protein extracts taken from total lymph node cells of WT, Rc3h1^Mins/Mins (Mins/Mins), Malt1^TBM/TBM (TBM/TBM), and combined Malt1^TBM/TBM; Rc3h1^Mins/Mins (TBM/TBM; Mins/Mins) mice. Spontaneous Roquin cleavage and upregulation of IκB_NS is prevented in TBM/TBM; Mins/Mins mice. (B) Representative flow cytometry analysis of Roquin cleavage pregated on CD4⁺ T cells (n = 6–10). (C) Kaplan–Meier plot showing survival of WT, TBM/TBM, and TBM/TBM; Mins/Mins mice (n = 5–51). Combining Malt1-insensitive Roquin-1 rescues survival of mice with constitutive MALT1 activity. (D) Representative spleen sizes and quantification of spleen weights and numbers of splenocytes. (E) Quantification of frequencies and numbers of CD69⁺ CD3⁺ cells in spleens. (F) Representative plots and quantifications of peripheral CD4⁺ T cell activation from spleens of WT, Mins/Mins, TBM/TBM and TBM/TBM; Mins/Mins mice showing naive (CD62L^hi CD44^–), central memory (CD62L^hi CD44⁺), and effector memory (CD62L^low CD44⁺) T cells (n = 6–10). (G) Representative plot and quantification of the direct Roquin target IκB_NS in splenic CD4⁺ T cells (n = 6–10). (H) Quantification of the direct Roquin target expression Ox40, IRF4, or ICOS expression in splenic CD4⁺ T cells, respectively (n = 6–10). Error bars represent mean ± SEM. CM: central memory, EM: effector memory, MFI: median fluorescence intensity. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.

Correlating Roquin cleavage and target derepression with TCR strength. (A) Representative plots and (B) quantification of Roquin cleavage (20G6) and CD69 expression in T cells stimulated with the indicated anti-CD3 and fixed anti-CD28 (10 µg/mL) concentrations for 18 h. (B) Quantifications display MFI (median fluorescence intensity) of CD69, cleaved Roquin (20G6), and IκB_NS (4C1) (n = 5). Data presented as mean ± SD. Statistical analysis was performed using an unpaired Student’s t test, two-tailed. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (C) In vitro coculture of naive WT OT-II CD4⁺ T cells with OVA_323–339 peptide-loaded bone marrow derived dendritic cells (BMDCs) for 18 h followed by flow cytometry to detect Ox40 and IκB_NS (4C1) expression for OT-II T cells (pregated on Vα₂⁺/Vβ₅⁺ TCR). Ox40, IκB_NS (4C1), and surface CD69 expression were analyzed in stimulations with increasing concentrations of WT OVA_323-339 peptide. (D) Quantification of Roquin target expression in OT-II T cells from cocultures shown in C, depicting the fold change (fc) in gMFI values of Ox40 or IκB_NS (4C1) after stimulation in comparison to unstimulated cells. Data are presented as mean ± SEM (n = 3–6). (E) Representative plots of Ox40 and IκB_NS (4C1) expression in iDKO CD4⁺ T cells retrovirally reconstituted with WT or mutant GFP-Roquin-1 constructs. (F) Relative quantification of target expression as in (E) in GFP^– cells and within 7 equal-sized gates corresponding to increasing GFP-Roquin-1 expression. WT and mutant GFP-Roquin-1 constructs are color-coded, and individual gMFI values (per target) were normalized to maximum levels observed in iDKO CD4⁺ T cells without retroviral transduction. Data are presented as mean ± SEM (n = 3–4). Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 3.

