Aberrant RNA sensing in regulatory T cells causes systemic autoimmunity

Domnica Luca; Sumin Lee; Keiji Hirota; Yasutaka Okabe; Junji Uehori; Kazushi Izawa; Anna-Lisa Lanz; Verena Schütte; Burcu Sivri; Yuta Tsukamoto; Fabian Hauck; Rayk Behrendt; Axel Roers; Takashi Fujita; Ryuta Nishikomori; Min Ae Lee-Kirsch; Hiroki Kato

doi:10.1126/sciadv.adk0820

. 2024 Mar 1;10(9):eadk0820. doi: 10.1126/sciadv.adk0820

Aberrant RNA sensing in regulatory T cells causes systemic autoimmunity

Domnica Luca ^1,^†, Sumin Lee ^2,^3,^†, Keiji Hirota ^1,⁴, Yasutaka Okabe ^5,⁶, Junji Uehori ⁷, Kazushi Izawa ⁸, Anna-Lisa Lanz ^9,¹⁰, Verena Schütte ¹, Burcu Sivri ¹, Yuta Tsukamoto ¹, Fabian Hauck ^9,¹⁰, Rayk Behrendt ¹¹, Axel Roers ¹², Takashi Fujita ^1,^2,³, Ryuta Nishikomori ¹³, Min Ae Lee-Kirsch ^14,¹⁵, Hiroki Kato ^1,^*

^¹Institute of Cardiovascular Immunology, Medical Faculty, University Hospital Bonn, University of Bonn, Bonn, Germany.

^²Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

^³Laboratory of Regulatory Information, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan.

^⁴Laboratory of Integrative Biological Science, Institute for Life and Medical Sciences, Kyoto University, Kyoto, Japan.

^⁵Laboratory of Immune Homeostasis, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan.

^⁶Center for Infectious Disease Education and Research, Osaka University, Osaka, Japan.

^⁷Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.

^⁸Department of Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan.

^⁹Division of Pediatric Immunology and Rheumatology, Department of Pediatrics, Dr. von Hauner Children's Hospital, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

^¹⁰Munich Centre for Rare Diseases (M-ZSE), University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

^¹¹Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany.

^¹²Institute of Immunology, University of Heidelberg, Heidelberg, Germany.

^¹³Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan.

^¹⁴Department of Pediatrics, University Hospital Carl Gustav Carus and Medical Faculty, Technische Universität Dresden, Dresden, Germany.

^¹⁵University Center for Rare Diseases, University Hospital Carl Gustav Carus and Medical Faculty, Technische Universität Dresden, Dresden, Germany.

Corresponding author. Email: hkato@uni-bonn.de

^†

These authors contributed equally to this work.

Roles

Domnica Luca: Conceptualization, Formal analysis, Investigation, Project administration, Validation, Visualization, Writing - original draft, Writing - review & editing

Sumin Lee: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review & editing

Keiji Hirota: Conceptualization, Methodology

Yasutaka Okabe: Investigation, Resources, Writing - review & editing

Junji Uehori: Resources

Kazushi Izawa: Conceptualization, Investigation, Resources

Anna-Lisa Lanz: Resources

Verena Schütte: Project administration

Burcu Sivri: Investigation, Validation

Yuta Tsukamoto: Investigation, Project administration, Validation

Fabian Hauck: Resources, Writing - review & editing

Rayk Behrendt: Conceptualization, Methodology, Resources

Axel Roers: Methodology, Resources

Takashi Fujita: Conceptualization, Project administration, Resources, Supervision

Ryuta Nishikomori: Conceptualization, Funding acquisition, Investigation, Resources, Supervision, Validation

Min Ae Lee-Kirsch: Funding acquisition, Investigation, Resources, Validation, Writing - review & editing

Hiroki Kato: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing - original draft

PMCID: PMC10906915 PMID: 38427731

Abstract

Chronic and aberrant nucleic acid sensing causes type I IFN–driven autoimmune diseases, designated type I interferonopathies. We found a significant reduction of regulatory T cells (T_regs) in patients with type I interferonopathies caused by mutations in ADAR1 or IFIH1 (encoding MDA5). We analyzed the underlying mechanisms using murine models and found that T_reg-specific deletion of Adar1 caused peripheral T_reg loss and scurfy-like lethal autoimmune disorders. Similarly, knock-in mice with T_reg-specific expression of an MDA5 gain-of-function mutant caused apoptosis of peripheral T_regs and severe autoimmunity. Moreover, the impact of ADAR1 deficiency on T_regs is multifaceted, involving both MDA5 and PKR sensing. Together, our results highlight the dysregulation of T_reg homeostasis by intrinsic aberrant RNA sensing as a potential determinant for type I interferonopathies.

Dysregulated regulatory T cells contribute to the onset of systemic autoimmunity in patients with type I interferonopathies.

INTRODUCTION

Type I interferonopathies, including Aicardi-Goutières syndrome (AGS), are rare monogenic autoinflammatory diseases commonly characterized by continuous production of antiviral type I interferons (IFN-I) and a striking variety of symptoms (1, 2). AGS is caused by mutations in genes that are involved in nucleic acid metabolism or sensing, including loss-of-function mutations in TREX1, SAMHD1, RNASEH2A-C, and ADAR1 and gain-of-function mutations in IFIH1, which encodes the double-stranded RNA (dsRNA) sensor MDA5 (3–7). While the AGS-causing genes function as components of innate immune pathways, some patients with AGS develop signs of systemic lupus erythematosus (SLE), a paradigm autoimmune disease (8, 9). ADAR1 catalyzes the editing of adenosine to inosine in dsRNA and thereby prevents the recognition of self-RNA by MDA5 (10). ADAR1 deficiency causes the aberrant production of IFN-I with up-regulation of IFN-stimulated genes (ISGs) designated IFN signature. In murine models, ADAR1 deficiency causes embryonic lethality, which is delayed by concurrent deletion of MDA5 or MAVS (10–12). Furthermore, ADAR1 deficiency activates PKR, OAS/RNase L, and ZBP1 (13–16), leading to transcriptional arrest and cell death via apoptosis or necroptosis. Naturally occurring regulatory T cells (T_regs) express the transcriptional factor FOXP3 and are indispensable for maintaining immune tolerance (17). T_reg loss or dysfunction caused by mutations in FOXP3 or other T_reg signature genes results in severe or fatal autoimmune disease in humans and mice (18–23). In mouse models of viral infection and inflammatory disease, IFN-I can diminish the immunosuppressive capacity of T_regs (24, 25). However, the contribution of T_regs to the pathogenesis of type I interferonopathies remains unclear. Here, we investigated the T_reg population in patients with AGS carrying mutations in ADAR1 or IFIH1 and analyzed the changes potentially resulting in pathogenesis.

RESULTS

Patients with AGS have a decreased frequency of peripheral effector T_regs

To examine the T_reg population in peripheral blood mononuclear cells (PBMCs) from patients with ADAR1 or IFIH1 mutations (table S1), we gated three primary fractions out of the CD4⁺ T cell population (Fig. 1A), based on the expression level of CD25 and CD45RA: CD25^lowCD45RA⁺ suppressive resting T_regs (Fr. I), CD25^hiCD45RA⁻ highly suppressive effector T_regs (Fr. II), and CD25^lowCD45RA⁻(FOXP3^low) nonsuppressive T cells (Fr. III), as reported previously (26). We found no difference between the percentages of resting T_regs (Fr. I); however, the effector T_regs (Fr. II) that are considered the primary suppressive T_regs were significantly decreased in patients with AGS compared to controls (Fig. 1B). The FOXP3^low nonsuppressive T cells (Fr. III) were also significantly reduced (Fig. 1B). Notably, we closely analyzed the T_reg populations of two patients (1 and 4) at different time points and observed a continual reduction of effector T_regs in Fr. II (fig. S1A). Considering that all patients were children at the time of analysis, we compared PBMCs from healthy adults (aged 20 to 50 years) and children (aged 2 to 18 years) and found a similar percentage of 0.5 to 2% of effector T_regs in Fr. II in both groups (fig. S1B). The inhibition of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, primarily by blocking JAK1 and JAK2, is a favorable treatment for some patients with type I interferonopathies (27–30). The inhibition of JAK3, which forms a dimer with JAK1 in interleukin-2 (IL-2)/IL-2R signaling, has been reported to reversibly down-regulate the expression of FOXP3 in T_regs (31). Notably, we detected no significant difference in fractions I to III as well as CD4⁺CD25⁺FOXP3^hi cells between untreated and JAK inhibitor–treated patients with AGS (fig. S1C).

Fig. 1. — (A) Representative flow cytometry (FC) plots of CD25 and CD45RA expression on CD4⁺ T cells from controls or patients with AGS. Fr. I: CD25^lowCD45RA⁺ suppressive resting T_regs, Fr. II: CD25^hiCD45RA⁻ highly suppressive effector T_regs, and Fr. III: CD25^lowCD45RA⁻ (FOXP3^low) nonsuppressive T cells. (B) Summarized percentages of Fr. I, Fr. II, and Fr. III from all controls and patients with AGS analyzed in this study. (C to F) Representative histograms of CTLA-4 and PD-1 expression on Fr. I (solid line) and Fr. II (dotted line) and summarized mean fluorescence intensity (MFI) values from controls (black) and patients with AGS (yellow), relative to Fr. I in healthy controls (HC). The samples from patients with AGS have been analyzed one at a time (in two instances, two at a time), together with control samples from healthy donors. The dot plots shown here contain pooled data from respective analyses. Samples from patients 1 and 4 have been analyzed at four [three for CTLA-4 and PD-1 expression; (D) and (F) and fig. S1D] and two different time points (fig. S1A), and the mean is represented as one symbol in pooled-data dot plots (B, D, and F); otherwise, each symbol represents one individual. Statistics were calculated using Student’s t test with Welch’s correction (B) and one-way ANOVA (D and F); *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns, not significant, P > 0.05.

T_regs express cytotoxic T lymphocyte–associated protein 4 (CTLA-4) that is crucial for their suppressive function, primarily by blocking CD80/CD86 signaling on antigen-presenting cells (32, 33). Consistent with previous studies, CTLA-4 was highly expressed in effector T_regs and less expressed in resting T_regs in healthy controls (26), whereas there was no difference in its expression between controls and patients with AGS (Fig. 1, C and D).

Programmed cell death protein 1 (PD-1) is known to inhibit T cell antigen receptor signaling, which is required for T_reg functions (34), and its blockade has been reported to augment T_reg suppressive capacity (35–37). We similarly found its expression to be higher on Fr. II compared to Fr. I of healthy controls (Fig. 1, E and F). On Fr. II of effector T_regs, PD-1 expression was significantly increased in patients with AGS compared to healthy controls (Fig. 1, E and F). Notably, in patients 1 and 4, PD-1 expression was continually increased at different time points we have analyzed (fig. S1D). This suggests a possible attenuation of effector T_reg suppressive activity along with the significant reduction of Fr. II in patients with AGS.

Adar1 deletion in T_regs causes T_reg loss and a scurfy-like lethal phenotype in mice

The availability of samples from patients with AGS is limited; therefore, we used murine models to gain deeper mechanistic insights on the effects of ADAR1 deficiency and constitutive MDA5 signaling in T_regs. Because systemic deletion of Adar1 results in embryonic lethality (11), we aimed to assess the intrinsic effect of ADAR1 ablation in T_regs and generated mice with T_reg-specific Adar1 deletion. We intercrossed Adar1^fl mice with Foxp3^YFP-Cre mice and generated Foxp3^Cre/Cre Adar1^fl/fl female or Foxp3^Cre/Y Adar1^fl/fl male mice, herein referred to as Foxp3^ΔAdar1. The Foxp3^ΔAdar1 mutant mice exhibited growth retardation (Fig. 2, A and B) compared to littermate control mice—Foxp3^Cre/Cre or Foxp3^Cre/Y Adar1^+/+, referred to as Foxp3-WT (wild type). Mutant mice died within 4 weeks of birth (Fig. 2C) and exhibited scaly skin on the tail, ears, and eyelids. By 3 weeks of age, these mice also exhibited general splenomegaly, lymphadenopathy (fig. S2, A and B), and thymic atrophy. On the basis of histologic evaluation, Foxp3^ΔAdar1 mice exhibited severe tissue damage with immune cell infiltration in the skin dermis, liver parenchyma, lung interstitium, kidney, and intestine (Fig. 2D and fig. S2C). We also noted the up-regulation of chemokines and cytokines, including Cxcl10, in the spleen, liver, kidneys, and lymph nodes, along with the up-regulation of Il-6 in the spleen and lymph nodes (fig. S2D). These phenotypes resemble those of the scurfy mice with Foxp3 mutations (33). Considering that autoimmune symptoms in scurfy mice are caused by the loss of T_regs (38), we subsequently examined the T_reg compartment and found an almost complete depletion of FOXP3⁺ T_regs in the spleen and lymph nodes of 3-week-old Foxp3^ΔAdar1 mutant mice (Fig. 2, E and F, and fig. S2, E to G). In contrast, the T_reg-specific deletion of another AGS-related gene, Trex1, using the same Foxp3^YFP-Cre mice, did not change the percentage and total number of T_regs in the spleen (Fig. 2, G and H, and fig. S2H). We detected a larger percentage of splenic FOXP3⁺ T_regs in mice at an earlier stage of growth, such as 1-week-old Foxp3^ΔAdar1 mice, compared to 3-week-old mice, albeit lower than in age-matched littermate controls (fig. S3A). Furthermore, 1-week-old Foxp3^ΔAdar1 mice exhibited intact thymuses, and we found no differences in the percentages of thymic FOXP3⁺ T_regs as well as CD4⁺, CD8⁺, and CD4⁺CD8⁺ T cells (fig. S3, A and B). These data suggest that ADAR1 deficiency did not impair thymic T_reg development and its distribution to secondary lymphoid organs. We analyzed the activation status of T cells by staining CD44 and CD62L. This revealed that both CD4⁺ and CD8⁺ T cells from Foxp3^ΔAdar1 mice exhibited a clear shift from a naïve (CD44⁻CD62L⁺) to an effector phenotype (CD44⁺CD62L⁻) (Fig. 2, I and J, and fig. S4, A and B). In contrast, the naïve and effector T cells in Foxp3^ΔTrex1 mice were similar to those in littermate controls (fig. S4, C and D).

Fig. 2. — (A and B) Pictures and body weight measurements of 3-week-old *Foxp3*-WT (n = 44) and *Foxp3*^ΔAdar1 (n = 36) mice. (C) Survival graphs of *Foxp3*-WT and *Foxp3*^ΔAdar1 mice (n = 7, but all *Foxp3*^ΔAdar1 mice developed a severe phenotype and were sacrificed at latest 4 weeks after birth). (D) Representative H&E staining images. S. intestine, small intestine. Scale bars, 100 μm. (E to H) Representative FC plots and summarized percentages (%) of CD4⁺FOXP3⁺ T_regs in the spleens. (I and J) Representative FC plots and summarized percentages of naïve (CD44⁻CD62L⁺), effector (CD44⁺CD62L⁻), and memory (CD44⁺CD62L⁺) CD4⁺ T cells in the spleens. (K) ELISA heatmap of indicated cytokines measured in supernatants from enriched CD4⁺ T cells (pg/ml), stimulated overnight with anti-CD3/anti-CD28 antibody–coated beads. (L) Representative FC plots of FOXP3⁺ versus GATA3⁺CD4⁺ T cells after overnight culture (without stimulation). Panels (E) to (J) are representative of ≥3 independent experiments with ≥3 mice per group. Panels (K) and (L) are representative of two experiments with two mice per group. In dot plots, each symbol indicates an individual mouse. Statistics were calculated using Student’s t test; ****P ≤ 0.0001; ns, not significant, P > 0.05.

Defective control of T helper cell 2 (T_H2)–related cytokines has been reported in the scurfy mutant (39); thus, we stimulated splenic CD4⁺ T cells using anti-CD3/anti-CD28 antibody–coated beads and monitored cytokine production. The Foxp3^ΔAdar1 mutant cells showed higher production of T_H2-related proinflammatory cytokines, such as IL-4, IL-5, IL-10, and IL-13 (Fig. 2K). Consistent with this, approximately 30% of CD4⁺ T cells from Foxp3^ΔAdar1 mice were GATA3 positive compared to <1% of Foxp3-WT CD4⁺ T cells (Fig. 2L). These data indicate that Foxp3^ΔAdar1 mice exhibit scurfy mouse–like lethal autoimmune symptoms as a consequence of T_reg loss and abnormal activation of effector T cells.

Constitutive MDA5 activation in T_regs causes T_reg loss and autoimmunity in mice

ADAR1 deficiency constitutively activates the cytoplasmic dsRNA sensor MDA5 (10). We hypothesized that the T_reg-specific constitutive activation of MDA5, by expressing the gain-of function mutant G821S of the Ifih1 gene (referred to as MDA5 G821S) (40), would result in T_reg population loss and the onset of autoimmune diseases, similar to that observed in Foxp3^ΔAdar1 mice. To examine the effect of constitutive MDA5 signaling on T_regs, we established a conditional MDA5 G821S expression system in mice (fig. S5A). In the absence of Cre recombinase, truncated, nonfunctional MDA5 proteins were expressed from the mutated allele by the insertion of a stop cassette, whereas MDA5 G821S was expressed by the deletion of the stop cassette in the presence of Cre recombinase (fig. S5B). As previously reported, mice that systemically express the MDA5 G821S mutant in this system exhibited severe growth retardation and autoimmune disorders including lupus-like nephritis (40). In the absence of Cre expression, MDA5 G821S^fl/+ mice did not exhibit any phenotypes and were comparable to WT mice. Next, for the specific expression of MDA5 G821S in T_regs, we intercrossed MDA5 G821S^fl/+ mice with Foxp3^YFP-Cre mice to generate Foxp3^Cre/Cre MDA5 G821S^fl/+ female or Foxp3^Cre/Y MDA5 G821S^fl/+ male mice, herein referred to as Foxp3-GS mice. Foxp3-GS mice also exhibited growth retardation and reduced body weight compared to littermate Foxp3-WT mice (Fig. 3, A and B). Approximately 50% of the Foxp3-GS mice survived until 8 weeks after birth, and some survived until almost 1 year (Fig. 3C), indicating a milder disease progression than that observed in Foxp3^ΔAdar1 mice, which did not survive >4 weeks after birth (Fig. 2C). We primarily used adult Foxp3-GS mice (aged 8 to 12 weeks) along with Foxp3-WT littermate controls for further analyses. Histologic evaluation of organs revealed that Foxp3-GS mice exhibited severe tissue inflammation with immune cell infiltration of the lungs, small intestine and colon, nephritis with immunoglobulin G (IgG) deposition, as well as the presence of antinuclear antibodies (ANAs) in the sera (Fig. 3, D and E). The up-regulation of Isg56, Ifn-β, Il-6, and Cxcl10 was also detected in the kidneys (Fig. 3F). Notably, we did not detect ANAs in the sera of Foxp3^ΔAdar1 mice, possibly due to their young age of 3 weeks old at the time of analysis (fig. S6). We subsequently examined the T_reg compartment in Foxp3-GS mice and found a reduction compared to littermate controls (Fig. 3, G and H). Although adult Foxp3-GS mice surviving >8 weeks generally exhibited milder loss of peripheral T_regs and autoimmune phenotypes than Foxp3^ΔAdar1 mice, these data reveal that intrinsic and constitutive MDA5 signaling in T_regs leads to a reduction of the T_reg population and triggers the onset of autoimmune symptoms.

Fig. 3. — (A and B) Pictures and body weight measurements of 4-week-old *Foxp3*-WT (n = 12) and *Foxp3*-GS (n = 9) mice. (C) Survival graph of *Foxp3*-WT (n = 12) and *Foxp3*-GS (n = 14) mice. (D) Representative H&E staining images of indicated organs. Scale bars, 100 μm. (E) Representative H&E staining images of kidneys, immunofluorescence staining images of IgG (green) in the kidneys (4′,6-diamidino-2-phenylindole (DAPI), blue), and immunofluorescence staining of L929 cells using sera from *Foxp3*-WT and *Foxp3*-GS mice. (F) Relative mRNA expression of indicated genes. (G and H) Representative FC plots, summarized percentages (%), and total numbers (#) of CD4⁺YFP⁺(FOXP3⁺) T_regs in spleens and small intestines. Panels (F) to (H) are representative of ≥3 independent experiments with ≥3 mice per group. In dot plots, each symbol indicates an individual mouse. Statistics were calculated using Student’s t test; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001; ns, not significant, P > 0.05.

Adar1 deletion in T_regs activates the PKR/eIF-2α pathway contributing to cell death

Next, we investigated whether cell death is involved in T_reg reduction in Foxp3-GS and Foxp3^ΔAdar1 mice. Flow cytometric analysis of annexin V and 7-aminoactinomycin D (7AAD) revealed that Foxp3-GS mice exhibited a higher frequency of apoptotic T_regs than control Foxp3-WT mice (Fig. 4A). We also found up-regulated mRNA expression of the pro-apoptotic gene Noxa in CD4⁺YFP⁺(FOXP3⁺) T_regs sorted from the spleens of Foxp3-GS and Foxp3^ΔAdar1 mice compared to controls (Fig. 4B). The expression of another pro-apoptotic gene Puma, as well as Isg56, was also up-regulated in Foxp3-GS T_regs, while the expression of other pro-apoptotic BH3-only genes, including Bim and Bad, and anti-apoptotic genes Bcl-2, Mcl-1, and Bcl-xL was comparable (fig. S7). Because the T_reg population in Foxp3^ΔAdar1 mice is extremely reduced, we induced T_reg differentiation by culturing enriched naïve CD4⁺ T cells with IL-2 and transforming growth factor–β (TGF-β) to further examine the apoptotic events. Ex vivo induced T_regs (iT_regs) from Foxp3^ΔAdar1 mice expressed FOXP3 similar to Foxp3-WT iT_regs but showed significant cell death that was rescued by treatment with a pan-caspase inhibitor, Q-VD-OPH (Fig. 4, C and D). To further investigate the effect of extrinsic factors on T_regs, we examined mice with Adar1 deletion or MDA5 G821S mutant expression specifically in CX3CR1-positive immune cells, indicated as Cx3cr1^ΔAdar1 and Cx3cr1-GS. We found that the FOXP3⁺ T_reg population was only mildly affected in both models. Notably, we confirmed ISG signature in the spleen of Cx3cr1-GS mice (Fig. 4, E and F, and fig. S8). These data indicate that intrinsic signaling in T_regs caused by ADAR1 deficiency or chronic MDA5 activation leads to apoptotic cell death and loss of the T_reg population, in both Foxp3-GS and Foxp3^ΔAdar1 mice.

Fig. 4. — (A) Annexin V⁺7AAD⁺ T_reg percentages in the spleens and mesenteric lymph nodes (MLNs). (B) Relative mRNA expression of *Noxa* in sorted CD4⁺YFP⁺(FOXP3⁺) T_regs. (C and D) Percentages of CD4⁺FOXP3⁺ ex vivo induced T_regs (iT_regs) at day 3 and their viability at day 5. iT_regs were induced from *Foxp3*-WT (n = 3) or *Foxp3*^ΔAdar1 (n = 5) naïve CD4⁺ T cells and treated or not with caspase inhibitor. (E and F) Representative FC plots and summarized percentages of CD4⁺FOXP3⁺ T_regs in the spleens of 30-week-old *Cx3cr1*^ΔAdar1, 10-week-old *Cx3cr1*-GS, and respective *Cx3cr1*-WT control mice. (G) Body weight measurements of 3-week-old *Foxp3*-WT (n = 44), *Foxp3*^ΔAdar1 (n = 36), and *Mavs*^−/−*Foxp3*^ΔAdar1 (n = 20) mice. (H) Survival graphs of *Foxp3*-WT (gray), *Foxp3*^ΔAdar1 (blue, n = 7), and *Mavs*^−/−*Foxp3*^ΔAdar1 mice (orange, n = 5). (All *Foxp3*^ΔAdar1 and *Mavs*^−/−*Foxp3*^ΔAdar1 mice to date developed a severe phenotype and were sacrificed at latest 4 or 8 weeks old, respectively.) (I to L) Representative FC plots and summarized percentages of CD4⁺FOXP3⁺ T_regs in the spleens. (M to P) Representative histograms and plots of phospho–eIF-2α mean fluorescence intensity in CD4⁺FOXP3⁺ T_regs from spleens. Panels (A), (B), (I), (J), (M), and (N) are representative of ≥3 independent experiments with ≥3 mice per group; panels (C) to (F) are representative of two independent experiments with ≥2 mice per group. In dot plots, each symbol indicates an individual mouse. Statistics were calculated using Student’s t test or one-way ANOVA; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant, P > 0.05.

Given that concurrent deletion of Ifih1 and/or Mavs delays the embryonic lethality of systemic Adar1^−/− mice (10–12), we examined a potentially similar effect and generated Mavs^−/−Foxp3^ΔAdar1 mice. At the age of 3 weeks, Mavs^−/−Foxp3^ΔAdar1 mice showed improved body weight and appearance(Fig. 4G and fig. S9A), and significant reduction of inflammatory cytokines in their kidneys, compared to age-matched Foxp3^ΔAdar1 mice (fig. S9B). However, their condition deteriorated at approximately 4 weeks after birth, and they died by 8 weeks after birth (Fig. 4H). Although Mavs^−/−Foxp3^ΔAdar1 mice exhibited mild improvement in terms of growth retardation and survival compared to Foxp3^ΔAdar1 mice, at 3 weeks old, they already showed a significant loss of T_regs (Fig. 4, I and J, and fig. S9, C to E) and a significant shift from naïve to effector CD4⁺ and CD8⁺ T cells, similar to that observed in Foxp3^ΔAdar1 mice (fig. S9F), indicating that an MDA5-independent pathway is also critically involved in T_reg homeostasis in Foxp3^ΔAdar1 mice.

It is known that ADAR1 deficiency activates PKR encoded by Eif2ak2 (13), and to examine its involvement in the T_reg cell death, we intercrossed Foxp3^ΔAdar1 mice with Eif2ak2^−/− knockout mice. Foxp3^ΔAdar1 mutant mice with systemic heterozygous PKR deficiency, Eif2ak2^+/−Foxp3^ΔAdar1, showed an improvement of their appearance and body weight; especially the skin of their ears, tails, and overall fur coat were comparable to those of littermate controls, in contrast to the severe scurfy-like appearance of age-matched Foxp3^ΔAdar1 mutant mice (fig. S10, A and B). Eif2ak2^+/−Foxp3^ΔAdar1 mice retained a larger fraction of T_regs, compared to that observed in Foxp3^ΔAdar1 mutant mice (Fig. 4, K and L). We also observed a down-regulation of proinflammatory cytokines and ISGs in organs such as kidney and liver (fig. S10C). PKR activation leads to eIF-2α phosphorylation, which subsequently blocks protein synthesis (13). The T_reg population in Foxp3^ΔAdar1 and Mavs^−/−Foxp3^ΔAdar1 mice exhibited high phosphorylation status of eIF-2α compared to controls (Fig. 4, M and N). The intensity of phosphorylated eIF-2α in T_regs from Eif2ak2^+/−Foxp3^ΔAdar1 mice was comparable to controls and significantly reduced compared to T_regs from Foxp3^ΔAdar1 mice (Fig. 4, O and P, and fig. S10D). Moreover, protein synthesis capacity monitored by puromycin incorporation was lower in T_regs from Foxp3^ΔAdar1 mice compared to controls (fig. S11, A and B). These data show evidence of PKR/eIF-2α–dependent protein synthesis shutoff, independently of MAVS signaling. ADAR1 deficiency also activates RNase L, leading to cell death (14); however, we did not observe 28S ribosomal RNA (rRNA) cleavage in RNA isolated from T_regs of Foxp3^ΔAdar1 mice, indicating that the OAS/RNase L pathway was not activated in these cells (fig. S12). Our data suggest that both MDA5/MAVS and PKR/eIF-2α pathways are involved in the dysregulation of T_reg homeostasis in Foxp3^ΔAdar1 mice and that ablation of the MDA5/MAVS pathway is not sufficient to rescue their phenotype.

DISCUSSION

We found a reduction of the T_reg population in patients with AGS caused by ADAR1 or IFIH1 mutations. In particular, the suppressive T_reg population in Fr. II was significantly reduced, and this T_reg population also significantly up-regulated the expression of PD-1, which may cause attenuation of T_reg function (35, 37). Together with murine data, we demonstrate the concept that dysregulated innate immune signaling due to ADAR1 deficiency or chronic MDA5 activation in T_regs is sufficient to cause autoimmunity as a consequence of T_reg loss. Thus, our findings indicate that along with constitutive IFN-I activation, T_reg loss and/or attenuation of T_reg function (25) are critically involved in the onset of autoimmune disease, and that systemic or local T_reg dysregulation may explain why patients with type I interferonopathies exhibit a variety of autoimmune manifestations. Considering that there are approximately 40 distinct genes associated with type I interferonopathies (1), it would be of interest to examine the T_reg populations in patients carrying mutations other than ADAR1 or IFIH1.

The limited availability of samples from patients with AGS or other type I interferonopathies, as well as the generally low frequency of T_regs in PBMCs, prompted us to use two murine models for further characterization. We found that ADAR1 deficiency leading to chronic activation of innate immune sensors MDA5 and PKR, as well as expression of constitutively active gain-of-function MDA5 in T_regs, induce apoptotic cell death and loss of peripheral T_regs, resulting in highly lethal autoimmune phenotypes. While we previously observed reduced T_reg total numbers in mice that systemically express the MDA5 G821S mutant protein (25), the T_reg numbers were only mildly affected in both Cx3cr1^ΔAdar1 and Cx3cr1-GS mice with ISG signature, indicating that the T_reg cell death is caused by intrinsic signaling rather than extrinsic effects from other immune cells. Several models with T_reg-specific deletion of anti-apoptotic genes such as MCL-1 and c-FLIP have been reported (41, 42), which cause similar T_reg loss and lead to lethal autoimmune phenotypes. Moreover, we demonstrate that ADAR1 deficiency in T_regs triggers PKR/eIF-2α–dependent protein synthesis shutoff, which is likely the major driving force toward cell death, given the severer and earlier onset of phenotype in Foxp3^ΔAdar1 mice compared to that of Foxp3-GS mice, as well as the larger frequency of T_regs retained in Eif2ak2^+/−Foxp3^ΔAdar1 mice. However, further investigation is needed to determine the long-term outcome of, especially homozygous, Eif2ak2 deletion in Foxp3^ΔAdar1 mice. It has been recently shown that simultaneous deletion of Ifih1 and Eif2ak2 is necessary to rescue the embryonic lethality of systemic ADAR1 p150-isoform knockout mice (16), suggesting that ablation of both RNA sensing pathways could be required to likewise rescue the Foxp3^ΔAdar1 mutant mice.

ADAR1 deficiency has also been reported to trigger ZBP1-induced necroptosis (15, 43–46) and p16-dependent senescence (47); therefore, the potential involvement of these mechanisms in the T_reg loss in this T_reg-specific ADAR1 deficient mouse model should be explored. Recently, mice with AGS-related Adar1 mutations have been reported to exhibit MDA5-dependent severe inflammation and AGS-like encephalopathy (48, 49). It is of interest to explore whether T_regs are affected by the respective mutations and their potential involvement in disease in these mice.

JAK inhibitors are used to treat type I interferonopathies, specifically aiming to reduce chronic IFN-I signaling, and they are beneficial to some extent, for example, in reducing inflammation and ameliorating skin lesions (50). JAK inhibitors reported to treat type I interferonopathies, so far, are not selective for individual JAKs. Moreover, considering that JAKs are critical in the signaling of different immunologically essential pathways, JAK inhibition has a broad immunosuppressive effect, leading to the increased risk of viral infections (51). JAK inhibitors also down-regulate FOXP3 expression in vitro and in vivo (31), and some studies have revealed that JAK inhibition resulted in a stark and long-lasting reduction of peripheral T_regs (52). Reducing the IFN-I response is currently the primary target for treating patients with type I interferonopathies, and there is a need for more precise inhibitors. However, it should also be considered that IFN-I is not the only culprit driving the pathogenesis and that its inhibition is insufficient to alleviate already developed autoimmune symptoms. T_reg adoptive transfer therapies have shown promising outcomes, for instance, in patients with type 1 diabetes (53) or in patients with amyotrophic lateral sclerosis, which is a neurological disease wherein FOXP3-expressing cells decrease with disease progression (54). On the basis of our findings, it is worthwhile to perform an in-depth functional analysis of T_regs in patients with type I interferonopathies caused by aberrant innate immune sensing, to potentially use them in combination with available treatments to improve clinical manifestations.

MATERIALS AND METHODS

Ethical statement

Blood samples were obtained with informed consent from patients with AGS and healthy donors, with approval from the Medical Ethics Committee of Kyoto University School of Medicine (R2831-2) and from the Ethics Committee of University of Dresden (TRR237/A11).

Collection and analysis of PBMCs

PBMCs were isolated from the buffy coats or whole blood samples via Ficoll-Paque density gradient centrifugation (GE Healthcare, #17-1440-02).

Mice

All animal experiments were performed according to the institutional and governmental guidelines of animal welfare in Germany (81-02.04.2019.A462) and Japan (Kyoto, 19323). Adar1^flox mice (55) (B6.129-Adar^tm1Knk/Mmjax, The Jackson Laboratory, #034619-JAX) were intercrossed with Foxp3^YFP-Cre mice (56) (B6.129(Cg)-Foxp3^{tm4(YFP/icre)Ayr}/J, The Jackson Laboratory, #016959) to generate homozygous male Foxp3^Cre/Y Adar1^fl/fl or female Foxp3^Cre/Cre Adar1^fl/fl mice with Adar1 deletion in FOXP3-expressing T cells, indicated as Foxp3^ΔAdar1 or Foxp3ΔAdar1. Trex1^flox mice were generated as previously described (57) and were intercrossed with Foxp3^YFP-Cre mice to generate homozygous male Foxp3^Cre/Y Trex1^fl/fl or female Foxp3^Cre/Cre Trex1^fl/fl mice with Trex1 deletion in FOXP3-expressing T cells, indicated as Foxp3^ΔTrex1 or Foxp3ΔTrex1. Mavs^−/− mice (58) were provided by S. Akira (Osaka University, Suita, Japan). These mice were intercrossed with Foxp3^ΔAdar1 mice to generate male Mavs^−/−Foxp3^Cre/Y Adar1^fl/fl or female Mavs^−/−Foxp3^Cre/Cre Adar1^fl/fl mice, indicated as Mavs^−/−Foxp3^ΔAdar1 or Mavs^−/−Foxp3ΔAdar1. Pkr^−/− mice (59) (here indicated as Eif2ak2^−/− mice) were provided by F. Weber (University of Giessen, Germany) with agreement from J. Pavlovic (University of Zurich, Switzerland) and were intercrossed with Foxp3^ΔAdar1 to generate Eif2ak2^+/−Foxp3^ΔAdar1 mice.

To generate conditional MDA5 G821S^fl/+ mice, a target construct containing loxP, an exon 13 fragment, a stop codon, poly A, and a PGK-Neo cassette with loxP sites was used; it was linked with the mutant exon 13 G821S (fig. S5A). The sequences containing a missense mutation in MDA5 exon 13 were amplified by polymerase chain reaction (PCR) and then inserted into the above construct. Then, the linearized targeting vector was transduced into murine hybrid embryonic stem (ES) cells via electroporation. Northern blotting was performed to confirm successful recombination in ES cells. Chimeric mice were bred with C57BL/6J mice for germline transmission (MDA5 G821S^fl/+ mice). MDA5 G821S^fl/+ mice were crossed with Foxp3^YFP-Cre mice to generate male Foxp3^Cre/Y MDA5 G821S^fl/+ or female Foxp3^Cre/Cre MDA5 G821S^fl/+ mice, indicated as Foxp3-GS mice. Adar1^flox mice were intercrossed with Cx3cr1^Cre mice (60) [B6J.B6N(Cg)-Cx3cr1^{tm1.1(cre)Jung}/J, The Jackson Laboratory, #025524] to generate Cx3cr1^Cre Adar1^fl/fl mice with Adar1 deletion specifically in CX3CR1-expressing immune cells, indicated as Cx3cr1^ΔAdar1 or Cx3cr1ΔAdar1. Cx3cr1^Cre mice were intercrossed with MDA5 G821S^fl/+ mice to generate Cx3cr1^Cre MDA5 G821S^fl/+ mice, indicated as Cx3cr1-GS.

Single-cell suspensions

Mouse spleens or lymph nodes were passed through 100-μm cell strainers (Sigma-Aldrich, #CLS431752-50EA) in fluorescence-activated cell sorting (FACS) buffer [phosphate-buffered saline (PBS; Thermo Fisher Scientific, #10010056) containing 5% fetal bovine serum (FBS) (Thermo Fisher Scientific, #10270106) and 2 mM EDTA (Merck, #93283)], incubated with ammonium-chloride-potassium lysing buffer (Thermo Fisher Scientific, #A1049201) for up to 5 min to lyse red blood cells, and then filtered once again through 70-μm cell strainers. To obtain single-cell suspensions from the small intestine, Peyer’s patches were first removed, and then the intestine was washed thoroughly with cold PBS and incubated in RPMI 1640 containing 3% FBS, 100 mM dithiothreitol (Thermo Fisher Scientific, #20290), and 0.5 mM EDTA, with shaking for 20 min at 37°C. After washing several times, the intestines were cut into small pieces and digested with deoxyribonuclease (0.5 mg/ml; Roche, #04716728001) and Liberase TL (1 mM/ml; Roche, #05401020001) in RPMI 1640 for 23 min at 37°C. The obtained single cells were filtered through 70-μm nylon screens in FACS buffer.

Cell culture and stimulation

CD4⁺ or naïve CD4⁺ T cells were isolated from whole splenocyte suspensions via negative selection (Miltenyi Biotec, #130-104-454 and #130-104-453) and cultured in RPMI medium (Thermo Fisher Scientific, #21875091) containing 10% FBS (Thermo Fisher Scientific, #10270106), 1× minimum essential medium nonessential amino acids (Gibco, #11140-035), 2 mM l-glutamine (Gibco, #25030-024), 1 mM sodium pyruvate (Gibco, #11360-039), penicillin (100 U/ml)–streptomycin (100 μg/ml) (Gibco, #15140-122), 25 mM Hepes (Pan-Biotech, #P05-01100), and 0.05 mM β-mercaptoethanol (Pan-Biotech, #P07-05020).

To induce T_reg differentiation, naïve CD4⁺ T cells were cultured on plates coated with α-CD3 (1 μg/ml) and α-CD28 (5 μg/ml) (eBioscience, #16-0031-81 and #16-0281-81) in the presence of IL-2 (50 ng/ml; BioLegend, #575406) and TGF-β (10 ng/ml; PeproTech, #100-21). The cells were then treated with 1 μM Q-VD-OPH pan-caspase inhibitor (MedChemExpress, #HY-12305). For enzyme-linked immunosorbent assay (ELISA; BioLegend, #740741), the enriched CD4⁺ T cells were stimulated overnight with mouse T activator CD3/CD28 beads (Thermo Fisher Scientific, #11-453-D) in a bead-to-cell ratio of 1:2. ELISA was performed using the LEGENDplex Mouse Th Cytokine Panel (BioLegend, #740741) according to the manufacturer’s instructions. To assess protein synthesis capacity, CD4⁺ T cells enriched by negative selection (Miltenyi Biotec, #130-104-454) were incubated with puromycin (10 μg/ml; Sigma-Aldrich, #P7255) for 45 min at 37°C, 5% CO₂ atmosphere, then stained intracellularly using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, #00-5523), and analyzed by flow cytometry.

Flow cytometry analysis

To assess cell viability, the cells were stained with fixable live/dead staining dye (BioLegend, #423114) diluted in PBS or 7AAD viability staining solution (BioLegend, #420403) for 15 min at room temperature (RT) protected from light. Thereafter, the cells were washed with PBS and incubated with Fc block diluted in FACS buffer (InVivoMAb anti-mouse CD16/CD32, Bio X Cell, #BE0307) for 15 min at 4°C protected from light. For surface staining, the cells were incubated with the desired antibody mix in FACS buffer for 20 to 30 min at 4°C protected from light. For intracellular staining, the cells were fixed and permeabilized using either the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, #00-5523) for transcription factor staining or the BD Cytofix/Cytoperm Kit (#554714) for cytosolic staining. Then, the cells were incubated with the desired antibody mix in corresponding 1× wash buffer for 20 to 30 min at 4°C protected from light. Anti–phospho–eIF-2α staining was performed using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, #00-5523), followed by staining with a secondary antibody conjugated to the preferred fluorochrome diluted in 1× wash buffer. The cells were then analyzed or sorted either on the BD Canto II, BD LSRFortessa, BD FACSAria Fusion, or BD FACSAria III systems and further analyzed using FlowJo Software (BD Biosciences). The anti-mouse antibodies used were as follows: CD4-allophycocyanin (APC) (RM4-5, BioLegend, #100516), CD44-peridinin chlorophyll protein (PerCP)/cyanine 5.5 (Cy5.5) (IM7, BioLegend, #103032), CD62L-phycoerythrin (PE) (MEL-14, BioLegend, #10407), Foxp3–Alexa Fluor 488 (AF488) (150D, BioLegend, #320012), green fluorescent protein (GFP)–AF488 (FM-264G, BioLegend, #338008), CD8–Brilliant Violet 650 (BV650) (53-6.7, BioLegend, #100741), Gata3-PE (16E10A23, BioLegend, #653803), GFP-AF488 (FM264G, BioLegend, #338008), phospho–eIF-2α (Ser⁵¹, Cell Signaling Technology, #3597), active caspase-3–PE (C92-605, BD, #561011), annexin V–PE-Cy7 (BioLegend, #640950), anti-rabbit IgG-PE (Cell Signaling Technology, #79408), and puromycin AF647 (Sigma-Aldrich, #MABE343-AF647). The anti-human antibodies used were as follows: CD4-APC (SK3, BioLegend, #344614), CD45RA-PerCP/Cy5.5 (HI100, BioLegend, #304122), CD25–PE-Cy7 (BC96, BioLegend, #302612), CD25-PE (M-A251, BD, #560989), CD152 (CTLA-4)–PE-Cy7 (BNI3, BioLegend, #369614), and CD279 (PD-1)-PE/Dazzle594 (EH12.2H7, BioLegend, #329940).

Histological staining

The organs were fixed with 4% paraformaldehyde (PFA) in PBS solution (Thermo Scientific Chemicals, J19943.K2) and then embedded with paraffin. Thereafter, 3-μm sections were prepared and stained with hematoxylin and eosin (H&E) using standard protocols.

IgG staining

First, 3-μm kidney sections were incubated with proteinase K (Invitrogen, #25530049) for antigen retrieval. Then, the sections were washed thrice (for 5 min each time) with PBS and incubated with PBS containing 20% donkey serum (Sigma-Aldrich, #D9663) and 0.05% Triton X-100 (Carl Roth, #3051.4) for 1 hour at RT. Thereafter, the sections were stained with IgG (Jackson ImmunoResearch, #715-606-151) diluted 1:50 in PBS containing 10% donkey serum for 1 hour at RT protected from light. After washing the sections once (1 min) with PBS, the sections were incubated with 4′,6-diamidino-2-phenylindole (Invitrogen, #D1306) diluted 1:1000 in PBS for 5 min at RT protected from light, followed by washing thrice (1 min each time) with PBS. The samples were imaged on the SP8 LIGHTNING confocal microscope (Leica).

ANA detection

L929 cells were seeded into an eight-well chamber (ibidi, #80826) and cultured overnight at 37°C in a 5% CO₂ atmosphere. Thereafter, the cells were washed with PBS and fixed with 4% PFA in PBS solution for 10 min at RT, washed again with PBS, and permeabilized with 0.1% Triton X-100 in PBS (PBST) for 20 min at RT. Then, the cells were incubated with blocking buffer [0.5% normal goat serum (Abcam, #ab7481) in PBST] for 1 hour at RT and further incubated with previously isolated mouse serum (diluted 1:150 in PBST) overnight at 4°C. The cells were washed twice with PBST and incubated with anti-mouse IgG (goat) secondary antibody diluted 1:1000 in PBST. After washing twice with PBST, the cells were kept in PBS and imaged on the SP8 LIGHTNING confocal microscope (Leica).

Assessment of RNA degradation

To assess RNA status, RNA was extracted from sorted CD4⁺YFP⁺ T cells using the Direct-Zol RNA Microprep Kit (Biozym, #R2061) and then analyzed using the Agilent 2200 TapeStation using High-Sensitivity RNA ScreenTape and Reagents according to the manufacturer’s instructions (Agilent Technologies, #5067-5579, #5067-5580, and #5067-5581).

Quantitative reverse transcription PCR

Whole organs were collected in TRIzol (Invitrogen, #15596) and homogenized using the gentleMACS tissue dissociator and tubes (Miltenyi Biotec, #130-096-427 and #130-093-237). Then, RNA was extracted using the phenol-chloroform method (Panreac AppliChem, #A1153,0100 and #A3691,1000). cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #43688). Reverse transcription PCR was performed using the Fast SYBR Green Master Mix or TaqMan Fast Advanced Master Mix on the Step One Plus Real-Time PCR System (Applied Biosystems, #4385614, #4444558, and #4376600). Relative RNA expression was determined using the ΔΔC_T method, in which ΔΔC_T = ΔC_T of the target gene from the sample of interest (e.g., Cxcl10 from Foxp3^ΔAdar1) − ΔC_T of the same gene from the control sample (e.g., Cxcl10 from Foxp3-WT), and ΔC_T = C_T (of the target gene) − 18S rRNA C_T (from the respective sample). The TaqMan probes (Thermo Fisher Scientific, #4331182) used were as follows: Isg56 (Ifit1) (#Mm00515153_m1), Ifnβ (#Mm00439546_s1), Il-6 (#Mm01210733_m1), and Cxcl10 (#Mm00445235_m1). The SYBR oligonucleotides used were as follows: Noxa [forward (F)–5′-GGAGTGCACCGGACATAACT-3′ and reverse (R)–5′-TTGAGCACTCGTCCTTCA-3′], Puma (F-5′-TGCTCTTCTTGTCTCCGCCG-3′ and R-5′-CATAGAGCCACATGCGAGCG-3′), Bad (F-5′-CGAAGGAGCGATGAGTT-3′ and R-5′-CCCACCAGGACTGGATAATG-3′), Bim (F-5′-GCCAAGCAACCTTCTGATGT-3′ and R-5′-CTGTCTTGCGGTTCTGTCTG-3′), Bcl-2 (F-5′-GGTCTTCAGAGAGACAGCCAGGAGAAATC-3′ and R-5′-GTGGTGGAGGAACTCTTCAGGATG-3′), Mcl-1 (F-5′-AAGCCAGCAGCACATTTCTGATGCC-3′ and R-5′-GTAATGGTCCATGTTTTCAAAGATG-3′), and Bcl-xL (F-5′-ACCAGCCACAGTCATGCCCGTCAGG-3′ and R-5′-GTAGTGAATGAACTCTTTCGGGAATGG-3′).

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 9 (9.5.1) for Windows (GraphPad Software, San Diego, CA, USA; www.graphpad.com). Student’s t test was used to compare the means of two groups, and ordinary one-way analysis of variance (ANOVA) was used to compare the means of three groups. P values are indicated as follows: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant, P > 0.05.

Acknowledgments

We thank the patients and their families. Blood samples were obtained with informed consent from patients with AGS and healthy donors, with approval from the Medical Ethics Committee of Kyoto University School of Medicine (R2831-2) and from the Ethics Committee of University of Dresden (TRR237/A11). We thank F. Hamsen, M. Shimizu, Y. Maruyama, and Y. Ohwada for patient sample preparation. We thank L. Surace (University of Bonn) for help with the protein synthesis assay. We also thank the members of the Flow Cytometry Core Facility (FCCF) and the Microscopy Core Facility (MCF) of the Medical Faculty at the University of Bonn for providing help, services, and devices as well as the Editage members (www.editage.com) for English language editing.

Funding: This work was supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) EXC 2151: ImmunoSensation2, project number 390873048 to H.K.; Open Philanthropy within the PANDEMIC ANTIVIRAL DISCOVERY PARTNERSHIP, grant number GV673603826 to H.K.; DFG CRC237, project number 369799452/B22 to H.K., CRC237 369799452/B21 to M.A.L.-K., CRC237 369799452/A11 to M.A.L.-K., CRC237 369799452/B19 to R.B., and CRC237 369799452/B17 to A.R.; BMBF (Bundesministerium für Bildung und Forschung) GAIN 01GM2206C to M.A.L.-K. and BMBF 01GM1910C to F.H.; JSPS Core-to-Core Program JPJSCCA20210008 to Y.O.; JSPS KAKENHI grant number JP22K07829 to R.N.; MHLW grant number JPMH20FC1407 to R.N.; AMED grant number JP18ek0109387 to R.N.; and DFG project numbers 216372401, 387335189, 216372545, and 387333827 (Devices of FCCF) and project number 388159768 (Devices of MCF).

Author contributions: Conceptualization: D.L., S.L., K.H., K.I., R.B., T.F., R.N., and H.K. Project administration: D.L., Y.T., V.S., T.F., and H.K. Funding acquisition: R.N., M.A.L.-K., and H.K. Investigation: D.L., S.L., Y.O., K.I., B.S., Y.T., R.N., and M.A.L.-K. Formal analysis: D.L. and S.L. Visualization: D.L. and S.L. Validation: D.L., S.L., B.S., Y.T., R.N., M.A.L.-K., and H.K. Methodology: S.L., K.H., R.B., and A.R. Writing—original draft: D.L., S.L., and H.K. Writing—review and editing: D.L., S.L., F.H., Y.O., and M.A.L.-K. Supervision: R.N., T.F., and H.K. Resources: S.L., Y.O., J.U., R.N., A.R., K.I., A.-L.L., F.H., R.B., T.F., M.A.L.-K., and H.K. Data curation and software: S.L.

Competing interests: H.K. is a paid consultant of Odyssey Therapeutics. The authors declare that they have no other competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The source data are available on the Zenodo repository, under the following DOI: 10.5281/zenodo.10261819.

Supplementary Materials

This PDF file includes:

Figs. S1 to S12

Table S1

sciadv.adk0820_sm.pdf^{(835.7KB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S12

Table S1

sciadv.adk0820_sm.pdf^{(835.7KB, pdf)}

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PERMALINK

Aberrant RNA sensing in regulatory T cells causes systemic autoimmunity

Domnica Luca

Sumin Lee

Keiji Hirota

Yasutaka Okabe

Junji Uehori

Kazushi Izawa

Anna-Lisa Lanz

Verena Schütte

Burcu Sivri

Yuta Tsukamoto

Fabian Hauck

Rayk Behrendt

Axel Roers

Takashi Fujita

Ryuta Nishikomori

Min Ae Lee-Kirsch

Hiroki Kato

Roles

Abstract

INTRODUCTION

RESULTS

Patients with AGS have a decreased frequency of peripheral effector Tregs

Fig. 1. Patients with AGS caused by ADAR1 or IFIH1 mutations have a decreased frequency of peripheral effector Tregs.

Adar1 deletion in Tregs causes Treg loss and a scurfy-like lethal phenotype in mice

Fig. 2. Adar1 deletion in Tregs causes Treg loss and a scurfy-like phenotype in mice.

Constitutive MDA5 activation in Tregs causes Treg loss and autoimmunity in mice

Fig. 3. Constitutive MDA5 activation in Tregs causes Treg loss and autoimmunity in mice.

Adar1 deletion in Tregs activates the PKR/eIF-2α pathway contributing to cell death

Fig. 4. Adar1 deletion in Tregs activates both the MDA5/MAVS and PKR/eIF-2α pathways contributing to cell death.

DISCUSSION

MATERIALS AND METHODS

Ethical statement

Collection and analysis of PBMCs

Mice

Single-cell suspensions

Cell culture and stimulation

Flow cytometry analysis

Histological staining

IgG staining

ANA detection

Assessment of RNA degradation

Quantitative reverse transcription PCR

Statistical analysis

Acknowledgments

Supplementary Materials

This PDF file includes:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Patients with AGS have a decreased frequency of peripheral effector T_regs

Fig. 1. Patients with AGS caused by ADAR1 or IFIH1 mutations have a decreased frequency of peripheral effector T_regs.

Adar1 deletion in T_regs causes T_reg loss and a scurfy-like lethal phenotype in mice

Fig. 2. Adar1 deletion in T_regs causes T_reg loss and a scurfy-like phenotype in mice.

Constitutive MDA5 activation in T_regs causes T_reg loss and autoimmunity in mice

Fig. 3. Constitutive MDA5 activation in T_regs causes T_reg loss and autoimmunity in mice.

Adar1 deletion in T_regs activates the PKR/eIF-2α pathway contributing to cell death

Fig. 4. Adar1 deletion in T_regs activates both the MDA5/MAVS and PKR/eIF-2α pathways contributing to cell death.