Eur. J. Immunol. 2011. 41: 3351–3360 DOI 10.1002/eji.201141629 Leukocyte signaling IL-33 synergizes with TCR and IL-12 signaling to promote the effector function of CD81 T cells Qianting Yang1,2, Gang Li1,3, Yibei Zhu1,3, Lin Liu1, Elizabeth Chen1, Hēth Turnquist4, Xueguang Zhang3, Olivera J. Finn1, Xinchun Chen2 and Binfeng Lu1 1 Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Shenzhen Clinical Centre for Infectious Diseases, Shenzhen Third People’s Hospital, Shenzhen, P. R. China 3 Department of Immunology, Institute of Medical Biotechnology, Soochow University, Suzhou, P. R. China 4 Department of Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 2 The effector functions of CD81 T cells are influenced by tissue inflammatory microenvironments. IL-33, a member of the IL-1 family, acts as a danger signal after its release during cell necrosis. The IL-33/ST2 axis has been implicated in various Th2 responses. Its role in CD81 T-cell-mediated immune response is, however, not known. Here we find that type 1 cytotoxic T (Tc1) cells cultured in vitro unexpectedly express high levels of the IL-33 receptor ST2. Interestingly, the expression of ST2 in Tc1 cells is dependent on T-bet, a master Th1/Tc1 transcription factor. In addition, IL-33 enhances TCR-triggered IFN-c production. IL-33 together with IL-12 can stimulate IFN-c production in Tc1 cells. Moreover, IL-33 synergizes with IL-12 to promote CD81 T-cell effector function. The synergistic effect of IL-33 and IL-12 is partly mediated by Gadd45b. Together, these in vitro data establish a novel role of IL-33 in promoting effector type 1 adaptive immune responses. Key words: CD81 T cells . Cellular activation . Cytokines . Signal transduction Supporting Information available online Introduction Interleukin-33 is a member of the IL-1 family of cytokines which also includes IL-1 (a and b) and IL-18 [1]. IL-1b and IL-18 are expressed as prodomains containing polypeptide precursors which are proteolytically cleaved by caspase-1 to generate the active forms of these cytokines [2]. IL-33 is different from IL-1b and IL-18 in that it cannot be processed by caspase-1; instead, IL-33 is cleaved by caspase-7 and -3 during apoptosis to functionally inactivate IL-33 [3–5]. IL-33 is constitutively expressed in the nuclei of blood vessel endothelial cells, fibroblastic reticular cells of lymphoid tissues, and tissue cells exposed to the external environment such as skin keratinocytes and stomach epithelial cells [6]. During necrotic processes, fulllength but biologically active IL-33 can be released [3, 5]. The fact that IL-33 may be released by necrotic cells during infection or trauma suggests that it may serve as an endogenous danger signal or ‘alarmin’ [6]. Ample evidence supports an important role of IL-33 in Th2 cell-mediated immune responses [1]. The IL-33 receptor complex consists of ST2 and IL-1RAcP, both of which are members of the IL-1 receptor family [7, 8]. ST2 is expressed by a number of cells Correspondence: Dr. Binfeng Lu e-mail: binfeng@pitt.edu These authors contributed equally to this work. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 3351 3352 Qianting Yang et al. involved in Th2-type responses such as Th2 cells [9, 10], DCs [11, 12], mast cells [13, 14], basophils [15, 16], and eosinophils [17]. IL-33 enhances IL-5 and IL-13 production by Th2 cells independently of IL-4 [7, 18]. Administration of either an antibody against ST2 or recombinant ST2 fusion protein inhibits eosinophilic airway inflammation and induces resistance to Leishmania major infection in BALB/c mice [9, 10]. In mice, IL-33 induces anaphylactic shock, in a T-cell-independent, mast cell-dependent manner [13]. Interestingly, IL-33 induces IL-13dependent cutaneous fibrosis [19]. In humans, the level of IL-33 is greatly increased in the blood of atopic patients during anaphylactic shock. Besides its expression on effector cells of Th2 immune responses, ST2 is also found on NK and NKT cells, which respond to IL-33 with increased IFN-g production, suggesting a role for IL-33/ST2 in innate Th1-type immune responses [15, 20]. Whether IL-33 plays a role in adaptive Th1-type immune response is not known. Here, we reveal that ST2 is highly expressed on type I cytotoxic (Tc1) cells. Its expression on Tc1 cells is mainly dependent on T-bet, a master transcription regulator of Th1 and Tc1 cells [21]. We have further found that IL-33 synergizes with TCR, IL-12 signaling, or both to drive IFN-g production in Tc1 cells and promote features of effector CD81 T cells. Our study establishes a novel role of IL-33 