Erythematosus Regulatory T Cells in Systemic Lupus Double ...

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Mechanistic Target of Rapamycin Complex 1 Expands Th17 and IL-4 + CD4 −CD8− Double-Negative T Cells and Contracts Regulatory T Cells in Systemic Lupus Erythematosus Hiroshi Kato and Andras Perl

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J Immunol 2014; 192:4134-4144; Prepublished online 28 March 2014; doi: 10.4049/jimmunol.1301859 http://www.jimmunol.org/content/192/9/4134

The Journal of Immunology

Mechanistic Target of Rapamycin Complex 1 Expands Th17 and IL-4+ CD42CD82 Double-Negative T Cells and Contracts Regulatory T Cells in Systemic Lupus Erythematosus Hiroshi Kato and Andras Perl

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ystemic lupus erythematosus (SLE) is a systemic autoimmune disease that leads to cutaneous, arthritic, renal, pulmonary, neurologic, and hematologic disease. Although dysregulated humoral immunity plays a crucial role in the pathogenesis, significant contribution of T cells has been increasingly recognized (1–3). A subset of TCR ab+ T cells that express neither CD4 nor CD8, known as CD42CD82 doublenegative T cells (DN T cells), constitute at most 5% of T cells in human and murine peripheral blood. Notably, DN T cells are increased in SLE patients (1, 4) and have been shown to secrete IL-4 (4) and assist B cells to produce anti-dsDNA Abs (1, 5). Lupus DN T cells secrete both IFN-g and IL-4, whereas healthy

Division of Rheumatology, Department of Medicine, State University of New York, Upstate Medical University, College of Medicine, Syracuse, NY 13210; Division of Rheumatology, Department of Microbiology and Immunology, State University of New York, Upstate Medical University, College of Medicine, Syracuse, NY 13210; and Division of Rheumatology, Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, College of Medicine, Syracuse, NY 13210 Received for publication July 12, 2013. Accepted for publication February 26, 2014. This work was supported by the National Institutes of Health (Grant AI072648), the Alliance for Lupus Research, and the Central New York Community Foundation. Address correspondence and reprint requests to Dr. Andras Perl, State University of New York, College of Medicine, 750 East Adams Street, Syracuse, NY 13210. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: AF, Alexa Fluor; DN T cell, CD42CD82 doublenegative T cell; HC, healthy control; mTOR, mechanistic target of rapamycin; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; pS6RP, phosphorylated S6 ribosomal protein; S6K1, S6 kinase 1; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index; T-DN T cell, true DN T cell; Treg, regulatory T cell. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301859

control (HC) DN T cells secrete IFN-g only (3). DN T cells from SLE patients expand significantly after anti-CD3 stimulation and produce significant amounts of IFN-g and IL-17 (6). IL-17+ and DN T cells are found in kidney biopsy specimens in patients with lupus nephritis. A series of these observations underscores the relevance of IL-4 and IL-17 to DN T cell pathogenicity in SLE. Regarding the roles of Th cell subsets in SLE, it has been controversial whether SLE is driven by Th1 or Th2 immunity given the various animal models showing discrepant findings. In humans, some studies showed increased IL-4, but decreased IFN-g, in lupus patients (7, 8), whereas others indicate the importance of IFN-g in diffuse proliferative lupus nephritis (9, 10). SLE patients with higher SLE disease activity index (SLEDAI) score have lower IFN-g but higher IL-4 expression than those with lower SLEDAI score (11). Frequency of polymorphism of IFN-gR gene was more frequent in lupus patients and was associated with skewing toward Th2 response (12). There is also a growing body of evidence highlighting the importance of IL-17 in SLE. SLE patients have increased serum IL-17 and frequency of Th17 cells (13–16). There was a positive correlation between plasma IL-17 or Th17 cell frequency and SLEDAI score (13–15, 17). Regulatory T cells (Tregs) play indispensable roles in maintaining peripheral tolerance. Although it is an appealing hypothesis that Treg defect contributes to dysregulated immune response in SLE, there have been contradictory observations concerning this notion. In SLE patients, the number of Tregs was shown to be reduced (18–23), unchanged (24, 25), or increased (26, 27). The suppressive function of Tregs was shown to be decreased in active SLE (22, 28, 29), decreased only in a portion of patients (24), or unimpaired (20, 25, 30). It is important to note that various methods have been used to phenotypically define Tregs, which may, in part, account for these discrepant findings. Other lines of evidence


