Activated T Cells Secrete an Alternatively Spliced Form of Common γ-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity

Article Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation Changwan Hong,1,5 Megan A. Luckey,1 Davinna L. Ligons,1 Adam T. Waickman,1 Joo-Young Park,1 Grace Y. Kim,1 Hilary R. Keller,1 Ruth Etzensperger,1 Xuguang Tai,1 Vanja Lazarevic,1 Lionel Feigenbaum,2 Marta Catalfamo,3 Scott T.R. Walsh,4 and Jung-Hyun Park1,* 1Experimental

Immunology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA 3Clinical and Molecular Retrovirology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA 4Department of Cell Biology and Molecular Genetics, Institute for Bioscience and Biotechnology Research, W. M. Keck Laboratory for Structural Biology, University of Maryland, Rockville, MD 20850, USA 5Present address: Department of Anatomy, Pusan National University School of Medicine, Yangsan 626-870, South Korea *Correspondence: [email protected] http://dx.doi.org/10.1016/j.immuni.2014.04.020 2Leidos

SUMMARY

The common g-chain (gc) plays a central role in signaling by IL-2 and other gc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of gc mRNA that results in protein expression and secretion of the gc extracellular domain. The soluble form of gc (sgc) is present in serum and directly binds to IL-2Rb and IL-7Ra proteins on T cells to inhibit cytokine signaling and promote inflammation. sgc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sgc. Thus, sgc expression is a naturally occurring immunomodulator that regulates gc cytokine signaling and controls T cell activation and differentiation.

INTRODUCTION Cytokines of the common g-chain (gc) family are critical for development of T lineage cells and depend on gc for cellular signaling (Rochman et al., 2009). gc deficiency results in a paucity of both mature B and T cells because of impaired signaling by the gc cytokine IL-7 (Noguchi et al., 1993). In fact, IL-7 signaling is necessary for developing lymphocytes to proceed through the pre-pro-B cell stage of B cell differentiation and the CD4 CD8 double-negative (DN) stage of thymopoiesis (DiSanto et al., 1995; Peschon et al., 1994; von Freeden-Jeffry et al., 1997). Impaired IL-7 signaling also profoundly impairs mature T cell survival and homeostasis (Schluns et al., 2000; Tan et al., 2001). Because gc is also required for IL-2 and IL-15 signaling, gc deficiency impairs development of FoxP3+ regulatory T cells and NK

cells (Heaney and Golde, 1996; Ma et al., 2006; Vosshenrich et al., 2005) and also alters CD4 T helper cell lineage fate and CD8 memory cell differentiation (Rochman et al., 2009). Thus, gc governs the generation, differentiation, and homeostasis of all lymphocyte subsets in the adaptive immune system. Although gc expression is necessary for gc cytokine signaling, gc alone cannot bind cytokines and cannot trigger downstream signaling (Minami et al., 1993). Rather, gc cytokines induce gc membrane proteins to complex with proprietary cytokine receptor subunits, such as the IL-7-specific IL-7 receptor a chain (IL-7Ra) and the IL-2-specific IL-2 receptor b-chain (IL-2Rb), to transduce cytokine receptor signals. Importantly, the magnitude and kinetics of gc cytokine responses are thought to be controlled by the proprietary cytokine-specific receptor subunits and not by gc (Rochman et al., 2009). For example, IL-7 stimulation affects IL-7Ra expression but does not affect gc expression (Park et al., 2004), and IL-2 stimulation affects IL-2Ra and IL-2Rb without affecting gc expression (Depper et al., 1985; Siegel et al., 1987). Consequently, modulation of gc expression is thought to be irrelevant to either the kinetics or magnitude of cytokine signaling. Instead, gc expression levels are thought to be developmentally set and to remain constant during T cell activation and differentiation (Rochman et al., 2009). In contrast to this prevailing view, we now report that modulation of gc expression actively contributes to the regulation of cytokine responses. Interestingly, gc exerts its regulatory effects not as a conventional membrane receptor protein but as a secreted protein induced by T cell stimulation. Specifically, we found that activated T cells expressed a gc mRNA splice isoform that resulted in production and secretion of soluble gc ectodomain proteins. Soluble gc inhibited cell signaling by gc cytokines and exacerbated inflammatory responses by promoting differentiation of pathogenic CD4+ Th17 cells both in vitro and in vivo. Conversely, the severity of Th17-cell-mediated inflammatory autoimmune disease was markedly diminished in mice lacking sgc. Thus, this study identifies a role for gc as an active immunoregulatory molecule whose alternatively spliced form is secreted to dampen signaling by gc cytokines and promote inflammatory T cell immune responses in vivo. Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc. 1

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

Figure 1. Soluble gc Proteins Are Products of Alternative Splicing (A) Activated T cells express soluble gc proteins. T cells were stimulated with a-TCR and a-CD28 in the presence or absence of the metalloprotease inhibitor TAPI2. Culture supernatants were harvested at indicated days and assessed for IL-2, TNF-a, and soluble gc. Results are the summary of three independent experiments. (B) Immunoblot detection of sgc proteins in WT and Il2rg / sera. Serum samples were immunoprecipitated (IP) with gc-ED-specific antibodies and probed with the same antibodies in immunoblots (IB). Data are representative of five independent experiments. (C) Immunoblot detection of the sgc C-terminal 9 aa epitope. a-gc-ED immunprecipitated samples were probed with custom-generated sgc C-terminal 9 aa-specific antibodies. Data are representative of five independent experiments. (D) Generation of T cells that exclusively produce membrane gc proteins. LN T cells from Il2rg / and mgcTg were assessed for gc surface (left) and mRNA expression (right). Cell surface gc expression (open histogram) versus control antibody staining (shaded histogram) is shown for Il2rg / and mgcTg LN T cells. Relative sgc and mgc mRNA expression were determined by qRT-PCR and normalized to expression in WT LN T cells, which was set to 100%. Data are summary of three independent experiments; ND indicates not detectable. (E) Serum sgc levels in WT, gcTg, Il2rg / , and mgcTg mice. Data are the summary of three independent experiments. Error bars represent mean and SEM.