in driving the effector function of CD81 T cells. Results IL-33 receptor ST2 is highly expressed in effector Tc1 cells To understand the molecular characteristics of Tc1 cells, we performed gene profiling studies and found that the IL-33 receptor ST2 was highly expressed in these cells (data not shown). To confirm our result, we performed a quantitative RTPCR (qRT-PCR) analysis on naı̈ve CD81 T cells polarized in Tc0, Tc1, Tc2, and Tc17 conditions for 4 days. The ST2 mRNA was not expressed in naı̈ve CD81 T cells (data not shown) and could be induced in CD81 T cells cultured in Tc0 and Tc17 conditions. The level of ST2 mRNA was four-fold higher in Tc1 cells compared with Tc0 cells, confirming our microarray analysis (Fig. 1A). Surprisingly, CD81 T cells cultured in the Tc2 condition showed minimum ST2 expression compared with those cultured in other conditions. These results suggest that IL-12 further increases ST2 expression, whereas IL-4 suppresses ST2 expression in CD81 T cells at the mRNA level. The ST2 protein could also be detected on the surface of Tc1 cells by flow cytometry (Fig. 1B). Additionally, we also found that TCR signaling further increased the levels of ST2 mRNA in Tc1 cells (Fig. 1C). In contrast, IL-1RAcP mRNA is expressed at similar levels among all subsets of effector CD81 T cells (Supporting Information Fig. 1). To examine whether ST2 expression on Tc1 cells is stable, we performed a second round of Tc1 polarization by stimulating Tc1 cells with anti-CD3 and anti-CD28 in Tc1 culture conditions for another 4 days. We found even higher levels of ST2 expression on & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Immunol. 2011. 41: 3351–3360 CD81 T cells following two rounds of Tc1 polarization (Fig. 1C–E). In contrast, CD81 T cells cultured in Tc2 conditions for two rounds expressed lower levels of ST2 mRNA and protein compared with those in the Tc1 condition (Fig. 1C–E). In addition, we have found similar differences in ST2 expression between human CD81 T cells cultured in Tc1 and Tc2 conditions (Supporting Information Fig. 2). Finally, important differences in ST2 expression were also observed between effector CD41 and CD81 T cells (Supporting Information Fig. 3). T-bet is required for ST2 expression in Tc1 cells T-bet and Eomes are two Tc1 master transcription regulators expressed in effector/memory CD81 T cells [21, 22]. T-bet is highly expressed in effector CD81 T cells and plays a predominant role in the function of effector CD81 T cells [23, 24]. In contrast, Eomes is highly expressed in both effector and memory CD81 T cells and plays a critical role in the survival of memory CD81 T cells [25]. Since ST2 is expressed in Tc1 cells, we decided to determine whether T-bet and/or Eomes regulate its expression. Naı̈ve CD81 T cells were isolated from wild type (WT), T-bet / , Eomes / , and T-bet/Eomes doubly deficient (DKO) mice and were polarized in vitro in Tc1 and Tc2 conditions. The levels of ST2 in cells cultured in these conditions were measured by qRTPCR. ST2 mRNA was highly expressed in WT effector Tc1 cells (Fig. 2A and B). Its level was unchanged in Eomes / effector Tc1 cells (Fig. 2A). In contrast, ST2 mRNA was greatly reduced in T-bet / Tc1 cells and was also reduced in DKO Tc1 cells (Fig. 2A). The surface expression of ST2 protein was also reduced in T-bet / Tc1 cells compared with WT Tc1 cells (Fig. 2B). These data suggest that T-bet is primarily required for ST2 expression in Tc1 cells, and Eomes seems not involved in regulating ST2 mRNA. We have shown previously that DKO CD81 T cells cultured in Tc2 conditions showed drastically increased Tc2 characteristics [26]. Because ST2 has been shown to be highly expressed in Th2 cells, we examined whether ST2 can be upregulated in DKO Tc2 cells. Surprisingly, ST2 expression was absent in DKO CD81 T cells cultured in the Tc2 conditions (Fig. 2A). It has been shown that IFN-g produced by T cells cultured in Tc1 conditions is involved in driving Th1-specific gene expression [27]. However, when we neutralized the function of IFN-g in Tc1 culture, ST2 expression was unchanged (Fig. 2C). Therefore, ST2 expression is not regulated by IFN-g in Tc1 cells. IL-33 synergizes with TCR signaling and/or IL-12 in promoting IFN-c production We have shown that ST2 is expressed in Tc1 cells, and ST2 mRNA level is further increased in Tc1 cells upon TCR stimulation. These data suggest that IL-33 might be involved in the function of Tc1 cells. Since IFN-g is a hallmark cytokine for Tc1 cells, we investigated whether IL-33 synergizes with TCR signaling in the production of IFN-g by Tc1 cells. We first stimulated Tc1 cells www.eji-journal.eu Leukocyte signaling Eur. J. Immunol. 