Decreasing Roquin activity and increasing TCR signal strength promote Th17 fate decisions. (A) Western blot analysis of Roquin-1 and Roquin-2 and IkB_NS in extracts from Th1-polarized CD4⁺ T cells cultured for 6 d. Depicted is a representative of three experiments. Quantification of (B) IκB_NS and (C) Ox40 expression determined in flow cytometry analyses of Th1 cells after 6d of in vitro culture. Data are presented as fold change of geometric mean fluorescence intensity (gMFIs) (n = 5–7). (D) Naive CD4⁺ T cells from WT, heterozygous Rc3h1^fl/+; Rc3h2^fl/+; Cd4-Cre, Rc3h1^fl/fl; Rc3h2^fl/+; Cd4-Cre or Rc3h1^fl/fl; Rc3h2^fl/fl; Cd4-Cre (i.e., double knockout; DKO) mice were activated with anti-CD3 and anti-CD28 antibodies and cultured for 3.5 d in vitro under Th17 polarizing conditions. T helper cell differentiation was assessed by i.c. cytokine staining of IL-17A production after P/I stimulation (n = 4) (D and E). (F) Naive WT OT-II transgenic T cells were cocultured with OVA_323–339 peptide-loaded BMDCs for 3.5 d in vitro under Th17 polarizing conditions. OT-II T cell differentiation (pregated on TCR Vα₂⁺Vβ₅⁺) in response to increasing concentrations of peptides is shown in a representative contour plot (F) or quantification (G) of IL-17A producing OT-II T after restimulation with P/I (n = 3–7). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (H and I) Naive CD4⁺ T cells from WT, Rc3h1^Mins/+, and Rc3h1^Mins/Mins mice were activated with anti-CD3 and anti-CD28 antibodies and cultured for 3.5 d in vitro under Th17 polarizing conditions. T helper cell differentiation was assessed by i.c. cytokine staining of IL-17A production after P/I stimulation. (J and K) In vitro Th17 differentiation of WT, Malt1^+/−, and Malt1^PM/− T cells as described in (H and I). (L and M) In vitro Th17 differentiation of WT, Nfkbid^fl/+;Cd4-cre, and Nfkbid^fl/fl;Cd4-cre T cells as described in (H and I) (n = 2–3). Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. Data are presented as mean ± SEM. i.c.: intracellular, P/I: PMA/Ionomycin. Statistical analysis was performed using an unpaired Student’s t test, one-tailed. *P < 0.05, **P < 0.01, ***P < 0.001, and **** P < 0.0001.

Fig. 4.

Malt1-insensitive Roquin-1 protects from experimentally induced autoimmune encephalomyelitis. (A–D) WT or (E) IL17A reporter mice were immunized with MOG peptide in complete Freund's adjuvant (s.c.) and injected with Pertussis toxin (PTX, i.v.) on day 0 and day 2. On the peak of EAE, mice were killed, and Roquin cleavage and/or IκB_NS expression were assessed. (A) Representative plots of CD4⁺ T cells in spleens and CNS of immunized mice. (B) Representative plots and quantifications of Roquin cleavage and IκB_NS expression in CD4⁺ T cells from CNS (red) and spleen (black) (n = 4). (C) Representative plots of CCR6⁺ (Th17) and CXCR3⁺ (Th1) CD4⁺ T cells from spleens and CNS of immunized mice. (D) Representative plots and quantification of Roquin cleavage in Th17 vs. Th1 cells in the CNS (n = 4). Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired Student’s t test, one-tailed. (E) Roquin cleavage in hNGFR⁺ (i.e., IL17A producing) or hNGFR^– CD4⁺ T cells in the CNS of mice at the peak of EAE (n = 4). (F) Average clinical EAE scores from day 9 to day 15 of WT and Mins/Mins mice immunized with MOG peptide as described in (A–C). (G) Quantification of total numbers of CNS-infiltrating CD4⁺ T lymphocytes in WT and Mins/Mins mice at day 15 (peak) of EAE isolated from spinal cords and brains (pooled per mouse) (n = 4). (H) Representative plots and quantifications of cytokine production by CNS infiltrating CD4⁺ T cells of WT and Mins/Mins mice at day 15 (peak) of EAE after 4 h of restimulation with P/I. (I) Naive CD4⁺ T cells from WT; 2D2 or Mins/Mins; 2D2 mice were adoptively transferred into Rag1^–/– recipients, and EAE was induced as described in (A–C). Average clinical EAE scores from day 9 to day 15 (n = 7). CNS: central nervous system, MFI: median fluorescence intensity, P/I: PMA/Ionomycin. Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired Student’s t test, one-tailed, for each time point. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.