The mechanistic target of rapamycin (mTOR) is activated in CD42CD82 double-negative (DN) T cells and its blockade is therapeutic in systemic lupus erythematosus (SLE) patients. Murine studies showed the involvement of mTOR complex 1 (mTORC1) and 2 (mTORC2) in the differentiation of Th1/Th17 cells and Th2 cells, respectively. In this study, we investigated the roles of mTORC1 and mTORC2 in T cell lineage development in SLE and matched healthy control (HC) subjects. mTORC1 activity was increased, whereas mTORC2 was reduced, as assessed by phosphorylation of their substrates phosphorylated S6 kinase 1 or phosphorylated S6 ribosomal protein and phosphorylated Akt, respectively. Rapamycin inhibited mTORC1 and enhanced mTORC2. IL-4 expression was increased in freshly isolated CD8+ lupus T cells (SLE: 8.09 6 1.93%, HC: 3.61 6 0.49%; p = 0.01). DN T cells had greater IL-4 expression than CD4+ or CD8+ T cells of SLE patients after 3-d in vitro stimulation, which was suppressed by rapamycin (control: 9.26 6 1.48%, rapamycin: 5.03 6 0.66%; p < 0.001). GATA-3 expression was increased in CD8+ lupus T cells (p < 0.01) and was insensitive to rapamycin treatment. IFN-g expression was reduced in all lupus T cell subsets (p = 1.0 3 1025) and also resisted rapamycin. IL-17 expression was increased in CD4+ lupus T cells (SLE: 3.62 6 0.66%, HC: 2.29 6 0.27%; p = 0.019), which was suppressed by rapamycin (control: 3.91 6 0.79%, rapamycin: 2.22 6 0.60%; p < 0.001). Frequency of regulatory T cells (Tregs) was reduced in SLE (SLE: 1.83 6 0.25%, HC: 2.97 6 0.27%; p = 0.0012). Rapamycin inhibited mTORC1 in Tregs and promoted their expansion. Neutralization of IL-17, but not IL-4, also expanded Tregs in SLE and HC subjects. These results indicate that mTORC1 expands IL-4+ DN T and Th17 cells, and contracts Tregs in SLE. The Journal of Immunology, 2014, 192: 4134–4144.


Materials and Methods Human subjects Twenty-seven SLE patients fulfilling the American College of Rheumatology diagnostic criteria were studied (45). Twenty-six of 27 patients were female. Age of study participants was 44.2 6 2.3 y (mean 6 SEM) in HC and 45.6 6 2.3 y in SLE. Disease activity was assessed by the SLE disease activity index (SLEDAI) scores (46), which ranged from 0 to 10 (mean 6 SEM: 5.19 6 0.62). Mean daily prednisone dose was 5.31 6 0.91 mg. Immunosuppressive drugs taken by the study subjects included hydroxychloroquine (n = 25), methotrexate (n = 3), mycophenolate (n = 5), cyclosporine (n = 1), and tacrolimus (n = 1). In each experiment, peripheral blood was obtained from SLE patients and HCs who have been matched for age within 10 y, ethnic background, and sex, and processed in parallel.

Isolation of CD3+ T cells and cell culture PBMCs were isolated by using Ficoll-Histopaque gradient (GE Health Care Bio-Sciences, Piscataway, NJ). CD3+ T cells were isolated by negative selection using untouched human T cell isolation kit (Life Technologies, Carlsbad, CA). Purity of CD3+ T cells was confirmed to be .97% (data not shown). The cells were cultured in RPMI 1640 culture media (Corning CellGro, Manassas, VA) with 10% FCS (Life Technologies, Eugene, OR), 1% penicillin/streptomycin, and 1% L-glutamine (all from Corning CellGro) for 3 d either in the presence or absence of plate-bound anti-CD3 (anti-TCR ε hybridoma from American Type Culture Collection, Manassas, VA) and soluble anti-CD28 (BD Biosciences, San Jose, CA) with or without 100 nM rapamycin (Biotica, Cambridge, U.K.). In some experiments, IL-4 (100 ng/ ml; catalog no. 200-04; Peprotech, Rocky Hill, NJ), IL-17 (10 ng/ml; catalog no. 14-8179; eBioscience, San Diego, CA), anti–IL-4 (5 mg/ml; catalog no. 500815; Biolegend, San Diego, CA), or IL-17 (10 mg/ml; catalog no. MAB 317; R&D Systems, Minneapolis, MN) was added to the culture media.

Surface and intracellular staining Negatively isolated CD3+ T cells were stained with PE Cy7-conjugated anti-CD4 and PerCP Cy5.5-conjugated anti-CD8 with or without PE or

brilliant violet 510–conjugated anti-CD25 (all from BD Biosciences). The cells were permeabilized as per the manufacturer’s instructions and stained with Alexa Fluor (AF)-488– or AF-647–conjugated anti–phosphorylated S6 ribosomal protein (anti-pS6RP) to assess mTORC1 (Cell Signaling, Danvers, MA), violet 450–conjugated anti-pAkt to assess mTORC2 (BD Biosciences), AF-647–conjugated anti-FOXP3 (Biolegend, San Diego, CA), and PE-conjugated anti–GATA-3 (eBioscience). For cytokine detection, FITC-conjugated anti–IFN-g, allophycocyanin-conjugated anti– IL-4, and PE-conjugated anti–IL-17 were used alone or together. For intracellular cytokine staining, cells were preincubated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 6 h, and with brefeldin A for 5 h (10 mg/ml; all from Sigma-Aldrich). Isotype control Abs included FITC-conjugated mouse IgG1, PE-conjugated mouse IgG1 k, PE-conjugated rat IgG2b k, brilliant violet 510–conjugated mouse IgG1, violet 450–conjugated mouse IgG1, and APC-conjugated mouse IgG1. All isotype control Abs were obtained from BD Biosciences except for PE-conjugated rat IgG2b k, which was obtained from eBioscience.