2011. 41: 3351–3360 C 20 20 15 15 RQ RQ A 25 10 5 5 0 10 TC0 TC1 TC2 0 Time 4d TC17 B 7d 2°+ 4d *** D 12 9 RQ TC0 (dotted) TC1 (solid) 4d+4h 6 3 0 TC2 (dotted) TC1 (solid) % of Max % of Max E TC17 (dotted) TC1 (solid) TC1 TC2 TC2 (dotted) TC1 (solid) ST2 ST2 Figure 1. Expression of ST2 in effector CD81 T cells. (A and B) Naı̈ve CD81 T cells were cultured in Tc0, Tc1, Tc2, and Tc17 conditions for 4 days. (A) Total RNA was made and subjected to real-time RT-PCR analysis of ST2 mRNA levels. RQ refers to relative quantity of ST2 mRNA, calculated by software provided by Applied Biosystem. RQ for Tc2 was set as 1. (B) Surface expression of ST2 was determined by flow cytometry. Isotype control is presented as the shaded area. (C) CD81 T cells were cultured in Tc1 conditions for 4 days (4d). These cells were either stimulated with anti-CD3 for 4 h (4d14 h), or cultured with the addition of fresh IL-2 (10 U/mL) for three more days (7d). At the end of the 7-day culture, cells were extensively washed and cultured in the Tc1 polarizing condition for an additional 4 days (second round for 4 days). Total RNA was made from cells in each group and subjected to real-time RT-PCR analysis of ST2 mRNA. RQ for Tc1 day 4 was set as 1. (D and E) Naı̈ve CD81 T cells were cultured in Tc1 or Tc2 polarizing conditions for 7 days. The cells were then subjected to a second round of Tc1 or Tc2 polarization for 4 days. (D) Total RNA was made and analyzed by real-time RT-PCR for the levels of ST2 mRNA. RQ for Tc2 was set as 1. (E) Surface expression of ST2 was determined by flow cytometry. Isotype control is presented as the shaded area. Data in A, C and D are presented as the mean7SEM of triplicate samples and are representative of three independent experiments. po0.001, two-tailed unpaired Student’s t-test. with different doses of anti-CD3 for 16 h in the presence or absence of IL-33. The addition of IL-33 to Tc1 cells stimulated with 1 and 0.5 mg/mL anti-CD3 mAbs resulted in higher frequencies of IFN-g producers and greater levels of secreted IFN-g protein (Fig. 3A and B). These data suggest that IL-33 synergizes with TCR signaling in the induction of IFN-g production in Tc1 cells. IL-18, a member of the IL-1 family protein, has been shown to synergize with IL-12 to stimulate IFN-g production in Th1 cells [28, 29]; we decided to determine whether IL-33 synergizes with IL-12 to induce IFN-g in Tc1 cells. Tc1 cells were cultured with IL-12, or IL-33 or IL-12 plus IL-33 for 24 h. IL-12 or IL-33 alone induced very few IFN-g1 Tc1 cells (o1%) and low levels of IFN-g protein (Fig. 3C and D). This result is consistent & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim with prior publication and our own observation which showed non-detectable levels of IFN-g when Th1 cells were stimulated with IL-12 alone [29]. In contrast, combining IL-12 and IL-33 drastically increased the frequency of IFN-g1 Tc1 cells (to about 28%) (Fig. 3C). IFN-g protein levels were also greatly increased when both IL-12 and IL-33 were added to the culture (Fig. 3D). Besides IFN-g production, IL-12 and IL-33 also significantly increased the number of cultured Tc1 cells (Fig. 3E). These data suggest that IL-12 and IL-33 synergize to drive effector function in Tc1 cells. Interestingly, when anti-CD3, IL-12, and IL-33 were added together, they synergized in inducing IFN-g production (Supporting Information Fig. 4). Thus, IL-33 synergizes with TCR signaling and IL-12 in promoting IFN-g production. www.eji-journal.eu 3353 Eur. J. Immunol. 2011. 41: 3351–3360 Qianting Yang et al. A 15 WT EKO TKO DKO RQ 10 5 0 TC1 TC2 B % of Max DKO (dotted) WT (solid) ST2 C 25 20 RQ 3354 15 10 5 0 α-IFN γ (μg / ml) 0 10 Figure 2. Role of Tbet and Eomes in ST2 expression in Tc1 cells. (A and B) Naı̈ve CD81 T cells from C57BL/6 WT (WT), T-bet / (TKO), Eomes / (EKO), and T-bet/Eomes doubly deficient (DKO) mice were cultured in Tc1 or Tc2 for 4 days. (A) Total RNA was made and subjected to realtime RT-PCR analysis for ST2. (B) Surface ST2 expression was analyzed by flow cytometry. (C) CD81 T cells cultured in Tc1 conditions with or without anti-IFN-g mAb (10 mg/mL) for 4 days. Total RNA was made and subjected to real-time RT-PCR analysis for ST2. Data in (A) and (C) are presented as the mean7SEM of triplicate samples. Results are representative of three independent experiments. Since T-bet is required for ST2 expression in Tc1 cells (Fig. 2), we determined whether this transcription factor