RESULTS Identification of a gc mRNA Splice Isoform that Produces a Secreted Form of gc T cell activation induces expression of soluble factors such as IL-2 that control immune responses both in trans and in cis (Malek, 2008). To further understand the role of such soluble factors, we analyzed culture supernatants of TCR- and CD28-stimulated T cells. We found that culture supernatants from activated T cells not only contained IL-2 and TNF-a, but surprisingly also contained large amounts of a secreted form of membrane gc proteins (Figure 1A). Although shedding is a classical mechanism of producing soluble forms of membrane proteins (Heaney and Golde, 1996), this was not the explanation for gc protein secretion, as shown by the fact that inhibition of membrane metalloproteases by TAPI2 treatment to prevent membrane protein shedding failed to prevent expression of soluble gc (Figure 1A). Instead, we found that activated T cells upregulated expression of a novel gc mRNA species that encoded a soluble form of gc (sgc; soluble gc) rather than the conventional membrane form of gc (Figures S1A–S1C available online). The gene encoding gc (Il2rg) is composed of eight exons, and the entire transmembrane (TM) domain is encoded in exon 6 (Cao et al., 1993). The new gc isoform was generated by alternative RNA splicing that excluded exon 6 to encode a gc protein product with the full-length gc extracellular domain (ED) but 2 Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc.

without the TM and intracellular domain (ID) (Figures S1A and S1B). Mechanistically, an open reading frame shift at the splice junction of exon 5 to exon 7 created a new 9 amino acid epitope followed by a stop codon. Notably, sgc transcripts were present at low levels in resting T and B cells and were highly expressed in immature thymocytes and NK cells as well as in activated T cells (Figure S1D). Interestingly, we also identified alternatively spliced sgc transcripts in human T cells, indicating an evolutionarily conserved mechanism of soluble gc expression and regulation (Figure S1E). To examine whether sgc is expressed in vivo, we tested serum for sgc proteins. C57BL/6 wild-type (WT) and gc-deficient (Il2rg / ) sera were immunoprecipitated with total gc-specific antibodies and then probed with gc extracellular domain-specific antibodies (a-gc-ED). As shown in Figure 1B, sgc proteins were detected in WT but not in Il2rg / serum. To determine whether serum sgc is a product of alternative splicing, we generated antibodies specific for the 9 residue C-terminal neoepitope, CLQFPPSRI (Figure 1C), and used these antibodies to probe anti-gc serum immunoprecipitates. Of note, the 9 residue neoepitope is not present in the mouse proteome (NCBI Blastp analysis, data not shown), and it is unique to the alternatively spliced sgc product. Immunoblot analysis showed immunoreactivity of CLQFPPSRI-specific antibodies with WT but not Il2rg / serum (Figure 1C). To further examine whether shedding contributes to sgc expression in vivo, we generated mice that were unable to

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

produce alternatively spliced sgc. We achieved this by generating a human CD2 mini-cassette-driven membrane gc transgene (gcTg) and then breeding the transgene into Il2rg / mice to generate Il2rg / gcTg mice (referred to as mgcTg mice). In mgcTg mice, all gc proteins are expressed from a prespliced, transgenic gc cDNA that encodes only the full-length membrane gc and cannot undergo splicing to produce the splice isoform. mgcTg T cells expressed large amounts of membrane gc mRNA and full-length gc protein, but no alternatively spliced sgc transcripts (Figures 1D). Strikingly, serum from such mgcTg mice lacked circulating sgc proteins, revealing that virtually all serum sgc is generated by alternative splicing, with little or no contribution from shedding (Figure 1E). Thus, mgcTg mice are sgc deficient, and serum sgc is the product of a posttranscriptional gc splice isoform. Serum sgc Expression Is Upregulated upon Immune Activation In Vivo Having identified sgc produced by activated T cells in vitro, we wished to know whether T cell activation would increase sgc expression in vivo. To this end, we assessed serum sgc amounts in BALB/c mice injected with CD3 antibodies (a-CD3) to induce acute polyclonal T cell stimulation in vivo. In fact, serum sgc titers were increased overnight after CD3 antibody injection concomitant with T cell activation even as total T cell numbers were not increased (Figures 2A, S2A, and S2B). Next, we wished to analyze serum sgc titer in mice with chronically activated T cells. Ctla4 / mice contain chronically activated T cells that mediate a lethal lymphoproliferative autoimmune disease (Waterhouse et al., 1995). We found the amount of serum sgc to be larger in Ctla4 / mice than in WT mice (Figure 2B). Importantly, elevated sgc titer was dependent on the presence of activated T cells and not on Ctla4 deficiency itself, because sgc expression was not elevated in nonautoimmune Ctla4 / Cd28 / mice that lack activated T cells and consequently are disease free (Figure 2B; Tai et al., 2007). To further assess sgc expression during in vivo immune activation, we also analyzed autoimmune Il2 / and CD25-deficient (Il2ra / ) mice. Both mouse strains have defective CD4+ regulatory T cell generation that results in massive in vivo T cell activation and lethal autoimmunity (Sadlack et al., 1993; Willerford et al., 1995). We found that both Il2 / and Il2ra / mice expressed increased amounts of serum sgc, confirming that sgc expression was elevated during in vivo immune activation (Figure 2C). Collectively, our results demonstrate that serum sgc protein levels are increased during T cell activation in vivo. Generation of sgc Transgenic Mice To understand the effect of increased sgc expression, we generated transgenic mice overexpressing sgc in T cells (sgcTg). In sgcTg mice, serum sgc levels were substantially increased (Figures 2D and 2E), but T cell numbers and phenotypes were unchanged from WT mice (Figures 2F, S2C, and S2D). To assess whether in vivo sgc upregulation had any effect on T cells, we stimulated sgcTg CD4+ T cells with PMA/ionomycin and examined their ex vivo cytokine expression profiles. gc cytokines play an instructive role in CD4 effector T cell differentiation (Zhu et al., 2010), and we wished to know how in vivo exposure to increased sgc would affect T cell function. Interestingly, sgcTg

CD4+ T cells contained higher proportions of IFN-g producers than did WT CD4+ T cells (Figure 2G). Expression of the Th2 cytokine IL-4, however, was not affected (Figure S2E). Notably, sgcTg CD4+ T cells contained a significantly larger population of IL-17-producing cells (p < 0.001), suggesting that increased sgc level promoted a Th1- and Th17-cell-prone proinflammatory environment (Figure 2G), but this could also reflect an increased presence of memory cells. Importantly, such settings were not created by defects in FoxP3+ CD4+ Treg cells as shown by the fact that both development and function of Treg cells were unaffected in sgcTg mice (Figures S2F and S2G). sgc Is Proinflammatory and Exacerbates EAE Pathology Because IL-17-producing Th17 cells are potent inducers of cell-mediated autoimmunity (Harrington et al., 2005; Park et al., 2005), we wished to know whether sgc would promote autoimmunity. Experimental autoimmune encephalomyelitis (EAE) is a cell-mediated neuroimmunological disease that is largely mediated by Th17 cells and serves as an experimental model for human multiple sclerosis (Goverman, 2009). Injection of MOG peptides results in the activation and brain infiltration of autoreactive lymphocytes, which, in our hands, induced paralysis that peaked around day 5 after onset of the disease. Strikingly, when assessing serum sgc expression in EAE-induced mice, we found increased sgc levels in diseased mice (Figure 3A). To understand whether increased sgc expression contributed to or was the consequence of EAE pathology, we assessed disease progression and found sgcTg mice to be significantly more susceptible to EAE than control mice (p < 0.001) (Figure 3B). In fact, in every parameter examined, sgcTg mice displayed a more pronounced phenotype than did WT mice. Specifically, we found in sgcTg mice that EAE severity worsened, disease progression accelerated, and disease recovery was substantially delayed (Figure 3C). Moreover, we found increased numbers of spinalcord-infiltrating CD4+ T cells, which correlated with dramatically increased percentages and numbers of Th1 and Th17 cells and IL-17, IFN-g double-producing CD4+ T cells (Figures 3D and 3E). Taken together, these results indicate that sgc enhances inflammatory immune responses and increases differentiation of pathogenic Th17 CD4+ effector cells in vivo. sgc Impairs IL-2 Signaling Having observed that sgc expression exacerbated T-cell-mediated autoimmune diseases in vivo, we wished to determine the effect of soluble gc proteins on the generation of inflammatory Th17 cells. Th17 cell differentiation is negatively regulated by IL-2 signaling (Laurence et al., 2007) and is increased by inhibition of IL-2 signaling (Chen et al., 2011; Pandiyan et al., 2007). Therefore, we wished to know whether increased Th17 cell generation in sgcTg mice was the result of diminished IL-2 signaling. To this end, we produced sgc as recombinant proteins (rsgc) (Figure S3). Importantly, we found that rsgc proteins were expressed as disulfide-linked homodimers (Figure 4A), and that the same was true for endogenously produced sgc proteins in serum (Figure S4A). In fact, alternative splicing of gc in both humans and mice creates a C-terminal cysteine residue that promotes dimerization of sgc proteins (Figures S1A and S1E). Therefore, unlike membrane gc proteins, sgc proteins exist as dimers. Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc. 3

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

Figure 2. sgc Expression Is Induced by T Cell Activation In Vivo (A) Serum sgc levels in BALB/c mice injected overnight with a-CD3 antibodies. ELISA data are summary of two independent experiments with two mice per group. (B and C) Serum sgc levels in Ctla4 / and Ctla4 / Cd28 / , and in Il2 / and Il2ra / mice. ELISA data are summary of three independent experiments. (D) sgc protein expression in WT and sgcTg mice sera. Serum samples were immunoprecipitated with gc-ED-specific antibodies and probed with the same antibodies in immunoblots. Numbers in box indicate densitometric quantification of sgc expression. Data are representative of five independent experiments. (E) Relative sgc levels in WT and sgcTg serum. ELISA data are summary of five independent experiments. (F) LN cell profiles of WT and sgcTg mice. Data are representative of 17 independent experiments. (G) IFN-g and IL-17A expression in sgcTg CD4+ LN T cells. CD4+ LN T cells from WT and sgcTg mice were stimulated for 3 hr with PMA and ionomycin and assessed for IFN-g and IL-17A expression by intracellular staining. IFN-g, IL-17A profile is representative of six independent experiments (left). Bar graph shows percent IFN-g- or IL-17A-producing CD4+ T cells. Error bars represent mean and SEM.

Because gc has no affinity for IL-2 (Minami et al., 1993), soluble gc would not diminish IL-2 signaling by binding free IL-2. Thus, we wished to know whether sgc might diminish IL-2 signaling by binding to IL-2 receptor proteins on the cell surface. To test this idea, we performed surface plasmon resonance (SPR) studies with purified rsgc on immobilized IL-2Rb extracellular domain (ED) proteins. Strikingly, SPR analysis showed that 4 Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc.

sgc proteins directly bound to IL-2Rb-ED and that dimeric sgc proteins bound much more strongly (Kd of 16.6 ± 5.5 mM) than monomeric sgc (Kd of 695 ± 76 mM) (Figure 4B). Of note, we consider that the theoretical affinity of sgc binding measured by in vitro SPR study reflects only the minimum potential affinity of sgc for IL-2Rb and that sgc would bind to actual cell surface IL-2Rb with much higher affinity under physiological conditions.