Immunoblotting Using lysates of T cells cultured for 3 d as described earlier, we performed immunoblotting using anti-Akt, anti–pAkt-Ser473 (Cell signaling), anti– S6 kinase 1 (anti-S6K1), and anti-pS6K1 (Santa Cruz Biotechnology, Dallas, TX). The signal intensity was normalized to Actin (Millipore, Temecula, CA).

Statistical analysis Student t test was performed for comparison of phenotype between two groups. Two-way ANOVA was followed by Bonferroni’s posttest for multiple comparisons using Prism 5 software (GraphPad, La Jolla, CA), with p , 0.05 considered significant. Supplemental materials include Supplemental Figs. 1 and 2.

Results mTORC1 activity is prominently increased in DN T cells, whereas mTORC2 activity is reduced in CD8+ T cells in patients with SLE Freshly isolated DN lupus T cells contained greater frequency of pS6RPhi cells (29.40 6 6.37%) than corresponding HC cells (17.59 6 4.47%, p , 0.001; Fig. 1), where the frequency of pS6RPhi cells was higher in DN T cells than CD4+ or CD8+ T cells in HC and SLE (HC DN T cells: 17.59 6 4.47%, HC CD4+ T cells: 0.56 6 0.09%, p = 0.0004; HC CD8+ T cells: 2.90 6 0.50%, p = 0.0029; SLE DN T cells: 29.40 6 6.37%; SLE CD4+ T cells: 1.04 6 0.16%, p , 0.0001; SLE CD8+ T cells: 5.02 6 0.73%, p , 0.0001; Fig. 1). After 3-d in vitro culture, the frequency of pS6RPhi cells remained higher in DN T cells of SLE patients relative to HC donors (Fig. 1B). We next quantified mTORC2 activity by assessing the phosphorylation of Akt by flow cytometry (47). In marked contrast with pS6RP, the frequency of pAkt+ cells was reduced in freshly isolated SLE T cells, particularly in CD8+ T cells (CD3+ T cell: SLE, 4.58 6 1.43%; HC, 7.17 6 2.59%; p = 0.043, CD8+ T cells: SLE, 7.52 6 2.78%; HC, 14.26 6 4.84%; p = 0.019; Fig. 1C, 1D). After 3-d in vitro culture, the frequency of pAkt+ cells was reduced in DN lupus T cells (3.61 6 0.72%) relative to HC (6.72 6 1.63%; p = 0.035; Fig. 1D). Rapamycin suppresses mTORC1, but augments mTORC2, in human T cells Rapamycin inhibited mTORC1 in T cells cultured for 3 d in the presence of anti-CD3/CD28 based on pS6K1 expression detected by immunoblotting (Fig. 2A, 2B).We confirmed that these effects were truly attributed to rapamycin, but not DMSO in which rapamycin had been dissolved, by testing the effect of the same concentration of DMSO on all immunological phenotypes studied in this work (data not shown). Interestingly, rapamycin augmented mTORC2 based on pAkt expression in HCs and SLE patients (Fig. 2A, 2B). These data suggest that mTORC1, but not mTORC2, is a target of rapamycin treatment in SLE.


indicate negative correlation between Treg frequency or suppressive function and SLEDAI score (14, 20–22). Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase, which plays pivotal roles in metabolism, cell growth, proliferation, survival, and differentiation (31). mTOR has recently emerged as a key regulator of T cell proliferation and differentiation (32–36). mTOR complex 1 (mTORC1) is essential for Th1 and Th17 differentiation, whereas mTOR complex 2 (mTORC2) is indispensable for Th2 differentiation in mice (37). mTORC1 and mTORC2 inhibit Treg differentiation by suppressing Foxp3 expression (38). Consistent with this, rapamycin promotes the generation of Tregs both in vitro and in vivo (39–42). Our laboratory has shown increased mTOR in SLE T cells (43). Importantly, rapamycin alleviates some features of active SLE refractory to other immunosuppressive drugs (44). Despite such a growing body of insights into the relevance of mTOR in T cell lineage development, as well as the extensive observations about the aberrant T cell activation in SLE, roles of mTOR in T cell cytokine expression and Treg development in SLE remain unknown. In this article, we show that SLE T cells had increased activity of mTORC1, most prominently in DN T cells, but reduced activity of mTORC2, particularly in CD8+ T cells. IL-4 expression was increased in freshly isolated CD8+ T cells, as well as DN T cells of SLE patients, especially in those with higher disease activity. Although increased IL-4 production by CD8+ T cells was associated with rapamycin-resistant overexpression of GATA-3, it was mTORC1 dependent in DN lupus T cells. In marked contrast with IL-4, IFN-g expression was reduced in all SLE T cell subsets and was not dependent on mTORC1. SLE T cells expressed higher IL-17, most prominently in CD4+ T cells in an mTORC1-dependent manner. Frequency of CD4+CD25+FOXP3+ Tregs was reduced in SLE. Rapamycin expanded Tregs by blocking mTORC1 in these cells and by contracting Th17 cells, shedding new light on the therapeutic mechanisms of action of rapamycin in SLE.