is required for the IFN-g production driven by IL-12 plus IL-33. Naı̈ve CD81 T cells were isolated from WT Pmel-1 TCR transgenic mice [30] and T-bet KO Pmel-1 TCR transgenic mice and were stimulated either with cognate peptide plus APCs (Fig. 3F) in Tc1 conditions for 4 days. These cells were incubated with IL-12, or IL-33, or IL-12 plus IL-33 for an additional 24 h. Supernatants were analyzed for levels of IFN-g by ELISA. We observed that IL-12 plus IL-33 induced high amounts of IFN-g in WT Pmel-1 Tc1 cells. In contrast, no IFN-g was produced in T-bet KO Pmel-1 Tc1 cells cultured with IL-12 plus IL-33 (Fig. 3F). These results demonstrate that Tc1 cells generated via stimulation by a cognate peptide plus APCs are similarly subjected to a synergistic effect of IL-12 plus IL-33, and T-bet is required for IFN-g production driven by IL-12 plus IL-33, likely via regulation of ST2. Similar & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim results were obtained using Tc1 cells stimulated with plate-bound anti-CD3 and anti-CD28 without APCs (data not shown). In addition, the synergy between anti-CD3 and IL-33 was also dependent on T-bet, and IL-33 could not further increase IFN-g production induced by anti-CD3 in T-bet / CD81 T cells (Supporting Information Fig. 5). IL-33 promotes effector signatures but inhibits characteristics of resting CD81 T cells We have shown that IL-12 and IL-33 induced IFN-g production in Tc1 cells. Thus, we further investigated whether IL-12 and IL-33 regulate IFN-g gene expression. We performed real-time RT-PCR analysis for IFN-g mRNA. We found that IL-33 or IL-12 alone induced small amounts of IFN-g mRNA. IL-12 plus IL-33 induced a large amount of IFN-g mRNA in Tc1 cells (Fig. 4A). Therefore, IL-12 and IL-33 regulate IFN-g at the mRNA level. Since T-bet and Eomes are involved in IFN-g production in Tc1 cells, we also examined the levels of T-bet and Eomes mRNAs in Tc1 cells cultured in IL-12, or IL-33, or IL-12 plus IL-33 conditions. IL-12 and IL-33 individually did not further induce significant amounts of T-bet mRNA in Tc1 cells. However, IL-12 plus IL-33 induced much greater levels of T-bet mRNA (Fig. 4B). In contrast, both IL-12 and IL-33 modestly diminished Eomes mRNA levels (Fig. 4D). Together, IL-12 and IL-33 further reduced the level of Eomes mRNA (Fig. 4D). Therefore, IL-12 and IL-33 differentially regulate T-bet and Eomes in Tc1 cells. Besides T-bet and Eomes, Runx3 has also been implicated in regulating levels of IFN-g [23]. However, IL-12 and IL-33 did not seem to significantly regulate mRNA levels of Runx2 nor Runx3 in Tc1 cells (data not shown). Recent data showed that Blimp1 is a master regulator of the terminal differentiation of CD81 effector T cells [31–33]. In addition, we have found also T-bet and Eomes are involved in Blimp1 expression in CD81 T cells (Supporting Information Fig. 6). Because IL-12 and IL-33 synergistically induce T-bet, we therefore examined whether IL-12 and IL-33 induced Blimp1 in Tc1 cells. IL-33, but not IL-12, induced small amounts of Blimp1. IL-12 and IL-33 together, greatly elevated levels of Blimp1 (Fig. 4C). It is worth noting that IL-12 and IL-33 do not increase levels of granzyme B or perforin (Supporting Information Fig. 7). Thus, IL-12 and IL-33 together promote the effector function of Tc1 cells by inducing IFN-g, T-bet, and Blimp1. TCF-1 and LEF-1 are known transcription factors mediating WNT signals. We have shown that TCF-1 and LEF-1 are associated with naı̈ve and resting activated T cells and are drastically downregulated upon TCR stimulation in both naı̈ve and effector CD41 T cells [34]. Recently, TCF-1 was shown to be associated with central memory T cells and required for the maintenance of these cells via its regulation of Eomes [35]. IL-33, or IL-12, or IL-12 plus IL-33 downregulated both TCF-1 and LEF-1 mRNAs in Tc1 cells (Fig. 4E and F). TCF-1 and LEF-1 downregulation may contribute to the reduction of Eomes mRNA when Tc1 cells were cultured with IL-12 and IL-33. www.eji-journal.eu Leukocyte signaling Eur. J. Immunol. 2011. 