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

Figure 3. sgc Is Proinflammatory and Exacerbates EAE Disease Progression (A) Serum sgc in EAE-induced mice. sgc was assessed by immunoprecipitation and immunoblot before (day 0) and after (day 15) EAE induction in WT mice. Blot shows representative serum sgc expression of four independent experiments. (B) EAE induction in WT and sgcTg mice. EAE clinical scores are representative of seven independent EAE experiments, each with five mice per group. (C) EAE disease course in WT and sgcTg mice. Data are summary from seven independent experiments, each with five mice per group. (D) Th1 and Th17 cell subset analysis of spinal-cord-infiltrating CD4+ T cells in EAE-induced WT and sgcTg mice. IL-17A, IFN-g profile is representative of two independent experiments with two mice per group. (E) Spinal-cord-infiltrating CD4+ T cell numbers in EAE-induced mice. On day 14 after immunization, IFN-g- and/or IL-17A-producing CD4+ T cells were enumerated from spinal cords of WT and sgcTg mice. Data are representative for two independent experiments with two mice per group. Error bars represent mean and SEM.

To further assess whether sgc directly binds to T cells, we utilized flow cytometry. To do so, we examined TCRb+ mature thymocytes of mice with conditional deletion of gc in DP thymocytes (Il2rgfl/flE8IIICre) because they lack surface gc expression (Figure S4B; McCaughtry et al., 2012). Notably, incubation of Il2rgfl/flE8IIICre thymocytes with rsgc proteins revealed direct binding of sgc to the surface, which we visualized by staining with gc-specific antibodies (Figure 4C) and which was still

detectable at 0.06 mg (10 nM) (Figure 4D). More importantly, addition of IL-2 significantly increased (p < 0.0001) sgc binding to Il2rgfl/flE8IIICre cells (Figures 4C and 4D), indicating that sgc affinity to IL-2Rb is increased by IL-2. In sum, sgc proteins, especially dimeric sgc proteins, directly bind to surface IL-2Rb chains, especially in the presence of IL-2. To directly test whether sgc binding to IL-2Rb suppressed IL-2 signaling in T cells, we assessed IL-2-induced STAT5 and Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc. 5

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

Figure 4. sgc Inhibits IL-2 Signaling (A) Dimer formation of recombinant sgc proteins. Recombinant sgc proteins were expressed in insect cells and were resolved in SDS-PAGE under reducing (+DTT) and nonreducing conditions. sgc was detected with antibodies against the gc-ED. Immunoblot is representative of four independent experiments. (B) sgc binding affinities to IL-2Rb determined by SPR. Binding sensorgrams of monomeric (left) and dimeric (right) sgc to immobilized IL-2Rb. The insets show dose-response curves of the maximal responses (Rmax, depicted by the dashed boxes) for each sgc concentration. Plots of Rmax versus sgc concentrations were nonlinear fit to a single-site binding affinity model to determine Kd values. (C) Recombinant sgc binding to mature Il2rgfl/flE8IIICre thymocytes. Il2rgfl/flE8IIICre thymocytes (1.5 3 106 cells in 100 ml) were incubated for 3 hr with 2 mg rsgc in the presence or absence of IL-2 (200 ng) and assessed with gc antibodies for surface gc staining. Histograms show surface gc staining on TCRbhi thymocytes. (D) Titration of sgc binding to Il2rgfl/flE8IIICre thymocytes. Il2rgfl/flE8IIICre thymocytes were treated with increasing amounts of rsgc in the presence or absence of IL-2 (200 ng) as described above. Data are the summary of three independent experiments (mean and SEM). (E) Recombinant sgc impairs proximal IL-2 signaling. CD8+ T cells were stimulated with IL-2 in the presence (4 mg/ml rsgc) or absence (Veh; vehicle) of rsgc and assessed for pSTAT5 (left) or pAKT contents (right). Line graphs show fold induction of IL-2-induced STAT5 or AKT phosphorylation over cells treated with Veh alone. Data are the summary of four independent experiments (mean and SEM). (F) Recombinant sgc inhibits IL-2-induced T cell proliferation. CFSE-labeled WT CD8+ LN T cells were stimulated with IL-2 in the presence or absence of rsgc. CFSE dilution was analyzed at the indicated time points. Histograms are representative of five independent experiments.

AKT phosphorylation in the presence or absence of rsgc (Malek, 2008). We found that rsgc effectively suppressed downstream IL-2 signaling (Figure 4E) and that rsgc-induced impairment of IL-2 signaling reduced IL-2’s biological effects on T cells. In mice, naive CD8+ T cells respond to IL-2 by vigorous proliferation (Cho et al., 2010). We confirmed such IL-2-induced prolifer6 Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc.

ation in purified CD8+ T cells and found that rsgc suppressed IL-2-driven proliferation in the same cells (Figure 4F), demonstrating a direct inhibitory role of sgc in IL-2 signaling. Thus, by binding to IL-2Rb proteins on the cell surface, sgc prevents association with membrane gc proteins to impair IL-2 signaling (Figure S4C).

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

Figure 5. sgc Promotes Th17 Cell Differentiation of CD4+ LN T Cells (A) sgc promotes IL-17A expression. Sorted naive CD4+ T cells were stimulated under Th17-cell-polarizing conditions and assessed for intracellular IL-17A and IFN-g expression. Where indicated, recombinant human IL-2 (rhIL-2), neutralizing mouse IL-2-specific antibodies (a-mIL-2), recombinant sgc (4 mg/ml rsgc), or vehicle (Veh) were added at the beginning of the culture. Data are representative of ten independent experiments. (B) Summary of IL-17A+CD4+ T cell differentiation under modified Th17-cell-polarizing conditions. Each symbol represents an individual experiment under the indicated condition as described above. (C) IL-17A secretion in WT and sgcTg CD4+ T cells. Culture supernatants were collected at day 4 of Th0 or Th17 cell differentiation cultures. ELISA data are representative for five independent experiments. (D) Redundant effect of rsgc and mIL-2-specific antibodies on Th17 cell differentiation. Naive CD4+ T cells were stimulated under Th17-cell-polarizing conditions in the presence of rsgc or mIL-2-specific antibodies. Each symbol represents an individual mouse. (E) sgc effect on IL-17A expression under IL-2-deficient conditions. Naive CD4+ T cells were stimulated under Th17-cell-polarizing conditions to assess intracellular IL-17A and IFN-g expression. Where indicated, rsgc were added at the beginning of the differentiation culture. Contour plots are representative of four independent experiments (left). Bar graph shows percent increase of IL-17A-producing CD4+ T cells by rsgc treatment compared to vehicle control. Error bars represent mean and SEM.

sgc Promotes Th17 Cell Differentiation by Suppressing IL-2 Signaling To assess the IL-2 inhibitory effect of sgc on Th17 cell differentiation, we analyzed Th17 cell generation by using in vitro cultures in the presence of rsgc. As previously described (Laurence et al., 2007), recombinant IL-2 potently suppressed, whereas antagonizing IL-2 antibodies substantially enhanced, Th17 cell differentiation (Figures 5A and 5B). Importantly, sgc treatment or sgc overexpression had the same effect as IL-2 antibodies as it increased both the proportion of IL-17-producing cells and over-

all IL-17 expression (Figures 5B and 5C). In fact, coincubation of rsgc and antagonizing IL-2 antibodies did not have additive effects, indicating that the Th17-cell-promoting mechanisms of rsgc and IL-2 antibodies were redundant (Figure 5D). To further demonstrate that the sgc effect on Th17 cells was due to its inhibition of IL-2 signaling, we analyzed sgc effects on CD4+ T cells from Il2 / mice. We stimulated naive Il2 / CD4+ T cells under Th17 cell conditions with or without rsgc and found that unlike the effect of sgc on WT T cells, sgc failed to increase IL-17 expression in Il2 / T cells (Figure 5E). Identical Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc. 7

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

Figure 6. sgc Deficiency Is Protective against Inflammatory Autoimmune Diseases (A) EAE induction in WT and mgcTg mice. EAE clinical scores are summary of two independent EAE experiments, each with four mice per group. (B) Th1 and Th17 cell subset analysis of spinal-cord- and brain-infiltrating CD4+ T cells in EAE-induced WT and mgcTg mice. Contour plots show representative IL-17A, IFN-g profiles from four mice per group. (C) Spinal-cord- and brain-infiltrating CD4+ T cell numbers in EAE-induced mice. On day 15 after immunization, IFN-g- and/or IL-17A-producing CD4+ T cells were enumerated from spinal cords and brains of WT and mgcTg mice. Data are representative of four mice per group. (legend continued on next page)

8 Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc.

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

results were observed in Il2 / sgcTg CD4+ T cells where sgc transgene expression failed to enhance Th17 cell differentiation compared to Il2 / cells (Figure 5E). Altogether, these results document that sgc promotes Th17 cell differentiation by specifically impairing IL-2 signaling. sgc Deficiency Ameliorates Th17-Cell-Mediated Inflammatory Autoimmune Diseases Because sgc exacerbated IL-17 expression and inflammation, we wished to know whether the absence of sgc would dampen inflammatory responses that otherwise occurred in WT mice. To this end, we assessed EAE induction in mgcTg mice that express only transgenic membrane gc receptors and lack endogenous sgc proteins (Figure 1E). Strikingly, EAE disease progression in mgcTg mice was dramatically reduced compared to WT mice, with delayed disease onset and lower clinical scores (Figure 6A). Analysis of spinal-cord- and brain-infiltrating T cells further revealed a significant reduction in inflammatory CD4+ T cell numbers and greatly diminished percentages of IL-17-IFN-g double producers, demonstrating a protective effect of sgc deficiency in mgcTg mice (Figures 6B and 6C). Importantly, the severity of inflammatory reactions was markedly reduced in mgcTg mice despite the presence of normal numbers and appearances of peripheral T cells (Figures S5A and S5B). To further assess the ameliorative effect of sgc deficiency, we utilized the CD4+CD45RBhi T cell transfer model of inflammatory bowel disease (IBD) (Powrie et al., 1993). Adoptive transfer of naive T cells into immunodeficient Rag2 / mice results in severe autoimmune inflammatory colitis that can be monitored by body weight loss and increased IBD clinical scores (Table S1). Notably, we found that Rag2 / host mice injected with mgcTg CD4+ T cells had markedly diminished IBD, whereas Rag2 / mice injected with WT CD4+ T cells suffered from significant body weight loss, destruction of colon tissues, and rapid deterioration of health (Figures 6D, 6E, S5C, and S5D). Importantly, adoptively transferred mgcTg CD4+ T cells were still activated in vivo and produced large amounts of IFN-g, but it was specifically the expression of proinflammatory IL-17 that was dramatically diminished in these cells (Figure 6F). Thus, sgc-deficient T cells were unable to induce severe autoimmune IBD because of reduced Th17 cell generation. Conversely, T cell expression or overexpression of sgc increased the severity of inflammatory disease (Figure S5D). These results reveal that sgc exacerbates in vivo inflammatory immune responses and contributes to the severity of in vivo autoimmune pathology. sgc Overexpression Impairs T Cell Development and Naive T Cell Homeostasis Although sgc directly binds to LN T cells (Figure 7A), resting T cells do not express high levels of IL-2Rb. These data suggested that sgc also bound to receptors for cytokines other than IL-2. Specifically, we considered that sgc might bind to IL-7 receptors because they are highly expressed on all resting

T cells. SPR analysis showed that this was indeed the case. We found that sgc proteins directly interacted with mouse IL-7Ra-ED proteins and that dimeric sgc showed a dramatic increase of IL-7Ra-ED binding activity (Kd = 3.9 mM) over monomeric sgc proteins (Kd = 50.8 mM) (Figure 7B and Table S2). To further demonstrate sgc binding to surface IL-7Ra proteins, we incubated Il2rgfl/flE8IIICre thymocytes with rsgc proteins and assessed rsgc binding to thymocyte subpopulations. TCRbhi mature thymocytes express high levels of IL-7Ra and strongly bound sgc, whereas DP thymocytes do not express surface IL-7Ra and did not bind sgc (Figure 7C). To directly show that recombinant sgc specifically bound to IL-7Ra proteins, we examined gc-deficient TCRbhi thymocytes that were additionally deficient in IL-7Ra (McCaughtry et al., 2012). Indeed, IL-7Ra deficiency substantially reduced sgc binding to sgc-deficient TCRbhi thymocytes (Figure 7D), documenting that sgc directly binds and interacts with surface IL-7Ra proteins. IL-7 is critical for thymopoiesis and impaired IL-7 signaling results in reduced thymus cellularity (von Freeden-Jeffry et al., 1995). Notably, increased sgc expression in sgcTg mice resulted in decreased total thymocyte numbers (Figure 7E). CD25 is highly expressed on immature DN thymocytes but dilutes out with proliferation so that CD25 is normally absent on DP thymocytes. However, sgcTg DP thymocytes still expressed significant amounts of surface CD25, which indicated impaired IL-7 signaling and impaired expansion of DN cells (Figure S6A; Crompton et al., 1994). These results demonstrate that sgc blocks in vivo IL-7 signaling. In further support of an sgc effect on IL-7 signaling, we observed increased T cell apoptosis and reduced percentages and numbers of naive T cells in sgcTg mice (Figures 7F and 7G), even as their T cells expressed identical levels of IL-7Ra and other gc family receptors to WT mice (Figure S6B). Moreover, adoptive transfer of WT T cells into congenic sgcTg or WT host mice revealed that survival of CD44lo naive donor T cells was substantially reduced in sgcTg hosts compared to WT hosts (Figure S6C). Finally, to directly demonstrate that sgc blocks IL-7-induced survival, we stimulated naive T cells with IL-7 in vitro and assessed the effect of recombinant sgc on cell survival. As shown in Figure S6D, rsgc effectively blocked IL-7-induced T cell survival in vitro. Thus, sgc inhibited IL-7 signaling during both T cell development and peripheral T cell homeostasis. DISCUSSION Here we report a regulatory mechanism of gc signaling that tunes T cell responsiveness to IL-2 and IL-7 and potentially to other gc cytokines as well. This regulatory pathway was based on posttranscriptional generation and secretion of the gc extracellular domain that directly bound to gc cytokine proprietary receptors such as IL-2Rb and IL-7Ra, especially in the presence of cytokines, and interfered with cytokine signaling. Importantly, secreted gc proteins required disulfide-linked homo-dimerization

(D) Body weight change of IBD-induced mice. Rag2 / mice were injected with each 0.5 million WT or mgcTg CD4+CD45RBhi T cells and monitored for change in body weights. Data show results from four mice per group. (E) Colon histology from Rag2 / host mice injected with naive CD4+ T cells from the indicated donor mice at 6 weeks after T cell transfer. (F) Th1 and Th17 cell subset analysis in CD4+ LN T cells from IBD-induced WT and mgcTg mice (left). Data show results from three mice for each group (right). Error bars represent mean and SEM.

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Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

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10 Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc.

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Immunity Soluble gc Receptor Controls Cytokine Signaling

for high-affinity binding and inhibition of cytokine signaling. During steady-state conditions, sgc interfered primarily with IL-7-dependent processes, resulting in impaired thymopoiesis and naive T cell survival. During T cell activation, sgc inhibited IL-2 signaling and promoted the development of proinflammatory Th17 cell responses as demonstrated by increased Th17 cell differentiation in vitro and by exacerbation of EAE disease in sgc transgenic mice in vivo. Conversely, in vivo sgc deficiency suppressed Th17 cell differentiation and dramatically reduced the severity of inflammatory autoimmune disease, resulting in markedly improved disease outcomes. Thus, this study identifies sgc proteins as endogenous immunomodulators that exacerbate in vivo inflammatory diseases by directly binding to cytokine receptors and dampening T cell responses to gc cytokines. Cytokine signaling in T cells is tightly controlled at many levels. Expression of cytosolic inhibitory molecules, such as SOCS, limits the magnitude and duration of cytokine signaling (Yoshimura et al., 2007). Upregulation of surface cytokine receptor expression, on the other hand, positively affects cytokine signaling. Secretion of gc proteins now adds another layer of regulatory complexity because sgc expression is upregulated upon T cell activation but, unlike the upregulation of other IL-2 receptor chains that enhance IL-2 signaling, upregulation of sgc suppresses IL-2 signaling. Moreover, soluble gc is unique in its regulatory mechanism because it suppressed IL-2 signaling without binding to the IL-2 molecule itself. This is in contrast to other chains of the IL-2 receptor complex that regulate IL-2 responsiveness by direct interaction with IL-2 (Minami et al., 1993). Because IL-2 signaling requires membrane gc association with IL-2Rb, sgc binding to IL-2Rb proteins probably blocks IL-2 signaling by preventing recruitment of membrane gc proteins and assembly of signaling-competent IL-2 receptor/membrane gc complexes. The secreted gc protein is structurally identical to the membrane gc receptor, except for absent transmembrane and intracellular domains. Because ligand binding is mediated by the extracellular domain, it was initially unclear how soluble gc could compete with membrane gc for binding to proprietary receptor proteins. The mechanism was partly revealed by SPR analysis, which showed that dimeric sgc proteins possessed >40-fold increased affinity of binding to IL-2Rb proteins compared to monomeric sgc proteins, which was further enhanced in the presence of cytokines. Thus, serum sgc proteins, which are mostly dimers, are vastly superior in binding to IL-2Rb than

membrane gc proteins, which are monomers and do not display any meaningful affinity to IL-2Rb (Rickert et al., 2004). As a result, sgc proteins effectively outcompete membrane gc protein for binding to proprietary cytokine receptors IL-2Rb and IL-7Ra. Therefore, upon dimerization, sgc proteins acquire binding specificities that make them distinct from monomeric sgc and membrane gc proteins. In this regard, we think it is not a coincidence that alternative splicing of sgc pre-mRNA creates a new C-terminal cysteine residue that promotes disulfide linkage and dimerization of sgc proteins. Indeed, we found such de novo C-terminal cysteine residues in both mouse and human sgc proteins, indicating that dimerization of sgc proteins is an evolutionary conserved mechanism of regulating cytokine signaling. Soluble cytokine receptors have been observed in previous studies (Baran et al., 1988; Mortier et al., 2004; Nielsen et al., 1998). Expression of soluble cytokine receptors had been associated with inflammation, autoimmunity, and cancer (Heaney and Golde, 1996), but it was uncertain whether their expression was the cause or the consequence of immune activation. In theory, soluble cytokine receptors might either potentiate or antagonize cytokine signaling (Heaney and Golde, 1996). Soluble IL-6Ra, for example, directly binds IL-6 and potently enhances IL-6 signaling (Rose-John, 2012), whereas soluble IL-15Ra sequesters IL-15 and prevents it from binding to membrane IL-15 receptors (Mortier et al., 2004). gc proteins, however, do not show significant binding affinity to free cytokines, so it was not immediately obvious what role soluble gc would play in cytokine signaling. By using recombinant sgc proteins and sgc transgenic mice, we have demonstrated that sgc antagonizes IL-2 and IL-7 signaling and increases inflammatory immune responses. Because T cell activation induces sgc production and sgc in turn induces inflammatory cytokine expression, sgc production is a mechanism to reinforce proinflammatory immune responses downstream of T cell activation. In fact, sgc expression would limit IL-2 responses and promote effector T cell differentiation. Thus, our present results provide an explanation for earlier reports on soluble gc expression in which a significant correlation was found in Crohn’s disease patients between disease activity and serum gc levels (Nielsen et al., 1998). According to our present findings, increased soluble gc expression would dampen gc cytokine signaling, which would enhance inflammatory cell activation to worsen the disease outcome. In agreement with our perspective, soluble gc is highly expressed under inflammatory conditions in both humans and mice. Soluble gc was

Figure 7. sgc Overexpression Impairs T Cell Development and Homeostasis (A) Surface gc chain staining on WT and sgcTg CD4+ T cells. Data are the summary of four independent experiments. (B) sgc binding affinities to IL-7Ra determined by surface plasmon resonance. Binding sensorgrams and Kd of monomeric (left) and dimeric (right) sgc proteins to immobilized IL-7Ra proteins. (C) Cell surface gc staining of Il2rgfl/flE8IIICre thymocytes incubated with recombinant sgc. Il2rgfl/flE8IIICre thymocytes were incubated for 2 hr with 4 mg rsgc or Veh (BSA in PBS) and assessed with gc antibodies for surface gc staining. Histograms show surface gc staining on DP and TCRbhi thymocytes. (D) Cell surface gc staining of Il2rgfl/flE8IIICre and Il7rafl/flIl2rgfl/flE8IIICre thymocytes incubated with rsgc. Surface gc staining was assessed as mean fluorescence intensity (MFI) in TCRbhi thymocytes. Data are summary of two independent experiments. (E) Total thymocyte numbers in WT and sgcTg mice. Results are the summary of eight independent experiments. (F) T cell apoptosis in WT and sgcTg LN T cells. Caspase-3 activity was determined in naive CD44lo CD4+ or CD8+ LN T cells via CaspGLOW apoptosis kits. Histograms show caspase-3 activity in indicated cells (left). Bar graphs show summary of three independent experiments (right). (G) Naive and memory phenotype analysis of WT and sgcTg LN T cells. Contour plots show CD62L/CD44 and CD122/CD44 profiles and percentages of CD4+ and CD8+ LN T cells, respectively (left). Bar graph shows naive CD4+ and CD8+ LN T cells numbers from WT and sgcTg mice (right). Data are the summary of ten independent experiments. Error bars represent mean and SEM.

Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc. 11

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

abundantly expressed in synovial fluid of patients with rheumatoid arthritis and also highly induced in mice challenged with L. major (Meissner et al., 2001; Nishio et al., 2001). We also found that EAE disease outcome and in vivo inflammatory responses were exacerbated in sgcTg mice. Collectively, these data indicate a role for sgc as an active inducer of pathogenic T cells and inflammation. Because sgc promotes inflammation, it is interesting to speculate whether neutralization of serum sgc expression would ameliorate inflammatory disease and dampen destructive T cell responses. The striking reduction in disease severity of EAE or IBD-induced mgcTg mice lacking sgc observed here suggests that depletion of sgc might be an effective therapy in patients suffering from inflammatory immune diseases. In this regard, the 9 aa C-terminal neoepitope CLQFPPSRI (and the 7 aa neoepitope RCPEFPP in humans) of sgc provides an excellent target for intervention with neutralizing antibodies. These peptide epitopes are generated only upon alternative splicing of gc pre-mRNA exon 5 to exon 7, and they are found only in sgc and not in membrane gc proteins. In contrast to conventional gc-specific antibodies, C-terminal sgc-specific antibodies will neutralize only alternatively spliced sgc proteins, making this epitope an attractive target to clear sgc from circulation without affecting membrane gc receptor function. Discovery of the unique C-terminal sgc epitope was enlightening because it helped us identify the molecular origin of sgc. Specifically, the 9 aa C-terminal sequence is not found in membrane gc proteins so that its presence in serum directly demonstrated that sgc proteins are products of alternative RNA splicing. Soluble cytokine receptors are usually produced by one of the following two major mechanisms; proteolytic cleavage and shedding of membrane cytokine receptors or alternative splicing of cytokine receptor pre-mRNA (Levine, 2004). TNF receptors are classical examples of cytokine receptors that are cleaved by transmembrane metalloproteinases and shed from cells (Hwang et al., 1993). Soluble IL-6 receptors, on the other hand, are usually generated by alternative splicing (Horiuchi et al., 1994). Our data document that sgc is exclusively produced by alternative splicing. It is conceivable that genetic variations might also contribute to increased sgc expression. In fact, IL-7Ra gene polymorphisms can result in increased expression of soluble IL-7Ra (sIL7Ra) (Gregory et al., 2007), and sIL7Ra expression has been proposed as a significant risk factor for multiple sclerosis in humans (Harley, 2007; Lundstro¨m et al., 2013). Specifically, these SNPs led to exclusion of exon 6 during IL-7Ra pre-mRNA splicing and resulted in the deletion of the transmembrane domain to produce soluble IL-7R proteins (Goodwin et al., 1990; Gregory et al., 2007). It would be interesting to assess gc genes for genetic variations that increase exon 6 exclusion and to test whether such SNPs are associated with increased risk for MS or other autoimmune disease. Potentially, such gc genetic variations could predispose carriers to excessive inflammatory responses by increased levels of sgc. In conclusion, the present study has identified sgc as an endogenous regulatory mechanism that dampens T cell signaling to gc cytokines and results in enhanced activity of Th17 cells. Enforced sgc expression constrained T cell survival under homeostatic conditions and promoted a proinflammatory environment under immune activation. Reciprocally, in vivo sgc 12 Immunity 40, 1–14, June 19, 2014 ª2014 Elsevier Inc.

deficiency suppressed inflammatory Th17 cell differentiation and effectively reduced the severity of Th17-cell-mediated autoimmune disease. Thus, sgc represents a naturally occurring immunomodulator of T cells and introduces a target for intervention in inflammation and disease. EXPERIMENTAL PROCEDURES Animals C57BL/6 (B6) mice and B6.Ly5.2 mice were obtained from the Jackson Laboratory. Ctla4 / , Ctla4 / Cd28 / , Il2 / , Il2ra / , Rag2 / , and Il2rg / mice were bred in our colony. Il2rgfl/flE8IIICre and Il7rafl/flIl2rgfl/flE8IIICre mice were kindly provided by A. Singer (NCI). The gc- and sgc-transgenic constructs were generated by ligating a murine gc or sgc cDNA into human CD2 (hCD2) enhancer-promoter-based vectors, respectively, and injected into fertilized B6 oocytes to generate gcTg and sgcTg mice. Animal experiments were approved by the National Cancer Institute Animal Care and Use Committee (ACUC), and all mice were cared for in accordance with US NIH guidelines. Surface Plasmon Resonance Purified mouse IL-2Rb ectodomain expressed from yeast was purchased from MyBiosource and used without further modification. IL-2Rb was aminecoupled to a CM5 sensor chip after buffer exchange into PBS (pH 7.4) via a NAP-5 (GE Healthcare) column by similar methods described previously (McElroy et al., 2012). Concentration series of monomer and dimer forms of insect-cell-derived rsgc were injected over immobilized IL-2Rb at a flow rate of 25 ml/min via HBS-EP (pH 7.4). Surface plasmon resonance (SPR) experiments were performed at 25 C with a Biacore 3000 instrument (GE Healthcare). SPR sensorgrams were double-referenced and trimmed with BIAevaluation 4.1 (GE Healthcare). Dose-response curves (Rmax versus [rsgcmonomer or dimer]) were fit with Prism5.0 (GraphPad). Experiments were performed in triplicate. Detection of Soluble Cytokine and Receptor Levels Serum sgc was detected in a sandwich ELISA with gc-specific polyclonal antibodies (R&D Systems) as capture antibodies and biotin-conjugated gc-specific monoclonal antibodies (4G3; BD) as detection antibodies. Recombinant sgc protein was used as positive control. IL-2, IFN-g, TNF-a, and IL-17A were analyzed by ELISA according to the manufacturer’s instructions (R&D Systems). In Vitro CD4+ T Helper Cell Differentiation Naive CD4+ T cells were electronically sorted by gating on CD62L+CD44loCD25 cells. Sorted cells were stimulated with plate-bound a-CD3 and a-CD28 and cultured under nonskewing Th0 cell conditions (medium alone) or were differentiated into Th17 cells with human TGF-b1 (5 ng/ml; Peprotech), mouse IL-6 (30 ng/ml; BD), anti-mouse IL-4 (10 mg/ml), and anti-mouse IFN-g (10 mg/ml). Where indicated, human IL-2 (100 U/ml; NCI) or anti-mouse IL-2 (10 mg/ml; Becton Dickinson) or mouse rsgc (4 mg/ml) was added to the cell culture. Cell Proliferation Assay Naive CD8 responder T cells were labeled with CFSE (carboxyfluorescein diacetate succinimidyl ester; Invitrogen) and incubated with human IL-2 (10 mg/ml; Peprotech) for 2 or 3 days. CD4+CD45RBhi T Cell Transfer-Induced Colitis CD4+ T cells were purified from WT, sgcTg, and mgcTg mice with the EasySep mouse CD4+ T cell isolation kit (StemCell Technologies). CD4+CD45RBhi cells were then electronically sorted by FACS. Rag2 / mice were injected with 5 3 105 CD4+CD45RBhi T cells and monitored for 7 weeks for body weight change and clinical signs of colitis. Experimental mice showing clinical signs of severe colitis and a body weight loss of >20% were sacrificed according to the NCI ACUC guidelines. Statistical Analysis Statistical differences were analyzed with Student’s two-tailed t test. Clinical score comparisons between two groups were performed with nonparametric Mann-Whitney test. Curves of body weight change were fitted with nonlinear

Please cite this article in press as: Hong et al., Activated T Cells Secrete an Alternatively Spliced Form of Common g-Chain that Inhibits Cytokine Signaling and Exacerbates Inflammation, Immunity (2014), http://dx.doi.org/10.1016/j.immuni.2014.04.020

Immunity Soluble gc Receptor Controls Cytokine Signaling

regression and group difference was examined by slope and intercept. p values of less than 0.05 were considered significant. *p < 0.05, **p < 0.01, ***p < 0.001, and NS (not significant) (Student’s two-tailed t test). SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, two tables, and Supplemental Experimental Procedures and can be found with this article online at http:// dx.doi.org/10.1016/j.immuni.2014.04.020. ACKNOWLEDGMENTS We thank A. Singer, C. Mackall, and M. Kimura for critical review of this manuscript. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research; and the NIH (AI072142 to S.T.R.W.). Received: September 25, 2013 Accepted: April 9, 2014 Published: June 5, 2014

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