The ERM Moesin Regulates CD8+ Regulatory T Cell Homeostasis and Self-Tolerance

This information is current as Hiroki Satooka, Daisuke Nagakubo, Tomomi Sato and of September 28, 2021. Takako Hirata J Immunol published online 4 October 2017 http://www.jimmunol.org/content/early/2017/10/04/jimmun ol.1700074 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 4, 2017, doi:10.4049/jimmunol.1700074 The Journal of Immunology

The ERM Protein Moesin Regulates CD8+ Regulatory T Cell Homeostasis and Self-Tolerance

Hiroki Satooka,* Daisuke Nagakubo,* Tomomi Sato,*,† and Takako Hirata*

The ––moesin (ERM) are a family of membrane-associated proteins that link membrane proteins with filaments in the cell cortex and regulate many cellular processes, including cell shape determination, membrane transport, and signal transduction. Lymphocytes predominantly express two ERM members, ezrin and moesin. Mutations in the moesin in humans are associated with primary immunodeficiency with profound lymphopenia, and moesin-deficient mice exhibit a similar lymphopenia phenotype. In this study, we show that aging moesin-deficient mice develop a systemic lupus erythematosus–like autoimmune phenotype, which is characterized by elevated serum autoantibody levels and glomerulonephritis. Younger moesin- deficient mice exhibited elevated basal levels of several Ig isotypes and enhanced Ab affinity maturation upon immunization. + + +

Germinal center B cells and follicular helper T cells spontaneously accumulated in unimmunized mice, and CD8 CD44 CD122 Downloaded from Ly49+ regulatory T (CD8+ Tregs) cells, which inhibit the expansion of follicular helper T cells, were severely reduced in these mice. Isolated CD8+ Treg cells from moesin-deficient mice showed impaired proliferation in response to IL-15, which was accompanied by defects in STAT5 activation and IL-15Ra internalization, suggesting that moesin plays a key role in IL-15–mediated signaling. These findings underscore the importance of moesin in IL-15–dependent CD8+ Treg cell homeostasis and, thus, the control of self- tolerance. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/ he ezrin–radixin–moesin (ERM) proteins are a family of mutation was identified by recent newborn screening for primary widely distributed membrane-associated proteins that link immunodeficiency (9). Although moesin is ubiquitously expressed T plasma membrane proteins with actin filaments in the cell in cells of hematopoietic origin (7), in humans and mice with cortex, thereby regulating many fundamental cellular processes, moesin mutations, naive T cells are the most markedly affected including cell-shape determination, membrane-protein localiza- lymphocyte subset, which exhibits particularly low numbers of tion, membrane transport, and signal transduction (1–3). Because naive CD8+ T cells. of their high sequence similarity, ERM proteins are generally T cell lymphopenia is often associated with autoimmunity (10– thought to be functionally redundant. Lymphocytes predominantly 12). Many patients with lymphopenia-associated primary im-

express two ERM proteins, ezrin and moesin (4). Although their munodeficiency, such as Wiskott–Aldrich syndrome and Omenn by guest on September 28, 2021 functions are mostly redundant during TCR- and BCR-mediated syndrome, develop autoimmunity (13, 14). A number of mech- lymphocyte activation (5, 6), we previously found that moesin anisms have been proposed to link lymphopenia and autoim- gene–inactivated mice exhibit lymphopenia in the peripheral munity. For example, homeostatic proliferation of lymphocytes blood and lymph nodes (LNs), indicating that moesin has a unique in response to lymphopenia is one mechanism that may con- role in lymphocyte homeostasis (7). Recently, whole-exome se- tribute to autoimmunity, because homeostatic proliferation fa- quencing of DNA from primary immunodeficiency patients re- vors the expansion of autoreactive T cells. Another possible vealed the presence of hemizygous mutations in the moesin gene, mechanism involves the induction of defective regulatory which is located on the X (8). Seven patients with T (Treg) cells (11). early-onset and persistent lymphopenia were found to harbor one Treg cells play crucial roles in controlling the balance between of two different mutations, both of which were predicted to be effective immune responses and tolerance by regulating the ex- disease-causing mutations. An additional case with the same pansion and function of effector T cells. Among effector T cells, follicular helper T (Tfh) cells are a distinct subset of CD4+ T cells that provide cognate help to B cells during germinal center (GC) *Department of Fundamental Biosciences, Shiga University of Medical Science, + Otsu, Shiga 520-2192, Japan; and †Department of Pediatrics, Shiga University of reactions (15, 16). Upon exposure to a foreign Ag, naive CD4 Medical Science, Otsu, Shiga 520-2192, Japan T cells differentiate into Tfh cells, which help B cells differentiate ORCID: 0000-0002-1740-6168 (T.H.). into Ab-producing plasma cells and long-lived memory B cells. Received for publication January 17, 2017. Accepted for publication September 7, Although Tfh cells are required for Ab responses against foreign 2017. Ags, their unrestrained accumulation results in the production of This work was supported by Japan Society for the Promotion of Science KAKENHI autoantibodies, which leads to the development of autoimmune Grants 25460588 and 16K08831. diseases, such as systemic lupus erythematosus (SLE). CD4+ and Address correspondence and reprint requests to Prof. Takako Hirata, Department of CD8+ Treg cells have been identified as negative regulators of Fundamental Biosciences, Shiga University of Medical Science, Seta-Tsukinowa- + cho, Otsu, Shiga 520-2192, Japan. E-mail address: [email protected] GC reactions. CD4 Treg cells expressing Foxp3 are a well- The online version of this article contains supplemental material. characterized Treg subset that is critical for the maintenance of + Abbreviations used in this article: gc, common g-chain; CGG, chicken g-globulin; immune homeostasis (17). A subpopulation of CD4 Treg cells ERM, ezrin–radixin–moesin; GC, germinal center; LN, lymph node; NP, nitrophenyl; that coexpresses Bcl6 and CXCR5 has been identified as follicular PAS, periodic acid–Schiff; SLE, systemic lupus erythematosus; Tfh, follicular helper regulatory T (Tfr) cells in humans and mice. These cells limit the T; Tfr, follicular regulatory T; Treg, regulatory T. expansion of Tfh cells and inhibit GC reactions (18). CD8+ Treg Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 cells are characterized by the surface expression of CD44, CD122,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700074 2 MOESIN REGULATION OF CD8+ Treg CELL HOMEOSTASIS and Ly49 in mice, and they regulate the activity of Tfh cells Flow cytometry through recognition of the unconventional MHC class I molecule The mAbs used for flow cytometric analyses were purchased from BD Qa-1 expressed on the Tfh cell surface (19, 20). In humans, HLA- Biosciences, eBioscience, or BioLegend and included those to CD4 E–restricted CD8+ T cells function similarly to murine Qa-1– (RM4-5), CD8 (53-6.7), CD21 (7E9), CD23 (B3B4), CD25 (PC61.5), CD44 restricted CD8+ Treg cells in maintaining self-tolerance (21). (IM7), CD45RB (C363.16A), CD45R/B220 (RA3-6B2), CD93 (AA4.1), Defective Treg cell homeostasis leads to excessive Tfh cell ac- CD95/Fas (Jo2), CD122 (5H4), CD132 (TUGm2), Bcl6 (BCL-DWN), CXCR5 (2G8), Foxp3 (FJK-16s), GL-7 (GL-7), Ly49 (14B11), ICOS cumulation and autoimmunity. In a mouse model of Wiskott– (C398.4A), IgD (11-26c.2a), IgM (RMM-1), IL-15Ra (DNT15Ra), PD-1 Aldrich syndrome, decreased numbers and impaired function of (RMP1-30), and p-STAT5 (SRBCZX). Single-cell suspensions were in- CD4+ Treg cells may contribute, in part, to the development of cubated with anti-CD16/CD32 for 10 min, followed by staining with mAbs autoimmunity (22). Defective CD8+ Treg cell activity also leads to for 30 min on ice and washing. Data were acquired on a FACSCalibur or FACSCanto II (both from BD Biosciences) and analyzed using FlowJo the development of SLE-like autoimmunity (19, 20). (TreeStar). For CXCR5 staining, biotinylated anti-CXCR5 was detected The maintenance of the peripheral T cell pool is regulated by with allophycocyanin-labeled streptavidin. For IL-15Ra staining, the cells complex homeostatic mechanisms, including TCR signaling from were incubated with a PE-labeled anti–IL-15Ra mAb and then stained contact with MHC and signaling by the common g-chain (gc) with biotinylated anti-rat IgG (Jackson ImmunoResearch), followed by family of cytokines, such as IL-2, IL-7, and IL-15 (23). Homeo- PE-labeled streptavidin. For intracellular staining, the cells were fixed and + permeabilized using a Foxp3/Transcription Factor Staining Buffer Kit stasis of CD4 Treg cells is regulated by contact with IL-2. IL-2 (eBioscience) for Bcl6 and Foxp3 and a BD Cytofix/Cytoperm Fixation/ + signals are crucial during CD4 Treg cell development in the thy- Permeabilization Kit for moesin as described previously (26). For p-STAT5 mus, as well as homeostatic proliferation and survival in peripheral staining, the cells were fixed with 1.5% formaldehyde and permeabilized

LNs. In contrast, CD8+ Treg cells depend on IL-15 for their pro- with methanol. Downloaded from liferation and survival (24), and IL-15–deficient mice exhibit de- Immunohistochemistry and immunofluorescence microscopy fective CD8+ Treg cell function (20). In this study, we report that moesin deficiency led to an SLE-like Paraffin-embedded kidney sections were stained with H&E. For periodic acid–Schiff (PAS) staining, paraffin-embedded sections were stained autoimmune disease in aging mice. Tfh cells and GC B cells with 0.5% periodic acid and Schiff solutions, and the nuclei were stained spontaneously accumulated in the spleen and LNs of moesin- with Mayer’s hematoxylin. Images of H&E- and PAS-stained kidney deficient mice, leading to the production of anti-dsDNA Abs sections were captured with an Eclipse 90i microscope (Nikon). For C3 http://www.jimmunol.org/ and the deposition of complement C3 and IgG in the kidneys. In and IgG staining, frozen kidney sections were fixed with ice-cold meth- addition, CD8+ Treg cells, which inhibit the expansion of Tfh anol and acetone, respectively, and stained with FITC-labeled anti-mouse C3 (MP Biomedicals) and Alexa Fluor 555–labeled anti-mouse IgG cells, were severely reduced in these mice. Notably, we found that (BioLegend). For staining of spleen sections, the frozen sections were + an ex vivo culture of CD8 Treg cells from moesin-deficient mice fixed with acetone and stained with biotinylated anti-CD90.2/Thy1.2 exhibited a significant attenuation of IL-15–induced proliferation, (eBioscience), followed by staining with Alexa Fluor 594–labeled which was accompanied by defects in STAT5 activation and streptavidin (Invitrogen) and Alexa Fluor 488–labeled anti-CD45R/B220 (BD Biosciences). IL-15Ra internalization. Collectively, our results reveal a novel + + For sorted CD8 Treg cell staining, surface and intracellular IL-15Ra role for moesin in the regulation of IL-15–dependent CD8 Treg was stained using a successive staining protocol, as described previously cell homeostasis and the maintenance of self-tolerance. (27). IL-15–stimulated CD8+ Treg cells were fixed with 4% paraformal- by guest on September 28, 2021 dehyde and surface stained with goat Abs against IL-15Ra (R&D Sys- tems) and Alexa Fluor 488–labeled anti-goat IgG (Invitrogen). The cells Materials and Methods were then permeabilized with 0.25% Triton-X in PBS and stained with the Mice same goat anti–IL-15Ra Abs, followed by biotinylated anti-goat IgG (Abcam) and Alexa Fluor 555–labeled streptavidin (Invitrogen). Stained Moesin-deficient mice were produced as described previously (25) and cells were cytospinned onto glass slides at 800 rpm for 5 min. The fluo- backcrossed for .10 generations onto the C57BL/6 genetic background. rescence microscopy images were captured with an FV-1000D IX81 Male moesin-deficient (Msn2/Y) mice and littermate wild-type (Msn+/Y) confocal microscope (Olympus). For the ratio of surface/intracellular IL- mice were used for most experiments. In some experiments, female het- 15Ra, green and red fluorescence intensities were measured for each cell erozygous (Msn+/2) mice were used. The mice were housed at the Re- in captured images using the ImageJ Area measurement tool (National search Center for Animal Life Science at Shiga University of Medical Institutes of Health) (28, 29). The ratio was calculated by dividing the Science. All studies were approved by the Animal Research Committee of green fluorescence intensity by the red fluorescence intensity. Shiga University of Medical Science. Quantitative PCR Cell preparation and stimulation Total RNA was extracted using TRIzol Reagent (Invitrogen) and reverse transcribed with a High Capacity RNA-to-cDNA Kit (Applied Biosystems). Lymphocytes were isolated from spleens and LNs by mechanical disruption Quantitative real-time PCR was performed using KOD SYBR qPCR Mix between the frosted surfaces of two glass slides, followed by filtration (Toyobo) and a LightCycler 480 instrument (Roche). The primer pairs are through 100-mm nylon mesh twice. The collected cells were quantified 59-CGCCAGATGCAAGTGTTGTAT-39 and 59-TCCTGGGGATTATC- + + + with a hemocytometer. For proliferation assays, CD4 Treg (CD4 CD25 ) CAAGTCAAT-39 for STAT5a, 59-CGATGCCCTTCACCAGATG-39 and + + + + + and CD8 Treg (CD8 CD44 CD122 Ly49 ) cells were sorted using a 59-AGCTGGGTGGCCTTAATGTTC-39 for STAT5b, 59-CCCACAGTT- FACSAria (BD Biosciences) and cultured with plate-bound anti-CD3 (5 CCAAAATGACGA-39 and 59-GCTGCCTTGATTTGATGTACCAG-39 for mg/ml, 145-2C11) and soluble anti-CD28 (1 mg/ml, 37.51; both from IL-15Ra,59-TGGAGCCTGTCCCTCTACG-39 and 59-TCCACATGCAA- BioLegend) in complete RPMI 1640 for 9 d, with IL-2 (100 ng/ml; R&D GAGACATTGG-39 for CD122, and 59-GCAACAGAGATCGAAGCTGGA-39 Systems) or IL-15 (100 ng/ml; Miltenyi Biotec). The cells were counted and 59-AGATTGGGTTATAGCGGCTCC-39 for CD132. with a hemocytometer on the indicated days. For IL-15Ra–internalization and STAT5-activation assays, isolated Immunization and ELISA splenocytes were starved in serum-free RPMI 1640 for 2 h and then stimulated with IL-15 (100 ng/ml) or IL-2 (100 ng/ml) for the indicated Basal serum titers of the Ig isotypes were analyzed by ELISA. The ELISA times, and the cell surface IL-15Ra or p-STAT5 levels were determined by plates were coated with goat anti-mouse isotype Abs (Bethyl Laboratories) flow cytometry. For IL-15Ra staining for immunofluorescence micros- and blocked with 1% BSA in PBS, followed by the addition of serially copy, sorted CD8+ Treg cells were starved in serum-free RPMI 1640 for diluted serum samples and standards. The plates were incubated and 1 h. The cells were then stimulated with IL-15 (100 ng/ml) for the indi- washed, and HRP-conjugated goat anti-mouse isotype Abs (Bethyl Lab- cated times. oratories) were added to the wells. After further incubation and washing, The Journal of Immunology 3

3,39,5,59-tetramethylbenzidine was added as a substrate. The reactions were stopped with 2 M H2SO4. For affinity-maturation assays, nitrophenyl (NP)-coupled chicken g-globulin (CGG) (NP:CGG conjugation ratio, 39:1; NP39-CGG; Bio- source Technologies) was used as an immunogen. The mice were immu- nized i.p. with 100 mgofNP39-CGG precipitated in alum (Imject Alum; Thermo Fisher Scientific) and boosted with 50 mgofNP39-CGG in PBS on day 42. Sera were collected on days 7, 14, 28, 42, 49, and 63 after the first immunization, and the anti-NP IgM and IgG1 serum titers were measured by ELISA. The plates were coated with NP52-BSA or NP4-BSA (both from Biosource Technologies) and blocked with 1% BSA in PBS and then se- rially diluted serum samples were added. The bound Abs were detected with HRP-conjugated goat anti-mouse IgM or IgG1. The plates were de- veloped as described above. Serum anti-dsDNA Ab titers were measured with a mouse anti-dsDNA ELISA kit (Shibayagi), according to the manufacturer’s instructions.

Statistical analysis Statistical analysis was performed using the two-tailed Student t test. To determine survival rates, log-rank analysis was performed.

Results Downloaded from Aging moesin-deficient mice develop an SLE-like autoimmune phenotype Although Msn2/Y mice exhibit T and B cell lymphopenia in the peripheral blood, young Msn2/Y mice appear healthy (7). In this 2/Y study, we found that after 16 wk of age, several Msn animals http://www.jimmunol.org/ became lethargic and died. By 80 wk of age, 28% of Msn2/Y mice had died, whereas all of the littermate control Msn+/Y mice sur- vived, indicating that Msn2/Y mice exhibited a higher mortality than the controls (Fig. 1A). Because lymphopenia is often asso- ciated with autoimmunity, we examined the development of au- toimmune diseases in aging Msn2/Y mice. The analysis of sera from 8- to 72 wk-old mice showed that while the anti-dsDNA Ab titers were comparable between Msn+/Y and Msn2/Y mice at 8 wk 2 of age, they were significantly elevated in 72 wk-old Msn /Y mice by guest on September 28, 2021 (Fig. 1B). High titers of anti-dsDNA Abs are suggestive of auto- FIGURE 1. Aged moesin-deficient mice display an autoimmune phe- +/Y 2/Y immune disease, particularly SLE (30). SLE is a chronic auto- notype. (A) Survival curves of Msn and Msn mice. The survival of +/Y 2/Y immune disease that is characterized by the generation of Msn (n = 22) and Msn (n = 23) mice was evaluated for 80 wk. B autoantibodies and immune complexes, which, together with au- p = 0.0036, log-rank test. ( ) Anti-dsDNA Ab titers in sera from young (8-wk-old) and aged (72-wk-old) Msn+/Y and Msn2/Y mice. Data (mean 6 toreactive T cells, can cause damage to several organs, including SEM) represent the results from 10 to 15 mice per group. **p , 0.01. (C) skin, lung, and kidney (31, 32). This type of organ damage leads to Kidney sections from young or aged Msn+/Y and Msn2/Y mice stained with serious and fatal complications, such as lupus nephritis (33). A H&E and PAS. Scale bars, 50 mm. (D) Immunofluorescence staining of C3 histological analysis of the kidney sections stained with H&E and and IgG in kidney sections from young or aged Msn+/Y and Msn2/Y mice. PAS revealed increased cell numbers, matrix deposition, and Glomeruli are outlined. Scale bar, 20 mm. thickened basement membranes in the glomeruli of aged Msn2/Y mice (Fig. 1C). Furthermore, C3 and IgG deposition, which is a We next examined B cell responses after immunization with common feature of lupus nephritis, was observed in the glomeruli +/Y 2/Y 2 NP -CGG. Msn and Msn mice were immunized with alum- of aged, but not young, Msn /Y mice (Fig. 1D). No histopatho- 39 precipitated NP -CGG on day 0 and boosted on day 42. NP- logical changes were observed in the kidney of either young or 39 +/Y specific IgM Ab titers, measured with NP52-BSA–coated plates aged Msn mice (Fig. 1C, 1D). These results suggested that +/Y 2/Y moesin deficiency led to an SLE-like autoimmune disease in aged by ELISA, were similar between Msn and Msn mice in both mice. their primary and secondary immune responses (Fig. 2B). Al- though the total NP-specific IgG1 titers, measured with NP52- Moesin-deficient mice display altered humoral immune BSA–coated plates, were reduced in Msn2/Y mice at 4 wk after responses the primary immunization (Fig. 2C, left panel), high-affinity NP- To investigate the mechanisms underlying the autoimmunity specific IgG1, measured with NP4-BSA–coated plates, tended to development in Msn2/Y mice, we first examined the basal serum be higher in these mice after the secondary immunization (Fig. 2C, titers of the various Ig isotypes by ELISA as a parameter of right panel). The percentage and number of B cells producing NP- +/Y B cell activity. Although young Msn2/Y mice did not exhibit an specific IgG1 in the spleen were similar between Msn and 2 autoimmune phenotype, their serum IgM, IgG2b, IgG2c, and IgE Msn /Y mice (Supplemental Fig. 1). Although the ratio of high- levels were significantly higher and their IgA titers were sig- affinity/total NP-specific IgG1 gradually increased after immuni- nificantly lower than those of age-matched Msn+/Y mice zation in both genotypes, the increase was more prominent in (Fig. 2A). Msn2/Y mice, especially during the secondary response (Fig. 2D). 4 MOESIN REGULATION OF CD8+ Treg CELL HOMEOSTASIS

+/Y 2/Y FIGURE 2. Ab responses are altered in moesin-deficient mice. (A) Basal serum titers of Ig isotypes from young Msn and Msn mice. The following Downloaded from isotypes were quantified by ELISA: IgM, IgG1, IgG2b, IgG2c, IgG3, IgA, and IgE. Data (mean 6 SEM) represent the results from 12 mice per group. (B– +/Y 2/Y D) NP-specific Ab responses. Msn and Msn mice were immunized i.p. with NP39-CGG on day 0 and boosted on day 42. The mice were bled on days 0, 7, 14, 28, 42, 49, and 63. (B) Relative concentrations of NP-specific IgM were measured with NP52-BSA–coated plates. (C) Relative concentrations of total and high-affinity NP-specific IgG1 were measured with NP52-BSA– and NP4-BSA–coated plates, respectively. (D) The ratios of high-affinity anti-NP4 IgG1/total anti-NP52 IgG1 were plotted as a function of time. Data (mean 6 SEM) represent the results from eight mice per group. *p , 0.05, **p , 0.01, ***p , 0.001. http://www.jimmunol.org/

These results indicated that Ab affinity maturation was enhanced Furthermore, immunofluorescence analysis of the spleen revealed in Msn2/Y mice. that the overall organization of the T cell zones and B cell follicles was normal in Msn2/Y mice (Fig. 3C). GC B cells and Tfh cells are expanded in moesin-deficient mice During humoral immune responses, Ag is transported to the Because B cell activity was found to be enhanced in Msn2/Y mice, T cell zones and B cell follicles, which initiates the activation and we next examined whether B cell compartments are expanded in interaction of T and B cells, resulting in GC reactions. We found these mice. Although B cell numbers in the blood and LNs of that the number of GC B (B220+Fas+GL-7+) cells in the spleen of 2/Y 2/Y young Msn mice were reduced, their numbers in the spleen unimmunized young Msn mice was significantly increased by guest on September 28, 2021 were slightly increased compared with those in Msn+/Y mice (7). compared with that in control Msn+/Y mice (Fig. 4A). The number Thus, we analyzed B cell subsets in the spleen by flow cytometry. of GC B cells was also significantly higher in aged Msn2/Y mice Immature B cells are identified as B220+CD93+ cells, which than in age-matched Msn+/Y mice (Fig. 4B). Spontaneous GC for- mature from T1 (B220+CD93+IgMhiIgDlo) to T2 (B220+CD93+ mation and GC cell expansion are hallmarks of many autoimmune- IgM+IgDhi) subsets (34). The number of T1 B cells was slightly prone mouse strains (35). Because ERM proteins have been higher in Msn2/Y mice than in Msn+/Y mice, whereas that of T2 implicated in B cell signaling, we examined whether B cell pro- B cells was similar in the two types of mice (Fig. 3A, 3B). The liferation was affected by the moesin deficiency. Ex vivo stimu- number of mature B cells, including follicular (B220+CD932 lation of B cells from Msn+/Y and Msn2/Y mice with anti-IgM Abs, CD21intCD23hi) and marginal zone (B220+CD932CD21hiCD23lo) which ligate the BCRs, resulted in moderate increases in prolif- B cells, was also comparable in both types of mice (Fig. 3A, 3B). eration of both types of cells (Supplemental Fig. 2). Stimulation

FIGURE 3. The organization of B and T cell areas in the spleen is normal in moesin-deficient mice. (A) Flow cytometric analysis of B cell sub- populations in the spleen from young Msn+/Y and Msn2/Y mice. T1 (B220+CD93+IgMhiIgDlo), T2 (B220+CD93+IgM+IgDhi), follicular (FO; B220+ CD932CD21intCD23hi), and marginal zone (MZ; B220+CD932CD21hiCD23lo) B cell populations were identified. Numbers adjacent to the outlined areas indicate the percentage of cells in each box. (B) Numbers of T1, T2, FO, and MZ B cells in the spleen of young Msn+/Y and Msn2/Y mice. Data (mean 6 SEM) represent the results from four mice per group. *p , 0.05. (C) Immunofluores- cence staining of spleen sections. Frozen sections were stained for B220 (red) and Thy1.2 (blue). Scale bar, 250 mm. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 4. GC B and Tfh cells are expanded in the spleen of moesin- deficient mice. Identification of GC B cells in the spleen of young (A)or aged (B) Msn+/Y and Msn2/Y mice using flow cytometry. GC B cells were identified as B220+GL-7+Fas+ cells. Expression of GL-7 and Fas in B220+ B cells and the GC B cell percentages and counts are shown. Data (mean 6 SEM) represent the results from three (A) and six (B) mice per group. Identification of Tfh cells in the spleen of young (C) or aged (D) Msn+/Y by guest on September 28, 2021 and Msn2/Y mice using flow cytometry. Tfh cells were identified as CD4+ PD-1+CXCR5+ or CD4+ICOS+CXCR5+ cells. Expression of PD-1 (C)or ICOS (D) and CXCR5 in CD4+ T cells and the Tfh cell counts are shown. Data (mean 6 SEM) represent the results from four mice per group. Numbers adjacent to the outlined areas indicate the percentage of cells in each. *p , 0.05, **p , 0.01. via BCR-independent pathways also induced similar increases in the proliferation of Msn+/Y and Msn2/Y cells (Supplemental Fig. 2). These results suggested that the moesin deficiency in B cells did not affect their proliferative capacity.

During GC development, B cells receive cognate help from Tfh + cells, a subpopulation of effector T cells that migrate into the B cell FIGURE 5. Moesin deficiency leads to decreased numbers of CD8 Treg cells. Identification of CD4+ Treg cells in the spleen of young (A)or follicle and GC (36). In this study, we found that the number of B +/Y 2/Y + + + aged ( ) Msn and Msn mice using flow cytometry. Expression of Tfh (CD4 PD-1 CXCR5 ) cells was higher in the spleen of young + 2/Y +/Y Foxp3 and CD25 in CD4 T cells and the percentage and number of Msn mice than in that of age-matched Msn mice (Fig. 4C). CD4+ Treg cells are shown. Data (mean 6 SEM) represent the results + + + In aged mice, Tfh cells were identified as CD4 ICOS CXCR5 from four (A)andsix(B) mice per group. Identification of Tfr cells in the + + cells, because CD4 PD-1 cells accumulated in both genotypes, spleen of young (C)oraged(D) Msn+/Y and Msn2/Y mice using flow possibly as the result of an age-related expansion of PD-1+ T cells cytometry. Expression of Bcl6 and CXCR5 in CD4+CD25+Foxp3+ cells (37). We found that the number of Tfh cells was also increased in and the percentage and number of Tfr cells are shown. Data (mean 6 aged Msn2/Y mice (Fig. 4D). Taken together, our findings sug- SEM) represent the results from four mice per group. Identification of CD8+ Treg cells in the spleen of young (E) or aged (F) Msn+/Y and Msn2/Y gested that moesin deficiency led to spontaneous Tfh accumula- + tion and GC expansion that started at a young age. mice using flow cytometry. Expression of CD44 and CD122 in CD8 T cells and of CD122 and Ly49 in CD8+CD44+CD122+ cells and the Moesin deficiency impairs CD8+ Treg cell homeostasis percentage and number of CD8+ Treg cells are shown. Data (mean 6 SEM) represent the results from four (E) and six (F) mice per group. The expansion of Tfh cells is controlled by several subsets of Treg + Numbers adjacent to the outlined areas indicate the percentage of cells in cells. Foxp3-expressing CD4 Treg cells are a well-characterized each box. **p , 0.01, ***p , 0.001. subset that regulates various effector T cell subsets. We found that young and aged Msn2/Y mice had comparable CD4+ Treg (CD4+ CD25+Foxp3+) cell numbers to those of their age-matched Msn+/Y 6 MOESIN REGULATION OF CD8+ Treg CELL HOMEOSTASIS counterparts (Fig. 5A, 5B). In addition, the number of Tfr (CD4+ CD25+Foxp3+CXCR5+Bcl6+) cells, a specialized subset of CD4+ Treg cells that suppress Tfh and GC B cells, was similar between Msn+/Y and Msn2/Y mice (Fig. 5C, 5D). In contrast, the number of CD8+ Treg (CD8+CD44+CD122+Ly49+) cells, which also inhibit Tfh cell expansion and are essential for self-tolerance (19, 20), were reduced in number and proportion in the spleen and LNs of young Msn2/Y mice compared with those of Msn+/Y mice (Fig. 5E, Supplemental Fig. 3A). A more severe reduction in CD8+ Treg cells was observed in the spleen and LNs of the aged Msn2/Y mice compared with the control mice (Fig. 5F, Supplemental Fig. 3B). These results suggested that moesin regulates CD8+ Treg cell homeostasis. The lymphopenic phenotype observed in the blood and LNs of young Msn2/Y mice is particularly prominent in the naive CD8+ T cell subset (7). Patients with mutations in the moesin gene also present with profound lymphopenia, with a marked decrease in naive CD8+ T cell counts (8). Naive T cells of the CD4+ and

CD8+ subsets decline with aging, with a concomitant increase in Downloaded from memory T cells. In this study, we found that the number and proportion of naive CD8+ T(CD8+CD44loCD45RBhi) cells were significantly reduced in the spleen of aged Msn2/Y mice com- pared with that of Msn+/Y mice, whereas naive CD4+ T(CD4+ CD44loCD45RBhi) cell numbers were comparable in the two

types of mice (Fig. 6A, 6B). In contrast, similar numbers of http://www.jimmunol.org/ memory CD4+ (CD4+CD44hiCD45RBlo)andCD8+ (CD8+ CD44hi) T cells were observed in the spleen of Msn+/Y and Msn2/ Y mice (Fig. 6A, 6B). Thus, CD8+ Treg cells, which have a FIGURE 6. CD8+ Treg defects in moesin-deficient mice are CD8+ Tcell memory phenotype, appear to be a small subset of memory CD8+ intrinsic. Identification of naive and memory CD4+ (A)orCD8+ (B) T cells in T cells that is severely affected by the moesin deficiency. The + +/2 the spleen from aged mice using flow cytometry. Naive and memory CD4 analysis of T cell subsets in female Msn mice showed that the T cells were identified as CD44loCD45RBhi and CD44hiCD45RBlo cells, ratio of moesin-deficient/moesin-expressing cells was decreased respectively. Naive and memory CD8+ T cells were identified as CD44lo + + more substantially in CD8 T cells than in CD4 T cells and was CD45RBhi and CD44hi cells, respectively. Data (mean 6 SEM) represent most profoundly reduced in the CD8+ Treg subset (Fig. 6C), results from six mice per group. (C) Expression of moesin in the CD8+ Treg, by guest on September 28, 2021 2 indicating that moesin plays a cell-intrinsic role in regulating CD8+ T, and CD4+ T cell subsets in the spleen from Msn+/Y, Msn /Y,and +/2 CD8+ Treg cell homeostasis. Msn mice. Staining results with an anti-moesin Ab (open graphs) and control IgG (shaded graphs) are shown. *p , 0.05, **p , 0.01, ***p , 0.001. Defective proliferation of moesin-deficient CD8+ Treg cells in response to IL-15 CD8+ Treg cells depend on IL-15, but not on IL-2, for their The receptors for IL-15 and IL-2 consist of three subunits: the maintenance and proliferation in humans and mice (38, 39). To shared subunits CD132 (gc) and CD122 (IL-2Rb) and the unique investigate the role of moesin in CD8+ Treg cell homeostasis, subunits IL-15Ra and CD25 (IL-2Ra), respectively (40). The CD8+ Treg cells isolated from the spleen of aged Msn+/Y or Msn2/Y trans-presentation model suggests that IL-15 bound to IL-15Ra mice were cultured in the presence of anti-CD3, anti-CD28, and presents the cytokine to target cells and signals via the CD122/ IL-15 or IL-2. Sorted CD4+ Treg cells were cultured as a control. CD132 complex (41, 42). However, IL-15Ra has also been shown We found that CD8+ Treg cells from Msn+/Y mice proliferated in to interact with CD122 and CD132 to form a high-affinity heter- the presence of IL-15, whereas Msn2/Y cells did not (Fig. 7A). otrimeric receptor that binds IL-15 and transduces signals via CD8+ Treg cells of either genotype did not proliferate appreciably STAT5 phosphorylation (43). Thus, we examined the levels of when cultured with IL-2, although Msn+/Y cells survived better p-STAT5 as a measure of IL-15 signaling. Upon IL-15 stimulation, than Msn2/Y cells (Supplemental Fig. 4A). In contrast, Msn2/Y Msn2/Y CD8+ Treg cells displayed markedly reduced p-STAT5 CD4+ Treg cells proliferated in response to IL-2, albeit to a levels compared with Msn+/Y cells (Fig. 7C, left panel), whereas slightly lesser degree than Msn+/Y cells (Fig. 7B). Msn+/Y and the reduction was not as marked in response to IL-2 (Fig. 7C, right Msn2/Y CD4+ Treg cells failed to proliferate in response to IL-15 panel). The mRNA expression of STAT5 was similar in the two (Supplemental Fig. 4B). The defective growth of Msn2/Y CD8+ cell types (Fig. 7D). The mRNAs encoding the three subunits of Treg cells was not due to increased apoptosis, given that the the IL-15R were also expressed at comparable levels in Msn+/Y percentage of annexin V+ propidium iodide2 apoptotic cells was and Msn2/Y cells (Fig. 7E, left panel). Their cell surface expres- comparable between Msn+/Y and Msn2/Y cells after 3 d of culture sion levels were also similar in the two genotypes (Fig. 7E, right (Supplemental Fig. 4C). Notably, naive CD8+ and CD4+ T cells panels). from Msn2/Y mice showed a similar or even enhanced prolifera- Because IL-15–mediated signaling is reported to involve the tion rate compared with Msn+/Y cells when stimulated with internalization of IL-15Ra but minimally of CD122 and CD132 anti-CD3, anti-CD28, and IL-2 (Supplemental Fig. 4D). Taken (44), we next examined the change in IL-15Ra cell surface ex- together, these results suggested that the reduced number of CD8+ pression upon IL-15 stimulation. We observed that IL-15Ra sur- Treg cells in Msn2/Y mice was due to their attenuated proliferation face levels decreased rapidly in response to IL-15 stimulation in in response to IL-15. Msn+/Y and Msn2/Y CD8+ Treg cells, but the rate in decrease was The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Moesin regulates CD8+ Treg cell responsiveness to IL-15. Growth curves of CD8+ Treg cells in response to IL-15 (A) and CD4+ Treg cells in response to IL-2 (B). CD8+ and CD4+ Treg cells isolated from the spleen of aged Msn+/Y and Msn2/Y mice were cultured with anti-CD3 and anti-CD28 for 9 d, with IL-15 for CD8+ Treg cells or IL-2 for CD4+ Treg cells. Data are expressed as the mean 6 SEM (n = 3). (C) STAT5 phosphorylation in CD8+ Treg cells stimulated with IL-15 (100 ng/ml; left panel) and IL-2 (100 ng/ml; right panel). Data (mean 6 SEM) were normalized to Msn+/Y cells in the absence of IL-15 (n = 3). (D) Relative expression of STAT5a/b mRNAs in CD8+ Treg cells. The mRNA levels were assessed by real-time PCR. Data (mean 6 SEM) represent the results from five mice per group. (E) mRNA and protein levels of IL-15R subunits in CD8+ Treg cells. The mRNA levels were assessed by real-time PCR (left panel). Data (mean 6 SEM) represent the results from four or five mice per group. Cell surface expression of IL-15R subunits was assessed by flow cytometry (right panels). (F) Cell surface expression of IL-15Ra on CD8+ Treg cells stimulated with IL-15. Data (mean 6 SEM) are expressed as mean fluorescence intensity (MFI) (upper panel). Relative loss of surface IL-15Ra was expressed as MFI decrease normalized to the value of Msn+/Y cells at 60 min, where the MFI decrease was calculated by subtracting the MFI at the indicated times from the basal MFI (n = 3) (lower panel). (G) Immunofluorescence analysis of IL-15Ra localization. CD8+ Treg cells isolated from the spleen of Msn+/Y and Msn2/Y mice were stimulated with IL-15 (100 ng/ml) for the indicated times and stained for IL-15Ra before permeabilization (green, surface IL-15Ra) and after permeabilization (red, mostly intracellular IL-15Ra). Scale bar, 2 mm. (H) The ratio of IL-15Ra stained before permeabilization/after permeabilization. Data are expressed as the mean 6 SEM (n = 5 or 6). *p , 0.05, **p , 0.01, ***p , 0.001. reduced in Msn2/Y cells (Fig. 7F), suggesting that IL-15Ra in- Discussion ternalization is affected by the moesin deficiency. To distinguish In this study, we showed that moesin deficiency led to an SLE-like between surface and intracellular IL-15Ra, IL-15–stimulated autoimmune disease in aged mice, which likely increased their CD8+ Treg cells were first stained for surface IL-15Ra, per- mortality. The spleen of moesin-deficient mice exhibited the meabilized, and stained again for intracellular IL-15Ra. Immu- spontaneous accumulation of Tfh cells and expansion of GC nofluorescence microscopy of the cells stained in this manner B cells, causing autoantibody production and immune complex revealed that most of the fluorescence signals obtained after per- deposition in the kidney, two hallmarks of SLE. CD8+ Treg cells, meabilization did not colocalize with surface staining, thus largely which inhibit the expansion of Tfh cells and, thus, are essential for representing intracellular IL-15Ra (Fig. 7G). The ratio of surface/ self-tolerance, were severely reduced in these mice. Furthermore, 2 intracellular IL-15Ra was higher in IL-15–stimulated Msn /Y we found that moesin regulated the homeostasis of CD8+ Treg cells than in Msn+/Y cells (Fig. 7H), supporting the view that cells in a cell-intrinsic manner through its unique role in modu- moesin plays a role in IL-15Ra internalization. Taken together, lating IL-15–dependent signaling. our results suggest that moesin contributes to the maintenance of The two ERM proteins moesin and ezrin, which are generally CD8+ Treg cells by regulating the IL-15/IL-15Ra signaling axis. regarded as functionally redundant, are abundantly expressed 8 MOESIN REGULATION OF CD8+ Treg CELL HOMEOSTASIS in lymphocytes (5, 6). However, our previous study showed that on IL-2, given that moesin deficiency caused some impairment in deficiency of moesin alone in mice leads to impaired lymphocyte CD8+ Treg cell survival and CD4+ Treg cell proliferation in the homeostasis, which is characterized by peripheral blood lym- presence of IL-2. The impact of these defects on CD8+ Treg cell phopenia (7). In this study, we showed that moesin deficiency led homeostasis in vivo remains to be determined. to a systemic autoimmune phenotype, with a marked decrease in Signaling initiated by cell surface receptors are often regulated CD8+ Treg cells, further demonstrating a unique role for moesin by endocytosis, including receptor internalization and sorting for in vivo. A unique role for moesin has also been implicated in degradation or recycling. Although G protein–coupled receptors humans through the study of primary immunodeficiency patients and receptor tyrosine kinases have been well studied for their with mutations in the moesin gene (8, 9). These patients present endocytic regulation of signaling, it is becoming clear that sig- with profound lymphopenia, which closely resembles that in naling through cytokine receptors, including gc family cytokine moesin-deficient mice. In humans and mice with moesin muta- receptors, is also regulated by endocytosis (51). IL-15Ra has been tions, the naive T cell counts are particularly low, with more se- shown to be internalized in response to IL-15 in CD8+ T cells (44). vere reductions observed in the naive CD8+ T cell subset than in Because the cytoplasmic domain of IL-15Ra is critical for IL-15– the naive CD4+ T cell subset. mediated signaling but not for trans-presentation (52), it is pos- Animal model studies and clinical observations have shown that sible that the cytoplasmic domain regulates IL-15Ra endocytosis lymphopenic conditions are permissive for the development of and, thus, signaling. A recent study showed that moesin is asso- autoimmunity (10–12). Under normal circumstances, the size of ciated with early endosomes, which are located close to the the peripheral T cell pool is remarkably stable. However, under membrane, and controls early-to-late endosome transport (53). lymphopenia, a homeostatic proliferation of T cells occurs as a Although the detailed mechanism by which moesin regulates Downloaded from compensatory mechanism to restore their numbers. The homeo- IL-15–dependent pathways remains unknown, the decreased in- static T cell expansion is accompanied by a loss of TCR diversity ternalization of IL-15Ra observed in the absence of moesin may and emergence of autoreactive T cells and may lead to autoim- explain, in part, the attenuated IL-15–mediated signaling and is munity. Other mechanisms of autoimmunity development involve consistent with studies describing a role for moesin in cell surface the loss or dysfunction of Treg cells (45). CD4+ Treg cells receptor trafficking (26, 54, 55).

expressing Foxp3 are primarily produced by the thymus as a In summary, our data establish a novel role for moesin in http://www.jimmunol.org/ functionally mature T cell subpopulation and play key roles in the maintaining CD8+ Treg cell homeostasis and self-tolerance. maintenance of self-tolerance and the control of physiological and Further research in this area will lead to a greater understand- pathological immune responses. The depletion of CD4+ Treg cells ing of how CD8+ Treg cells function and influence the devel- leads to the activation of rare self-reactive T cells, inducing severe opment of autoimmunity, along with important insights into the autoimmune diseases in humans and mice (46–48). Although the pathophysiology of primary immunodeficiency patients with CD8+ lineage of Treg cells is less understood, recent studies in- moesin mutations. dicate that Qa-1–restricted CD8+ Treg cells contribute to the maintenance of self-tolerance. Qa-1 mutant mice, which are de- Acknowledgments fective in the inhibitory interaction between CD8+ Treg cells and by guest on September 28, 2021 + We thank S. Tsukita for providing moesin-deficient mice and M. Yamada their target Qa-1 Tfh cells, exhibit severe autoimmune disease for technical assistance. (19). In addition, defective activity of CD8+ Treg cells is associ- ated with the development of an SLE-like disease in B6-Yaa Disclosures mutant mice (20). Although the lymphopenia-induced proliferation The authors have no financial conflicts of interest. of T cells probably precipitated the autoimmunity in Msn2/Y mice, we speculate that impaired CD8+ Treg cell homeostasis further contributed to the development of autoimmunity in these mice. It References + remains unknown whether the moesin deficiency in CD8 Treg cells 1. Bretscher, A., K. Edwards, and R. G. Fehon. 2002. ERM proteins and : alone is sufficient to cause autoimmunity. 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Msn+/Y Msn/Y Total cells

0.15 25 20 0.1 15 Msn+/Y /Y 10 Msn B220+ 0.05 IgD/IgM/Gr-1 5 0 0

SUPPLEMENTAL FIGURE 1. Moesin deficiency does not alter the frequency of NP-specific IgG1+ cells. The frequency of NP-specific IgG1+ cells in the spleens of Msn+/Y and Msn−/Y mice 8 days after NP-CGG immunization was examined by flow cytometry using 4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP)-coupled BSA labeled with allophycocyanin. Representative contour plots used for identifying NIP+IgG1+ B cells and the corresponding cell percentages and counts are shown. Numbers adjacent to the outlined areas indicate the percentage of cells in each. Data (mean ± SEM) represent the results from 3 mice per group.

Satooka et al. Supplemental Figure 2

Msn+/Y

Msn/Y

SUPPLEMENTAL FIGURE 2. Moesin deficiency does not alter B cell proliferation. B cells were purified from the spleens of Msn+/Y and Msn−/Y mice, labeled with carboxyfluorescein succinimidyl ester (CFSE), and cultured in the absence (unstim) or presence of lipopolysaccharide (LPS; 5 μg/ml), anti-IgM (5 μg/ml), LPS (2.5 μg/ml) plus anti-IgM (5 μg/ml), or IL-4 (50 ng/ml) plus anti-IgM (5 μg/ml) for 72 h. Cell proliferation was measured by flow cytometry. Numbers above the bracketed lines indicate the percentage of proliferating cells in each gate.

Satooka et al. Supplemental Figure 3

SUPPLEMENTAL FIGURE 3. LN CD8+ Treg cells are decreased in moesin-deficient mice. CD8+ Treg cells in the LNs of young (A) or aged (B) Msn+/Y and Msn−/Y mice were identified by flow cytometry. Expression of CD44 and CD122 in CD8+ T cells and of CD122 and Ly49 in CD8+CD44+CD122+ cells and the corresponding cell percentages and counts are shown. Numbers adjacent to the outlined areas in the representative contour plots indicate the percentage of cells in each. Data (mean ± SEM) represent the results from 4 (A) and 6 mice (B) per group. *p < 0.05, **p < 0.01.

Satooka et al. Supplemental Figure 4

A B

2 ** 2 ** 1.6 ** ** 1.6 ** ** 1.2 1.2 Msn+/Y Msn+/Y /Y /Y 0.8 Msn 0.8 Msn

0.4 0.4

0 0 0 2 4 6 8 10 0 2 4 6 8 10

C D

10 5 50 40 ** 8 4 40 30 3 6 Msn+/Y 30 Msn+/Y 20 Msn/Y Msn/Y 4 2 20 10 2 1 10 0 0 0 0

SUPPLEMENTAL FIGURE 4. Moesin deficiency does not affect CD8+ Treg cell apoptosis or naive CD8+ T cell proliferation. (A and B) Growth curve of CD8+ Treg cells in response to IL-2 (A) and of CD4+ Treg cells in response to IL-15 (B). CD8+ Treg and CD4+ Treg cells were isolated from aged Msn+/Y and Msn−/Y mice, cultured with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (1 μg/ml) for 9 days with IL-2 (100 ng/ml) for CD8+ Treg or IL-15 (100 ng/ml) for CD4+ Treg cells. Data (mean ± SEM) represent the results from triplicate wells. **p < 0.01. (C) Treg cell apoptosis. Isolated CD8+ or CD4+ Treg cells were cultured with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) in the presence of IL-15 or IL-2 (100 ng/ml each), respectively. At 72 h, the cells were stained with annexin V-FITC and propidium iodide (PI), and apoptotic (annexin V+PI−) cells were identified by flow cytometry. Data (mean ± SEM) represent the results from triplicate wells. (D) Naive T cell proliferation. Naive CD8+ (CD8+CD44loCD62Lhi) and CD4+ (CD4+CD44loCD62Lhi) T cells were sorted from the spleens of Msn+/Y and Msn−/Y mice. Sorted cells were labeled with CFSE followed by culture with anti-CD3 (10 μg/ml), anti-CD28 (10 μg/ml), and IL-2 (2 ng/ml). At 72 h, the cells were analyzed by flow cytometry. Data (mean ± SEM) represent the results from triplicate wells. **p < 0.01.