41: 3351–3360 IL-33(-) IL-33(+) B IFN γ ng / ml 0 IFN γ 0.5 1 400 α-CD3 (μg / ml) 200 100 0 0.5 1 5 ** 15000 10000 5000 0 IL-33 IL-12 - + - + + + CD8 IL-12(-) C ** nothing IL-33 300 0 α-CD3 (μg/ml) 5 IL-12(+) D F 350 IFN γ ng / ml IL-33 (+) 250 200 150 100 50 CD8 IFN γ ng / ml 300 IFN γ IL-33 (-) E Number of Viable cells A 0 IL-33 IL-12 ND ND ND - + - + 250 150 100 50 + + WT TKO 200 0 IL-33 IL-12 ND ND ND - + - + ND ND ND ND + + - + - + + Control + Figure 3. IL-33 synergizes with TCR signaling or IL-12 to promote IFN-g production. Naı̈ve CD81 T cells were cultured in Tc1 conditions for 4 days and were then stimulated with IL-33 and anti-CD3 for 16 h alone or in combination. IFN-g production was detected by (A) flow cytometry and (B) ELISA. Naı̈ve CD81 T cells were cultured in Tc1 condition for 4 days and were subsequently stimulated with IL-33 or IL-12 alone or in combination for 24 h. IFN-g production was measured by (C) flow cytometry or (D) ELISA. (E) Naı̈ve CD81 T cells were cultured in Tc1 condition for 4 days and were subsequently stimulated with IL-33 or IL-12 alone or in combination for 48 h. Cell viability was determined by Trypan blue exclusion. (F) Naı̈ve CD81 T cells from Pmel-1 WT and Pmel-1 T-bet / (TKO) mice cultured with APCs and the gp100 peptide in Tc1 polarizing condition for 4 days. The activated cells were then stimulated with IL-33 or IL-12 alone or in combination for 24 h. WT Tc1 cells stimulated by anti-CD3 for 16 h were used as a positive control. IFN-g production was measured by ELISA. ND, non-detectable. Data are presented as the mean7SEM of triplicate samples and representative of three independent experiments. po0.01, two-tailed unpaired Student’s t-test. IL-7 is required for the survival of long-term memory T cells [36]. IL-7R can also identify a population of effector T cells that have a substantially greater potential to form memory CD81 T cells [37]. We thus examined IL-7R mRNA levels in Tc1 cells cultured with or without IL-33 and IL-12. Both IL-12 and IL-33 individually induced downregulation of IL-7R (Fig. 4G). Together, they further diminished the levels of IL-7R mRNA and protein (Fig. 4G, Supporting Information Fig. 8). Collectively, these data support the idea that IL-12 and IL-33 synergistically drive the characteristics of effector Tc1 cells. Gadd45b mediates IL-12/IL-33-driven IFN-c production in Tc1 cells We and others have previously shown that Gadd45b regulates the activity of the p38 MAP kinase in Tc1 cells and mediates the synergistic effect of IL-12/TCR as well as IL-12/IL-18 in Th1 cells [38–40]; thus, we investigated whether Gadd45b was involved in IFN-g production driven by IL-33 plus IL-12 in Tc1 cells. We & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim performed real-time PCR analysis and found that IL-33 induced Gadd45b expression and IL-12 plus IL-33 further increased Gadd45b mRNA levels in Tc1 cells (Fig. 5A). Gadd45b was partially involved in the regulation of ST2, and two-fold reduced levels of ST2 mRNA were observed in Gadd45b / Tc1 cells when compared with WT Tc1 cells (Supporting Information Fig. 9). In WT Tc1 cells, IL-12 and IL-33 further enhanced p38 activation (Fig. 5B and C). In contrast, Gadd45b deficiency reduced the levels of detectable active p38 in Tc1 cells (Fig. 5B and C). IL-12 and IL-33 failed to further increase the levels of active p38 in Gadd45b-deficient Tc1 cells (Fig. 5B and C). In addition, Gadd45b deficiency also dampened IL-12/IL-33-induced IFN-g mRNA (Fig. 5D) and protein levels in Tc1 cells (Fig. 5E) as well as anti-CD3/IL-33-induced IFN-g mRNA levels (Supporting Information Fig. 10). Consistent with a role of the p38 MAP kinase in IL-12/IL-33 stimulated IFN-g production, the addition of 10 mM of p38 inhibitor almost completely abolished IFN-g production (Fig. 5F). The cell viabilities were not significantly different in these culture conditions. Collectively, these data demonstrate an important role of the Gadd45b/p38 axis in www.eji-journal.eu 3355 Eur. J. Immunol. 2011. 41: 3351–3360 Qianting Yang et al. IFN γ 40 D *** 6.0 20 10 0 IL-33/IL-12 -/- B 4 +/- -/+ +/+ 0.0 IL-33/IL-12 -/- +/- -/+ +/+ RQ RQ +/- -/+ +/+ Blimp1 10.0 2.5 0.0 IL-33/IL-12 -/- +/- -/+ +/+ * * 2.5 0.0 IL-33/IL-12 -/- 5 4 3 2 1 0 IL-33/IL-12 -/- +/- -/+ +/+ TCF-1 *** RQ 7.5 5.0 5.0 F *** IL-7R ** ** LEF1 7.5 1 5.0 2.5 0.0 IL-33/IL-12 -/- 3.0 E T-bet *** 2 C * 1.5 3 0 IL-33/IL-12 -/- 7.5 * 4.5 RQ RQ 30 G Eomes RQ A RQ 3356 +/- -/+ +/+ +/- -/+ +/+ Figure 4. IL-33 and IL-12 synergistically drive the effector fate of Tc1 cells. Naı̈ve CD81 T cells cultured in Th1 condition for 4 days were then stimulated with IL-33 or IL-12 alone or combined together for (A–D) 4 h or (E–G) 24 h. mRNAs for (A) IFN-g, (B) T-bet, (C) Blimp1, (D) Eomes, (E) Lef1, (F) TCF-1, and (G) IL-7R were quantified by real-time PCR. Data are presented as mean7SEM of triplicate samples and are representative of three independent experiments. po0.05, po0.01, po0.001, two-tailed unpaired Student’s t-test. mediating IL-12/IL-33 synergistic effect on IFN-g production in Tc1 cells. Discussion The IL-33 