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mTOR SKEWS T CELL LINEAGE DEVELOPMENT IN SLE

IL-4 expression is increased in freshly isolated SLE T cells, most prominently in CD8+ T cells

IL-4 expression is greatest in DN T cells after 3-d in vitro stimulation, which is suppressed by rapamycin

In freshly isolated untouched T cells, lupus patients had a greater frequency of IL-4+ cells (5.77 6 1.00%) than HCs (3.88 6 0.39%; p = 0.019, Fig. 3A, 3B). IL-4+ cells were most prominently expanded in CD8+ T cell compartment (SLE: 8.09 6 1.93%, HCs: 3.61 6 0.49%, p = 0.01). In SLE patients with SLEDAI score $5, IL-4 production was increased not only in CD8+ T cells (SLE: 9.58 6 2.80%; HCs: 3.93 6 0.80%; p = 0.028), but also in DN T cells (SLE: 5.96 6 1.23%; HCs: 4.48 6 0.97%; p = 0.027).

We stimulated T cells with PMA/ionomycin for intracellular cytokine staining. However, such stimulation causes CD4 downmodulation (48). In our experiments, this was particularly significant in cells cultivated in the presence of anti-CD3/CD28, rendering the bona-fide DN T cell population obscure (Fig. 4A). To eliminate the effect of such CD4 downmodulation as much as possible, we defined a population of T cells as highlighted in Fig. 4A, which we designated as “true double-negative T cells” (T-DN T cells). After stimulation for 3 d in the presence of anti-CD3/CD28, T-DN


FIGURE 1. Increased mTORC1 and decreased mTORC2 activities in T cells of patients with SLE. Untouched T cells from HCs and SLE patients were stained with CD4, CD8, anti-pS6RP, and anti-pAkt Abs immediately after isolation on day 0 (d0) and after 3-d incubation (d3). (A) Representative flow cytometry histograms of pS6RP staining. Numbers in the histograms denote the frequency of pS6RPhi cells in each T cell subset. Blue and red histograms and numbers represent data from HC and SLE donors, respectively. In the histogram of DN T cells, CD3+ T cells are overlaid as dotted lines. (B) The left panel shows cumulative data from eight sets of patients and matched HCs on day 0 (d0; ***p = 0.0029, †p , 0.001, ††p = 0.0004, †††p , 0.0001), whereas the right panel shows data after 3-d incubation (d3; *p , 0.05, **p = 0.0109, †p , 0.001). (C) Representative flow cytometry histograms of pAkt staining. Numbers in the histograms denote the frequency of pAkt+ cells in each T cell subset. Blue and red histograms and numbers represent data from HC and SLE subjects, respectively. (D) The left panel shows cumulative data from nine sets of patients and matched HCs on day 0 (d0: *p = 0.043, ***p = 0.019, †† p , 0.001), whereas the right panel shows data after 3-d incubation (d3: **p = 0.035, †p , 0.01, ††p , 0.001).


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T cells contained a greater frequency of IL-4+ cells than CD4+ or CD8+ T cells (HC T-DN T cells: 9.18 6 0.96%; HC CD4+ T cells: 4.02 6 0.58%; p , 0.001; HC CD8+ T cells: 4.77 6 0.62%; p , 0.001; SLE T-DN T cells: 9.26 6 1.48%; SLE CD4+ T cells: 3.12 6 0.40%, p , 0.001; SLE CD8+ T cells: 4.66 6 0.51%, p , 0.001; Fig. 4B). Rapamycin suppressed IL-4 expression by T cells cultured for 3 d in the presence of anti-CD3/CD28, most robustly in T-DN T and DN T cells (HC T-DN T cells: control, 9.18 6 0.96%; rapamycin, 4.40 6 0.51%, p , 0.001; HC DN T cells: control, 6.69 6 0.98%; rapamycin, 3.40 6 0.27%, p , 0.001; SLE T-DN T cells: control, 9.26 6 1.48%; rapamycin, 5.03 6 0.66%, p , 0.001; SLE DN T cells: control, 6.54 6 1.13%; rapamycin, 3.04 6 0.32%, p , 0.001; Fig. 4B). Rapamycin also suppressed the total number of IL-4–producing CD3+, CD4+, and DN T cells (Supplemental Fig. 2A), which can be attributed to the reduced proliferation of all T cell subsets exposed to rapamycin treatment (Supplemental Fig. 2C). GATA-3 expression is increased in CD8+ lupus T cells and is resistant to rapamycin treatment The data so far identified mTORC1 as an important mediator of IL-4 production by DN lupus T cells. However, in freshly isolated T cells, IL-4 was most prominently increased in CD8+ lupus T cells (Fig. 3A, 3B), in which we did not observe a significant increase of mTORC1 activity (Fig. 1A, 1B). These findings prompted us to look into mechanisms other than mTORC1 possibly underlying elevated IL-4 production by CD8+ lupus T cells. Indeed, freshly isolated CD8+ lupus T cells expressed increased GATA-3 (SLE: 13.25 6 4.62%, HC, 3.35 6 0.64%, p , 0.01; Fig. 5A, 5B), where GATA-3 expression was greater in CD8+ T cells than CD4+ (4.14 6 1.28%; p , 0.001) and DN T cells (7.15 6 2.51%; p , 0.01; Fig. 5). After 3-d in vitro culture, GATA-3 expression remained higher in CD8+ lupus T cells than corresponding HC cells, where CD8+ T cells contained greater frequency of GATA-3+ cells than CD4+ and DN T cells of SLE patients (Fig. 5B). Unlike mTORC1, GATA-3 was not suppressed by rapamycin in TCR-stimulated T cells (Supplemental Fig. 1A, 1B). These

findings suggest that rapamycin-resistant GATA-3, but not mTORC1, is likely mediating greater IL-4 production by SLE CD8+ T cells, which would account for the rapamycin resistance of IL-4 production by CD8+ T cells (Fig. 4B). IFN-g expression is reduced in all T cell subsets in SLE and reversed in T-DN T cells by rapamycin The data so far suggest the involvement of mTORC1 in IL-4 expression by DN T cells in SLE. In view of the opposing relation between IFN-g and IL-4 in Th cell differentiation, we next assessed IFN-g expression by HC and SLE T cells, and sought to determine the role of mTORC1 in T cell IFN-g expression. The proportion of IFN-g+ cells was greater among CD8+ than CD4+ and DN T cells (Figs. 3, 4C). SLE T cells, including DN T cells, contained fewer IFN-g+ cells than corresponding HC cells (Figs. 3, 4C). After 3-d stimulation, rapamycin augmented IFN-g expression by T-DN T cells, thus reversing the depletion of IFN-g– producing cells in this T cell compartment of patients with SLE (Fig. 4C). SLE T cells show increased expression of IL-17 that is corrected by rapamycin Previous observations by others suggest the relevance of both IL-4 and IL-17 to lupus pathogenesis (49, 50). Therefore, we also assessed the IL-17 expression by various T cell subsets in HC and SLE, and sought to determine the role of mTORC1 in T cell IL-17 expression. In freshly isolated T cells, SLE patients had a greater frequency of IL-17+ cells (2.86 6 0.44%) than HC T cells (2.11 6 0.20%; p = 0.043; Fig. 3A, 3B), most prominently in CD4+ T cells (SLE: 3.62 6 0.66%, HC: 2.29 6 0.27%; p = 0.019). After 3-d in vitro stimulation by anti-CD3/CD28, IL-17 expression remained increased in CD4+ T cells (SLE: 3.91 6 0.79%, HC: 2.46 6 0.34%; p , 0.01, Fig. 4D). Importantly, rapamycin inhibited IL-17 expression by CD3/CD28stimulated lupus T cells, most robustly in CD4+ T cells from 3.91 6 0.79% to 2.22 6 0.60% (p , 0.001, Fig. 4D). Rapamycin also suppressed the total number of IL-17–producing


FIGURE 2. Rapamycin suppresses mTORC1 activity but augments mTORC2 activity in lupus and control T cells. Untouched T cells from HC and SLE donors were cultured for 3 d in the presence of anti-CD3/CD28 with (+) and without 100 nM rapamycin (2). After obtaining T cell lysates, we performed immunoblotting using anti-Akt, anti-pAkt, anti-S6K1, and anti-pS6K1. (A) Representative immunoblot staining. (B) Cumulative data from five sets of experiments are presented after normalizing the signal intensity to human b-actin (*p = 0.02, **p = 0.0096, ***p = 0.004, ****p = 0.002).

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cells within the CD3+ and CD4+ T cell compartments (Supplemental Fig. 2B). Rapamycin blocks mTORC1 and promotes FOXP3 expression in CD4+CD25+ T cells To further expand our view into the roles of rapamycin treatment in SLE, we assessed the frequency of Tregs, as well as the impact of rapamycin on Treg expansion in HC and SLE. In untouched T cells rested in vitro for 3 d, the frequency of Tregs, defined as CD4+CD25+FOXP3+ T cells, was reduced in SLE patients (1.83 6 0.25%) relative to HC donors (2.97 6 0.27%, p = 0.0012; Fig. 6A, 6B). After 3-d CD3/CD28 stimulation, rapamycin promoted Treg expansion in HCs from 19.77 6

1.84 to 27.26 6 1.84% (p = 1.7 3 1027) and in SLE patients from 19.56 6 1.55 to 26.72 6 1.35% (p = 1.7 3 1027; Fig. 6C). Although rapamycin suppressed the proliferation of all T cell subsets, the number of Tregs was preserved after 3-d rapamycin treatment (Supplemental Fig. 2C). Thus, the rapamycin-induced relative expansion of CD4+CD25+FOXP3+ T cells may be attributed to their lower proliferative rate. Rapamycin inhibited mTORC1 as assessed by the frequency of pS6RPhi cells, which was reduced from 62.02 6 2.30 to 3.54 6 0.25% in HC donors (p = 3.7 3 10218) and from 62.45 6 2.80 to 2.92 6 0.26% in SLE patients (p = 2.9 3 10216; Fig. 6D, 6E, Supplemental Fig. 1A). Importantly, rapamycin directly blocked mTORC1 within CD4+ CD25+FOXP3+ T cells as evidenced by the reduced frequencies


FIGURE 3. Increased IL-4 and IL-17 and reduced IFN-g expression in freshly isolated lupus T cells. (A) Representative flow cytometry dot plots of IFN-g versus IL-4 staining (upper panel) and IL-17 versus IL-4 staining in lupus T cells (lower panel). Control denotes staining with isotype control Abs. (B) The left panel shows cumulative data of IL-4 expression from 18 sets of patients and matched HCs (***p = 0.019, †p = 0.01, †††p , 0.001). The center panel shows cumulative data of IFN-g expression from 15 sets of patients and matched HCs (††p = 0.002, †††p , 0.001, ‡p = 3.1 3 1025, ‡‡p = 1.0 3 1025, ‡‡‡ p = 3.6 3 1026). The right panel shows cumulative data of IL-17 expression from 17 sets of patients and matched HCs (*p , 0.05, **p = 0.043, ***p = 0.019, †††p , 0.001). Paired t test was performed to compare patients and matched HCs. Two-way ANOVA was performed to compare different T cell subsets.


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FIGURE 4. Rapamycin reverses the increased production of IL-4 and IL-17 by lupus T cells in a subsetspecific manner after CD3/CD28 stimulation for 3 d. (A) Flow cytometry detection of DN and T-DN T cells after incubation of untouched T cells for 3 d in the presence or absence of anti-CD3/CD28 and subsequent 6-h PMA/ionomycin treatment. (B) Cumulative data of IL-4 expression from 13 sets of patients and matched HCs (*p , 0.05, **p , 0.01, ***p , 0.001). (C) Cumulative data of IFN-g expression from 13 sets of patients and matched HCs (*p = 0.037, **p = 0.035, ***p = 0.033, ****p = 0.001, †p , 0.001, ††p = 0.00026, †††p = 0.00017). (D) Cumulative data of IL17 expression from 17 sets of patients and matched HCs (*p , 0.05, **p , 0.01, ***p , 0.001). Twoway ANOVA was performed to compare different T cell subsets and to determine the impact of 100 nM in vitro rapamycin treatment on cytokine production.

of pS6RPhi cells from 75.58 6 1.91 to 6.22 6 0.56% in HC donors (p = 1.6 3 10217) and from 72.86 6 3.07 to 5.29 6 0.62% in SLE patients (p = 1.5 3 10213; Fig. 6D, 6E). Notably, the CD4+ CD25+FOXP3+ T cells expressed higher mTORC1 than CD4+, CD8+, and DN T cells in HCs (CD4+ CD25+FOXP3+ T cells:

75.58 6 1.91%; CD4+ T cells: 63.76 6 2.40%, p = 0.0004; CD8+ T cells: 62.16 6 2.63%, p = 0.00021; DN T cells: 38.93 6 2.94%, p = 4.8 3 10212) and SLE donors (CD4+ CD25+FOXP3+ T cells: 72.86 6 3.07%, CD4+ T cells: 64.11 6 2.87%, p = 0.023; CD8+ T cells: 62.45 6 3.03%, p = 0.012; DN T cells: 36.94 6 2.52%,

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p = 2.1 3 10211; Fig. 6E), which was not the case in freshly isolated T cells or T cells rested in vitro for 3 d (data not shown). IL-17 blockade expands Tregs In light of our earlier observations that rapamycin suppressed IL-4 expression in DN T cells and IL-17 expression in CD4+ T cells, we examined whether these cytokines modulate Treg expansion by using neutralizing Abs directed to IL-4 or IL-17. After CD3/CD28 stimulation, the neutralization of IL-17 significantly expanded CD4+CD25+FOXP3+ “Tregs” from 23.71 6 3.97 to 27.15 6 4.00% in HC donors (p , 0.05) and from 22.10 6 1.75 to 25.53 6 1.61% in SLE patients (p , 0.05), suggesting that IL-17 inhibited Treg expansion (Fig. 7A, 7B). However, the IL-17 neutralization had no impact on mTORC1 (data not shown), indicating that IL-17 restrains Treg expansion through mTORC1-independent mechanisms. Unlike IL-17, neither exogenous IL-4 nor neutralization of IL-4 had significant impact on Treg expansion after 3-d in vitro CD3/CD28 stimulation (Fig. 7), as also illustrated in Fig. 8.

Discussion Our data solidify the importance of mTOR in mediating the increased production of IL-4 by DN T cells in SLE patients, where DN T cell IL-4 expression appears to be mTORC1 dependent (Fig. 8). DN T cells assist B cells to produce autoantibodies in SLE. Importantly, murine CD8+ T cells activated in the presence of IL-4 convert to DN T cells, which produce Th2 cytokines and induce B cells to produce Igs (51). Human CD8+ T cells convert to DN T cells on in vitro stimulation (52). These observations present an intriguing paradigm where IL-4 serves as an autoamplification factor for DN T cells by committing CD8+ T cells to expand the pathogenic DN T cell population in SLE. This model may account for our findings that freshly isolated CD8 + T cells most prominently produced IL-4, whereas DN T cells produced higher IL-4 than CD4+ and CD8+ T cells after 3-d TCR stimulation in patients with SLE.

Although our data identify mTORC1 as an important driver of IL-4 production in DN lupus T cells, IL-4 expression was not prominently elevated in freshly isolated DN T as compared with CD8+ T cells in patients with SLE. We attributed the moderate elevation in IL-4 production by DN T cells in this study to a lower level of disease activity. SLE patients with higher SLEDAI had higher IL-4 expression than those with lower SLEDAI score (11). Previously, we found that patients with SLEDAI of 11.8 6 1.1 had elevated IL-4 production in DN T cells (53). The enrollment criteria for the earlier study included patients who were not responsive or tolerant to other medications. In this study, the average SLEDAI of study subjects was 5.19 6 0.62. In fact, we found that SLE patients with SLEDAI disease activity scores $5 also exhibited increased IL-4 expression by DN T cells. IL-4 expression was increased in freshly isolated SLE CD8+ T cells, although this subset did not express increased mTORC1. Alternatively, GATA-3 was increased in SLE CD8+ T cells, but not in SLE DN T cells. Although GATA-3 is a master regulator gene for Th2 cells (54, 55), it is also known to be expressed by CD8+ T cells (56). GATA-3 expression was increased in CD8+ T cells in patients with systemic sclerosis (57). Although IL-4 expression by DN T cells was rapamycin sensitive, IL-4 expression by CD8+ T cells was rapamycin resistant, as was GATA-3. These findings suggest that rapamycin-sensitive mTORC1 and rapamycin-resistant GATA-3 are likely driving IL-4 expression by SLE DN T and CD8+ T cells, respectively (Fig. 8). In contrast with mTORC1, mTORC2 activity was reduced in SLE T cells, most prominently in CD8+ T cells. Rapamycin effectively blocked mTORC1, whereas it augmented mTORC2. These findings are consistent with a role for mTORC1 in inhibiting mTORC2 (58) and the relative specificity of rapamycin for blocking mTORC1 (59, 60). In marked contrast with IL-4, IFN-g was reduced in SLE T cells and was not suppressed by rapamycin. It is important to note that there was a reciprocal pattern of IL-4 versus IFN-g expression by SLE CD8+ T cells; the former is increased, whereas the latter is


FIGURE 5. Increased expression of GATA-3 in CD8+ lupus T cells. Untouched T cells from HC and SLE donors were examined immediately after isolation on day 0 (d0) and after 3-d incubation in vitro (d3). (A) Representative flow cytometry histograms of GATA-3 staining on day 0. Numbers in the histograms denote the frequency of GATA-3+ cells in each T cell subset. Blue and red histograms and numbers represent data from HC and SLE donors, respectively. (B) The left panel shows cumulative data from 10 sets of patients and matched HCs on day 0 (d0: **p , 0.01, ***p , 0.001), whereas the right panel shows cumulative data from 6 sets of patients and matched HCs after 3-d culture (d3: *p , 0.05, **p , 0.01, ***p , 0.001). Data were analyzed by two-way ANOVA.


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decreased. In light of our data supporting the relevance of IL-4 to lupus pathogenesis, as well as the reciprocal pattern of expression and rapamycin responsiveness of IL-4 versus IFN-g, IFN-g may protect against SLE (Fig. 8). Whereas IL-4 expression was highest in freshly isolated, untouched CD8+ T cells and in DN T cells after 3-d in vitro stimulation, CD4+ T cells appear to be the major source of IL-17 in SLE. Given the potent inhibitory potential of IL-4 on Th17 differentiation, this is consistent with our observation that IL-4 expression was lowest in CD4+ T cells. It is quite intriguing that production of two potentially pathogenic cytokines in SLE, IL-4


FIGURE 6. Rapamycin suppresses mTORC1 and promotes the expansion of CD4+CD25+FOXP3+ Tregs in untouched T cells from SLE and matched HC donors. (A) Representative flow cytometry of cells cultured for 3 d without CD3/CD28 stimulation. Numbers in the dot plots represent the frequency of CD4+CD25+FOXP3+ T cells. Control denotes staining with isotype control Abs. (B) Depletion of Tregs among untouched T cells of SLE patients. Cumulative data from 17 sets of patients and matched HCs were analyzed with paired t test (***p = 0.0012). (C) Expansion of Tregs by rapamycin in vitro. Untouched T cells from SLE and matched HC donors were cultured for 3 d in the presence of anti-CD3/CD28 with or without 100 nM rapamycin. Cumulative data from 19 sets of patients and matched HC donors were analyzed by paired t test (‡p = 1.7 3 1027). (D) Representative detection of pS6RP in T cell subsets by flow cytometry. Control denotes staining with isotype control Abs. (E) Effect of rapamycin on mTORC1 measured by pS6RP expression in T cell subsets. Data show cumulative analysis of 23 sets of patients and matched HCs using paired t test (*p = 0.023, **p = 0.012, ***p = 0.0088, ****p = 0.0004, *****p = 0.00021, † p = 5.5 3 10 25 , ††p = 2.1 3 10 211 , ††† p = 1.3 3 10211, ††††p = 4.8 3 10212, ††††† p = 7.3 3 10213, ‡p = 1.5 3 10213, ‡‡ p = 2.9 3 10215, ‡‡‡p = 2.9 3 10216, ‡‡‡‡ p = 9.0 3 10217, ‡‡‡‡‡p = 6.8 3 10217, xp = 1.6 3 10217, xxp = 3.7 3 10218, xxxp = 1.8 3 10218).

and IL-17, are driven by different T cell subsets, DN T cells and CD4+ T cells, and that DN T cell IL-4 expression and CD4+ T cell IL-17 expression are particularly sensitive to rapamycin treatment (Fig. 8). Notably, we did not find significant proportion of IL-4+ IL-17+ cells in our experiments, which is consistent with our observation that these cytokines are expressed by different subsets of T cells in SLE. In contrast with our study, a study by others showed that the frequency of IL-17+ cells was highest in DN T cells both in HCs and in SLE patients (6). It is important to note that they used PBMCs, whereas we used untouched T cells. Hence the potential explanation of this discrepancy would be either the

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APCs in the PBMCs promoting DN T cell IL-17 expression or the APCs negatively regulating CD4+ and CD8+ T cell IL-17 expression. We reviewed that there had been contradictory reports whether Tregs are decreased in SLE patients. This discrepancy may be attributable to the variability of methods by which Tregs have been phenotypically defined (28, 61, 62). Given that systemic corticosteroid was shown to promote Treg expansion (63), it is important to consider the variability of frequency of immunosuppressive drug usage, as well as steroid dosage depending on the study

FIGURE 8. Blockade of mTORC1, but not GATA-3, by rapamycin reverses proinflammatory cytokine imbalance in lupus T cells. Production of IL-4 and IL-17 is increased in DN and CD4+ T cells, respectively, in an mTORC1-dependent manner. Lupus CD8+ T cells produce IL-4 in an mTORC1-independent but GATA-3 dependent manner. Depletion of CD4+CD25+ FOXP3+ Tregs is reversed by rapamycin through direct blockade of mTORC1. Increased production of IL-17 may indirectly restrain Tregs. IFN-g is reduced in SLE, which is mTORC1 independent. The components highlighted in red and blue denote increases and decreases in SLE, respectively. Green arrows denote the corrective effects of rapamycin.

population. Although frequency of Tregs was reduced in SLE after 3-d in vitro culture, this was restored by rapamycin treatment, providing another rationale for rapamycin treatment in SLE. We observed robust rapamycin-mediated mTORC1 suppression in Tregs, suggesting that mTORC1 inhibition is one of the salient mechanisms in human Treg expansion where Treg-intrinsic effect of rapamycin plays an essential role (Fig. 8). Importantly, neutralization of IL-17 expanded Tregs, comparably with what was observed in rapamycin-treated cells, thus suggesting that inhibition of IL-17 may contribute to rapamycin-mediated Treg ex-


FIGURE 7. IL-17 restrains CD4+CD25+FOXP3+ Treg expansion. Untouched T cells from matched HC and SLE donors were stimulated with anti-CD3/ CD28 for 3 d in the absence or presence of IL-4 (100 ng/ml), anti–IL-4 (5 mg/ml), IL-17 (10 ng/ml), anti–IL-17 (10 mg/ml), or rapamycin (100 nM). (A) Representative flow cytometry dot plots. (B) Cumulative data from six sets of patients and matched HC donors analyzed by two-way ANOVA (*p , 0.05, ***p , 0.001). Control denotes stimulation with anti-CD3/CD28 without exogenous cytokine, cytokine-neutralizing Abs, or rapamycin.


Acknowledgments We thank all patients and healthy volunteers who contributed to this study.

Disclosures The authors have no financial conflicts of interest.

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Supplementary Figure S1

A

CD3+ T

CD4+ T

CD8+ T

DN T

52.76 %

62.00 %

37.35 %

20.64 %

48.01 %

53.49 %

39.48 %

18.88 %

54.81 %

61.91 %

44.19 %

23.97 %

56 23 % 56.23

64 44 % 64.44

44 83 % 44.83

25 33 % 25.33

Rapa -

HC

Rapa +

GATA-3

Rapa -

SLE

Rapa +

pS6RP

B Effect of Rapa on GATA-3 by d3 activated T cells 80 HC Rapa (-) HC Rapa (+) **

%G GATA-3 +

60

SLE Rapa (-) SLE Rapa (+)

40

20

0 CD3+ T

CD4+ T

CD8+ T

DN T

FIGURE S1. Rapamycin does not influence expression of GATA-3. (A) Untouched T cells from matched HC and SLE donors were stimulated with anti-CD3/CD28 in the presence or absence of 100 nM rapamycin for 3 days. GATA-3 expression was analyzed by flow cytometry. The numbers in the representative dot plots denote the frequency of GATA-3+ cells. (B) Cumulative data from 9 sets of SLE patients and matched HC donors analyzed by two-way ANOVA (** p