receptor ST2 was originally reported as a Th2 marker that was expressed on Th2 cell lines but not Th1 cell lines [9, 10]. Administration of either a mAb against ST2 or ST2 fusion protein greatly inhibited the induction of a lung mucosal Th2 immune response [10]. Recently, NK cells and NKT cells were shown to express ST2 and respond to IL-33. In addition, IL-12 and IL-33 stimulate IFN-g production in NK cells and NKT cells. Therefore, the innate arms of Th1 immune response are also driven by IL-33. Consistent with this idea, NKT cells are important for suppressing IL-33-driven allergic responses [15, 20, 41]. Our study showed that ST2 is expressed at high levels in CD81 T cells cultured in Tc1polarizing conditions. Strikingly, CD81 T cells cultured in Tc2 conditions did not express ST2. Furthermore, ST2 expression in Tc1 cells is regulated by T-bet, a master transcription regulator of Th1 effector functions. Therefore, our data reveal a new role of IL-33/ ST2 axis in promoting adaptive effector Tc1 cell-mediated immune responses. IL-33 was originally named as NF-HEV (nuclear factor from high endothelial venules), as it was known to interact with nuclear & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim chromatin [42]. Similar to IL-1a and HMGB1, IL-33 protein is mainly expressed in cell nuclei and is not normally secreted but can be released during cellular necrosis [3, 5]. Therefore, IL-33 is considered as a novel alarmin, an endogenous ‘danger’ signal to alert the immune system upon tissue damage during trauma or infection [6]. By itself, IL-33 induces a small amount of IFN-g. However, IL-33 synergizes with IL-12 and TCR signaling to drive large amounts of IFN-g production. Therefore, the biological function of IL-33 is dependent on other cytokines such as IL-12, and thus IL-33 acts to amplify inflammatory responses rather than to determine the nature of the inflammation. It has been reported that IL-12, a signature cytokine of cellmediated immune responses, inhibits Eomes while upregulating T-bet in effector CD81 T cells during the peak of infection with Listeria monocytogenes. After the resolution of infection, Eomes levels rise, whereas T-bet expression declines in resting memory CD81 T cells [24]. Therefore, T-bet plays a dominant role in effector CD81 T cells and Eomes is more important for homeostasis of memory CD81 T cells. Our data showed that IL-12 and IL-33 synergistically increased T-bet expression, whereas it suppressed the levels of Eomes. In addition, IL-12 and IL-33 synergistically increased Blimp1 expression, another key transcription factor critical for effector fate of CD81 T cells. To address whether IL-33 is involved in Tc1 differentiation, we cultured naı̈ve CD81 T cells in the presence of IL-33 and compared with CD81 www.eji-journal.eu Leukocyte signaling Eur. J. Immunol. 2011. 41: 3351–3360 A D Gadd45b * 10 8 RQ RQ 4 2 7.5 5.0 2.5 - + - + B Gadd45b KO WT 15’ 30’ 60’ 0.0 IL-33 IL-12 + + 0 15’ 30’ 60’ E 750 IFN γ ng/ml 0 IL-33 IL-12 Time p-p38 tubulin 600 - + - + - + + 7500 *** WT KO 5000 *** *** *** 2500 0 15’ 30’ 60’ *** 0 15’ 30’ 60’ + - + - + + *** 450 300 150 F 250 IFN γ ng/ml Mean relative intensity Of p-p38 C - WT KO 200 ND ND ND 0 IL-33 IL-12 0 Time * WT KO 10.0 6 0 IFN γ 12.5 - + - + + + 150 100 ** 50 0 IL-12/IL-33 p38 i (μM) *** *** - + + + + - - 50 10 1 Figure 5. Role of Gadd45b in IL-33/IL-12-stimulated IFN-g production in Tc1 cells. (A) Naı̈ve CD81 T cells were purified from WT mice and cultured in Tc1 condition for 4 days. Then these cells were stimulated with IL-33, or IL-12, IL-12 plus IL-33 for 4 h. Gadd45b mRNA levels were measured by real-time PCR. (B and C) Naı̈ve CD81 T cells were purified from WT and Gadd45b-deficient mice and cultured in Tc1 condition for 4 days. Cells were then stimulated with IL-33 plus IL-12 for 15, 30, and 60 min. Phosphorylated (phospho)-P38 (pP38) level was measured by (B) western blot and (C) the mean relative intensity of pP38 was calculated. (D and E) TC1 cells generated as in (B) were stimulated with or without IL-33 or IL-12 for 24 h. (D) IFN-g mRNA was detected by real-time PCR, and (E) IFN-g protein was measured by ELISA. (F) Naı̈ve CD81 T cells were cultured in Tc1 conditions for 4 days. These cells were then cultured with IL-12 and IL-33 in the presence or absence of P38 inhibitor (50, 10, and 1 mM) for 24 h; IFN-g production was detected by ELISA. ND, undetectable. KO, Gadd45b / . Data are presented as mean7SEM of triplicate samples and are representative of three independent experiments. po0.05, po0.01, po0.001, two-tailed unpaired Student’s t-test. T cells cultured in the neutral condition for 4 days. We then washed the cultured cells extensively and subsequently re-stimulated these T cells with anti-CD3 or PMA/ionomycin without IL-33. We have found no difference in IFN-g production between cells from these cultures (data not shown). Therefore, IL-33 alone does not seem to promote early Tc1 differentiation. This is likely due to the fact that ST2 is induced around 72–96 h after the start of the Tc1 or Tc0 culture and is expressed at negligible levels during earlier T-cell differentiation (data not shown). Collectively, these data are consistent with the idea that IL-33 promotes the effector characteristics of Tc1 cells directly. It is intriguing that we observed lower levels of ST2 expression in CD81 T cells cultured in the Tc2 condition. In humans, ST2 has been shown to be expressed on the Tc2 cell lines but not on Tc1 cell lines [43]. It is very likely that the difference is due to the culture conditions. We used plate-bound anti-CD3 and anti-CD28 plus IL-12 and anti-IFN-g to activate and differentiate Tc1 cells. In & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chan et al. [43], allogeneic APCs were used to stimulate human T cells. In addition, we stimulated T cells for 4 days whereas in Chan et al. [43], they stimulated CD81 T cells for many rounds. It is possible that ST2 levels increase more after several rounds of T-cell stimulation. Consistent with this idea, it has recently been reported that ST2 is only highly expressed in Th2 cells after three rounds of stimulation [44]. Similarly, we have found that ST2 expression was quite low in Th2 cells polarized for one round (Supporting Information Fig. 3). The reason Chan et al. did not observe ST2 expression on Tc1 cells might have been due to the way their Tc1 lines were generated. Multiple rounds of stimulation might lead to the outgrowth of Tc1 lines that are different from our Tc1 lines due to massive apoptosis of Tc1 cells. Our study demonstrates that IL-33 can promote Tc1 immune response in vitro. In vivo, potentially through acting on Th2 cells or Th2-supporting cells, such as mast cells and basophils, IL-33 plays an important role in Th2 responses against pathogens such as www.eji-journal.eu 3357 3358 Eur. J. Immunol. 2011. 41: 3351–3360 Qianting Yang et al. Pneumocystis murina, the nematode Trichuris muris, Toxoplasma gondii, and respiratory syncytial virus [45–48]. Upon L. major infection mice treated with anti-ST2L antibody had enhanced Th1 responses and developed significantly smaller lesions compared with mice treated with control IgG. ST2-deficient mice show a normal host defense against lung infection with Mycobacterium tuberculosis [49]. In contrast, based on our study, it is possible that IL-33 is required for Tc1-driven immune responses against virus, intracellular bacteria, and tumors. In addition, IL-33 has the potential to be used as an adjuvant in vaccines to boost Tc1 immune responses. Our future studies will focus on the in vivo role of IL-33 in various Tc1-mediated diseases. Materials and methods Mice CD4-cre Eomes fl/fl/Tbet doubly deficient mice and CD4-cre Eomes fl/fl mice were described [26]. Pmel-1 TCR transgenic mice were purchased from the Jackson Laboratory. All animals were maintained under specific pathogen-free conditions. All animal work have been approved by the Institution Animal Care and Use Committee at University of Pittsburgh. CD81 T cells culture Lymphocytes were collected from spleens and lymph nodes obtained from C57BL/6WT, T-bet / (TKO), Eomes / (EKO), T-bet/Eomes doubly deficient (DKO) and Gadd45b / mice. Naı̈ve CD62L1 CD44 CD81 T cells were purified by FACS or magnetic beads based methods. The naı̈ve CD81 T cells were more than 98% pure and cultured in Tc1, Tc0, Tc2, and Tc17 conditions as indicated. Cells were stimulated with 5 mg/mL plate-bound antiCD3 (clone 145-2C11) and 5 mg/mL plate-bound anti-CD28 mAbs (clone 37.51) in complete RPMI (cRPMI, RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 50 mM 2-ME, 100 U/mL penicillin, 100 mg/mL streptomycin) in the presence of huIL-2 (20 U/mL, obtained from the BRB Preclinical Repository), IL-12 (3.4 ng/mL) plus anti-IL-4 (10 mg/mL, clone 11B11, from the BRB Preclinical Repository) for the Tc1 condition, or huIL-2 (20 U/mL) for the Tc0 condition, or huIL-2 (20 U/mL), IL-4 (2 ng/mL), and anti-IFN-g (10 mg/mL, clone XMG 1.2) for Tc2, or anti-IL-2Ra (10 mg/mL, clone PC61, ATCC), IL-23 (10 ng/mL), IL-6 (10ng/mL), TGF-b1 (1 ng/mL), anti-IFN-g (10 mg/mL, clone XMG 1.2), and anti-IL-4 (10 mg/mL, clone 11B11) for the Tc17 condition. After 48 h, cells were re-plated to new wells without anti-CD3 and anti-CD28 and with freshly added IL-2 (20 U/mL) for another 2 or 5 days. Alternatively, naı̈ve CD81 T cells were cultured with anti-CD3 in the presence of T-cell-depleted cell-cycle arrested splenocytes as APCs. Naı̈ve Pmel-1 TCR transgenic CD81 T cells were cultured with 1 mM gp10025–33 in the presence of APCs for 4 days in various polarizing conditions as mentioned above. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Upon being cultured in various polarization conditions, CD81 T cells were washed once with cRPMI and were subsequently stimulated for various time points with plate-bound anti-CD3 with or without IL-33 (10 ng/mL, Peprotech), or in the presence of IL-12 (3.4 ng/mL) with or without IL-33 (10 ng/mL). For the treatment with inhibitors, cells were cultured with P38 inhibitor (Enzo Life Sciences), MEK1 inhibitor PD 098059 (Calbiochem), NF-B inhibitor (Sigma) and JNK inhibitor (Calbiochem). Abs For flow cytometry, anti-CD4 (GK1.5), anti-CD8 (53–6.7), and anti-IFN- Ab (clone XMG1.2) were all purchased from eBioscience (San Diego, CA, USA), and anti-ST2 (B4E6) was from MD Bioproducts. Flow cytometric analysis was performed using an FACS ow cytometer (BD Biosciences, San Jose, CA, USA). For Western blot, anti-phosphorylated (phospho)-p38, anti-phosphoJNK, and anti-phospho-ERK were obtained from Cell Signaling. Real-time PCR Cells were lysed in Trizol (Invitrogen) and total RNA was extracted following manufacturer’s instructions. RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The real-time PCR was performed on with SYBR green kit (Applied Biosystems). Cycling conditions were 10 min at 951C, followed by 40 repeats of 951C for 15 s and 601C for 60 s. The primers were: T-bet sense, 50 -CGGGAGAACTTTGAGTCCATGT-30 , Tbet antisense, 50 -GCTGGCCTGGAA GGTCG30 ; Eomes sense, 50 -GGCCTACCAAAACACGGATA-30 , Eomes antisense, 50 -GACCTCCAGGGACAATCTGA-30 ; ST2 sense, 50 -CAAGTAGGACCTGTGTGCCC-30 , ST2 antisense, 50 -CGTGTCCAACAATTGACCTG-30 ; Lef-1 sense, 50 -AGAAATGAGAGCGAATGTCGTAG30 , Lef-1 antisense, 50 -TTTGCACGTTGGGAAGGA-30 ; Tcf-1 sense, 50 -AGCTTTCTCCACTCTACGAACA-30 , Tcf-1 antisense, 50 -AATCCAGAGAGATCGGGGGTC-30 ; Runx2 sense, 50 -ATGCTTCATTCGCCTCACAAA-30 , Runx2 antisense, 50 -GCACTCACTGACTCGGTTGG-30 ; Runx3 sense, 50 -GGTCACCACCGTTCCATC-30 , Runx3 antisense, 50 -ACTTCCTCTGCTCCGTGCT-30 ; IL-7R sense, 50 -TATGTGGGGCTCTTTTACGAGT-30 , IL-7R antisense, 50 -GCCTCGGCTTTAACTATTGTGT-30 ; Gadd45b sense, 50 -CAGATTCACTTCACCCTGATCC-30 , Gadd45b antisense, 50 -GTTGTGCCCAATGTCTCCG-30 ; Blimp1 sense, 50 -CATGGAGGACGCTGATATGAC-30 , Blimp1 antisense, 50 -ATGCCTCGGCTTGAACAGAAG-30 ; IFN-g sense, 50 -TCAAGTGGCATAGATGTGGAAGAA-30 , IFN-g antisense, 50 -TGGCTCTGCAGGATTTTCATG-30 . Statistical analysis We used the two-tailed unpaired Student’s t-test. We considered p-values o0.05 as being significant. www.eji-journal.eu Leukocyte signaling Eur. J. Immunol. 2011. 41: 3351–3360 11 Turnquist, H. R., Sumpter, T. L., Tsung, A., Zahorchak, A. F., Nakao, A., Nau, G. J., Liew, F. Y. et al., IL-1beta-driven ST2L expression promotes maturation resistance in rapamycin-conditioned dendritic cells. J. Immunol. 2008. 181: 62–72. Acknowledgements: The authors thank Dr. Penelope Morel and Dr. Jun Yang for careful reading of the manuscript. This work was supported by NIH AI063496. This work is also partly supported by NSFC grants 30528008 (to B. L. and X. Z.) and 30700728 (to Y. Z.). B. L. was partly supported by the young investigator award from Cancer Research Institute. Q. Y. and X. C. are supported by Eleven-Fifth Mega-Scientific Project on ‘Prevention and treatment of AIDS, viral hepatitis and other infectious diseases’ (2008ZX10003-012). G. 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TKO: T-bet /
Tc1: type 1 Full correspondence: Dr. Binfeng Lu, Department of Immunology, University of Pittsburgh School of Medicine, 200 Lothrop Street, E1047, East Biomedical Science Tower, Pittsburgh, PA 15261, USA Fax: 11-412-383-8098 e-mail: binfeng@pitt.edu 39 Yang, J., Zhu, H., Murphy, T. L., Ouyang, W. and Murphy, K. M., IL-18stimulated GADD45 beta required in cytokine-induced, but not TCRinduced, IFN-gamma production. Nat. Immunol. 2001. 2: 157–164. 40 Lu, B., Ferrandino, A. F. and Flavell, R. A., Gadd45beta is important for perpetuating cognate and inflammatory signals in T cells. Nat. Immunol. Received: 30/3/2011 Revised: 27/7/2011 Accepted: 25/8/2011 Accepted article online: 2/9/2011 2004. 5: 38–44. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu