Cytokine Expression on Thymocyte Apoptosis and Th1/Th2 Synergistic

Synergistic Effect of IL-2, IL-12, and IL-18 on Thymocyte Apoptosis and Th1/Th2 Cytokine Expression

This information is current as Maria Cecilia Rodriguez-Galán, Jay H. Bream, Andrew Farr of September 23, 2021. and Howard A. Young J Immunol 2005; 174:2796-2804; ; doi: 10.4049/jimmunol.174.5.2796 http://www.jimmunol.org/content/174/5/2796 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Synergistic Effect of IL-2, IL-12, and IL-18 on Thymocyte Apoptosis and Th1/Th2 Cytokine Expression1,2

Maria Cecilia Rodriguez-Gala´n,* Jay H. Bream,† Andrew Farr,‡ and Howard A. Young3*

In the periphery, IL-18 synergistically induces the expression of the Th1 cytokine IFN-␥ in the presence of IL-12 and the Th2 cytokines IL-5 and IL-13 in the presence of IL-2. Although the expression of these cytokines has been described in the thymus, their role in thymic development and function remains uncertain. We report here that freshly isolated thymocytes from C57BL/6 and BALB/c mice stimulated in vitro with IL-2-plus-IL-18 or IL-12-plus-IL-18 produce large amounts of IFN-␥ and IL-13. Analysis of the thymic subsets, CD4؊CD8؊ (DN), CD4؉CD8؉, CD4؉CD8؊, and CD4؊CD8؉ revealed that IL-18 in combination with IL-2 or IL-12 induces IFN-␥ and IL-13 preferentially from DN cells. Moreover, DN2 and DN3 thymocytes contained more IFN-␥؉ cells than cells in the later stage of maturation. Additionally, IL-18 in combination with IL-2 induces CCR4 (Th2-

associated) and CCR5 (Th1-associated) gene expression. In contrast, IL-18-plus-IL-12 speciﬁcally induced CCR5 expression. The Downloaded from IL-2-plus-IL-18 or IL-12-plus-IL-18 effect on IFN-␥ and IL-13 expression is dependent on Stat4 and NF-␬B but independent of Stat6, T-bet, or NFAT. Furthermore, IL-12-plus-IL-18 induces signiﬁcant thymocyte apoptosis when expressed in vivo or in vitro, and this effect is exacerbated in the absence of IFN-␥. IL-12-plus-IL-18-stimulated thymocytes can also induce IA-IE expression on cortical and medullary thymic epithelial cells in an IFN-␥-dependent manner. Thus, the combination of IL-2, IL-12, and IL-18 can induce phenotypic and functional changes in thymocytes that may alter migration, differentiation, and cell death of immature

T cells inside the thymus and potentially affect the Th1/Th2 bias in peripheral immune compartments. The Journal of Immu- http://www.jimmunol.org/ nology, 2005, 174: 2796–2804.

he development of T lymphocytes occurs primarily in the thymus and play a role during thymocyte proliferation, differenti- thymus, and this process is dependent, at least in part, on ation, and thymus involution (4–8). Interestingly, IL-18 is not T cytokines (1–3). The thymic epithelial cells (TEC)4 are strictly associated with Th1 responses and has also been shown to the principal source of these immunoregulatory molecules (3), and have the potential of inducing the Th2 cytokines IL-4 (9), IL-5, in addition to IL-7, TEC can produce proinflammatory cytokines and IL-13 (10, 11). Our group previously reported that, in NK cell such as IL-1␣, IL-1␤, and TNF-␣. Considering that no inflamma- and T cells, IL-18 can synergize with IL-2 for the induction of IL-5 tory reactions occur in the thymus, the function of these cytokines and IL-13 as well for IFN-␥ (10). Therefore, IL-18 can induce both by guest on September 23, 2021 in the thymus can differ considerably from that in the peripheral Th1 and/or Th2 responses depending on its surrounding cytokine immune system (1–3). Although known for their role in Th1 cell milieu (11, 12). polarization and for their strong synergism in IFN-␥ production, Based on the fact that IL-2 (13), IL-12 (5–7), and IL-18 (4, 8) IL-12 and IL-18 have recently been reported to be expressed in the have been previously reported to be expressed and also to play a role in intrathymic T cell development, we investigated the effects of IL-18-plus-IL-12 or IL-18-plus-IL-2 on the induction of Th1 *Laboratory of Experimental Immunology, Center for Cancer Research, National and Th2 cytokines from fresh thymocytes and on the specific T cell † Cancer Institute, Frederick, MD 21702; Lymphocyte and Cell Biology Section, Mo- populations that arise during thymic ontogeny. lecular Immunology and Inflammation Branch, National Institute of Musculoskeletal Diseases, Bethesda, MD 20892; and ‡Departments of Biological Structure and Im- Typically, T cells require prior activation signals to be optimally munology, University of Washington, Seattle, WA 98195 responsive to cytokine stimulation. In this report, we demonstrate Received for publication August 17, 2004. Accepted for publication December that freshly isolated thymocytes can produce large amounts of 23, 2004. IFN-␥ and IL-13 after stimulation with IL-18 in combination with The costs of publication of this article were defrayed in part by the payment of page IL-2 or IL-12, independent of other external stimuli (PMA, iono- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mycin, CD3, Con A, TCR engagement, etc.). This cytokine gene 1 This project has been funded in whole or in part with federal funds from the National induction is effective even at concentrations of IL-12 and IL-18 Cancer Institute, National Institutes of Health, under Contract No. N01-CO-12400. lower than that present during certain microbial infections (14). Fur- The TE-71 and ANV 41.2 cell lines were created under National Institute of Allergy thermore, IL-2-plus-IL-18 also induces CCR4 (Th2 phenotype) and and Infectious Diseases Grants AI 59575 and AI 24137 (to A.F.). CCR5 (Th1 phenotype) expression, whereas IL-12-plus-IL-18 trig- 2 The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to copyright covering the article. The content gers the expression of only CCR5. Considering that CCR4 and CCR5 of this publication does not necessarily reflect the views or policies of the Department are constitutively expressed in the thymus, the up-regulation of these of Health and Human Services, nor does mention of trade names, commercial prod- chemokine receptors expression could alter the natural environment ucts, or organizations imply endorsement by the U.S. Government. of this organ through changes in migration pattern (15), negative se- 3 Address correspondence and reprint requests to Dr. Howard A. Young, National Cancer Institute-Frederick, Building 560, Room 31-23, Frederick, MD 21702-1201. lection (16), as well as potentially releasing cells to the peripheral E-mail address: [email protected] immune compartment with a Th1 or Th2 pre-established bias. 4 Abbreviations used in this paper: TEC, thymic epithelial cell; ATOC, adult thymic Interestingly, distinct thymocyte subsets exhibit specific cytokine organ culture; m, mouse; LN, lymph node; RPA, RNase protection assay; DN, production profiles, suggesting a different capacity for cytokine ex- CD4ϪCD8Ϫ double negative; DP, CD4ϩCD8ϩ double positive; CD4ϩ, CD4ϩCD8Ϫ single positive; CD8ϩ, CD4ϪCD8ϩ single positive; 7AAD, 7-aminoactinomycin D; pression by these subsets during T cell maturation. Furthermore, using KO, knockout. mice deficient in Stat family members, T-bet, the NF-␬B p50 subunit,

and NFAT, we were able to identify the transcription factors essential tion assay (RPA), 5 ␮g of total cytoplasmic RNA were analyzed using the for the production of IFN-␥ and IL-13 by thymocytes. IL-12-plus- RiboQuan kits (BD Pharmingen) and [33P]UTP-labeled riboprobes as de- IL-18 induces cell death in adult thymic organ culture (ATOC) but not scribed previously (21). in suspension cultures, demonstrating that the thymic microenvironment is important during apoptosis induction triggered by these cy- Cytokine assays tokines. This effect is also seen in IL-12-plus-IL-18 cDNA-injected ␥ Supernatants from cells treated in vitro were collected and assayed for mice in vivo and is exacerbated in the absence of IFN- . Finally cytokine production by ELISA. The kits used were as follows: mouse IL-12-plus-IL-18-stimulated thymocytes induce IA-IE expression in IFN-␥ and mouse IL-13 (R&D Systems). cortical and medullary epithelial cell lines in an IFN-␥-dependent manner, demonstrating that the synergistic effect of IL-2, IL-12, and Flow cytometry analysis and cell sorting IL-18 may not only influence Th1/Th2 polarization and apoptosis induction in thymocytes but also may influence the thymic microen- Up to six-color analysis was performed on a BD Biosciences FACSort flow vironment in an indirect way. cytometer as previously reported (22). Anti-mouse CD16/CD32 mAb (2.4G2; BD Pharmingen) was used to block the nonspecific binding. To detect intracellular expression of cytokines, cells were cultured with the Materials and Methods different stimuli, and 5 ␮g/ml brefeldin A (BD Pharmingen) was added Cytokines and Abs during the last 8 h. Cells were then stained for surface markers, washed, and fixed with Cytofix/Cytoperm buffer (BD Pharmingen) for 15 min at Recombinant human IL-2 was obtained from Hoffmann-La Roche. rIL-18 4°C. Cells were washed with Perm Wash buffer (BD Pharmingen) and was obtained from Medical and Biological Laboratories, and IL-12 was incubated with the anti-mouse IFN-␥ Ab or isotype-matched Ab for 30 min obtained from PeproTech. FITC or PE anti-mCD8, PE or PE-Cy5 anti- Downloaded from ␥ at 4°C. Following two washings, cells were analyzed in the flow cytometer. mouse (m)CD4, PE anti-mIFN- , PE anti-mNK1.1, PE-Cy5 anti-mCD44, For flow cytometry cell sorting, cells were stained with CD4-PE and allophycocyanin or PE-Cy7 anti-mNK1.1, allophycocyanin-Cy7 anti- CD8-FITC, and CD4ϪCD8Ϫ double-negative (DN), CD4ϩCD8ϩ double- mCD25, and FITC anti-mMHC-class II Abs were all purchased from BD positive (DP), CD4ϩCD8Ϫ single-positive (CD4ϩ), and CD4ϪCD8ϩ sin- Pharmingen. gle-positive (CD8ϩ) thymocytes were sorted to a purity of Ͼ97–99% using The IL-2 and IL-13 neutralizing Abs were purchased from BD Pharm- a MoFlo sorter (DakoCytomation). ingen and R&D Systems, respectively. IL-12 neutralizing Ab was a gift from Dr. A. Sher (National Institute of Allergy and Infectious Diseases, http://www.jimmunol.org/ National Institutes of Health, Bethesda, MD). Anti-IL-2 was a gift from Dr. Statistical analysis J. Cote-Sierra (National Institute of Allergy and Infectious Diseases, Na- tional Institutes of Health). Testing for significance of differences was assessed by Student’s t test using Microsoft Excel statistical analysis computer program. Mice C57BL/6 mice (B6), IFN-␥Ϫ/Ϫ, Stat6Ϫ/Ϫ, T-betϪ/Ϫ, p50Ϫ/Ϫ, and BCL-2 transgenic mice on a B6 background, Stat4Ϫ/Ϫ mice on a 129 background, Results Ϫ/Ϫ NFAT1 and NFAT4 mice on a BALB/c background, and their control Thymocytes can produce large amounts of Th1 and Th2 littermates were used in this study. All gene-deficient and transgenic mice were backcrossed for Ͼ10 generations with B6, 129, or BALB/c mice and cytokines maintained under specific pathogen-free condition and used for experi- It has been previously reported that, in NK and T cells, the com- by guest on September 23, 2021 ments at 4–6 wk of age. bination of IL-18 with IL-12 or IL-2 synergizes to produce Th1 Hydrodynamic injection of IL-12 and IL-18 cDNA and Th2 cytokines, respectively (10, 11). In our experiments, in- The hydrodynamic gene transfer procedure was conducted as described jection with a mammalian expression plasmid for IL-12 or IL-18 previously (17). In brief, animals were separated into four different groups resulted in high circulating levels of these cytokines whether in- and injected by tail vein with the following: 15 ␮g of empty vector control jected individually or in combination. Interestingly, we found that cDNA; 5 ␮g of IL-12 cDNA (pscIL-12, p40-p35 fusion gene) plus 10 ␮g thymocytes from mice injected with IL-12-plus-IL-18 cDNA re- of IL-18 cDNA (pDEF pro-IL-18); IL-12 cDNA alone or IL-18 cDNA spond by up-regulating IFN-␥ expression in vivo (Fig. 1, A and B). alone in 1.6 ml of sterile 0.9% sodium chloride solution. All of the plas- ␥ mids are driven by human elongation 1-␣ promoter. Thymuses were har- IFN- expression is low at 24 h poststimulation (3-fold increase vested 24, 48, 72, and 96 h postinjection. over control) and increases gradually over time, reaching levels Cell cultures and cell lines almost 50 times higher than control at 96 h postinjection (Fig. 1, A and B). These findings led us to investigate the capacity of imma- Thymuses, spleens, and lymph nodes (LNs) were smashed, washed, and ture T cells in the thymus to produce Th1 and Th2 cytokines. We resuspended in supplemented medium (RPMI 1640 supplemented with developed an in vitro model where thymocytes were directly stim- 10% heat-inactivated FBS, 100 U/ml penicillin G sodium, 100 ␮g/ml strep- tomycin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate, 1ϫ essential ulated with IL-2-plus-IL-18 or IL-12-plus-IL-18 and assayed for amino acids, and 10 mM 2-ME). Splenocyte cell suspensions were de- IFN-␥, IL-13, and IL-5 mRNA expression and compared with pleted of red cells by treatment with ACK lysis buffer, washed, and resus- those from secondary lymphoid organs such as LNs and spleen pended in supplemented medium. Cells were counted and cultured at 2.5 ϫ 6 (Fig. 1C). As shown in Fig. 1C, the combination of IL-2-plus- 10 cells/ml at 37°C with medium alone or in the presence of IL-2 (100 ␥ IU/ml), IL-12 (100 ng/ml), IL-18 (100 ng/ml), IL-2-plus-IL-18, or IL-12- IL-18 synergizes to induce IFN- (Th1), IL-13, and IL-5 (Th2) in plus-IL-18 for 48 h, unless otherwise specified. all tissues analyzed, with the Th2 cytokines particularly strongly The ATOC methods used have been described in detail elsewhere (18). expressed in thymus compared with spleen and LNs (C and D). A 3 Briefly, thymi were cut into 1-mm fragments and cultured on top of neutralizing anti-IL-12 Ab added to the cultures did not change the ␮ Nucleopore filters (0.8 M; Corning) at the air-medium interface using levels of cytokines produced (data not shown), indicating the syn- plastic cylinders instead of sponges. Cultures were maintained in a fully ergy between IL-2-plus-IL-18 was not due to the expression of humidified incubator at 37°C with 5% CO2 for 24 h. Thymic lobe fragments were stimulated with the cytokines for 24 h and then dispersed into endogenous IL-12. Although IL-12-plus-IL-18 stimulation induces a cell suspension, counted, and used in the apoptosis assays. some IL-13 but not IL-5 expression (Fig. 1, C and D), it strongly The TE-71 cells most closely resemble medullary TEC and the ANV induces a Th1 response with high levels of IFN-␥ mRNA expres- 41.2 cells most closely resemble cortical TEC (19, 20). sion in all three tissues analyzed (C and E). Furthermore, the use mRNA analysis of a neutralizing anti-IL-2 Ab confirmed that endogenous IL-2 Total RNA was isolated using a single-step phenol/chloroform extraction production was not responsible for the expression of IL-13 in the procedure (TRIzol; Invitrogen Life Technologies). For the RNase protec- IL-12-plus-IL-18-treated thymic cultures (data not shown). 2798 CYTOKINES AND THYMOCYTE APOPTOSIS AND Th1 AND Th2 POLARIZATION

expression; in contrast, IL-18-plus-IL-12 provides a more rapid and potent stimulus for IFN-␥ production (Fig. 2, A–C). Evaluation of the sensitivity of thymocytes to these combinations of cytokines revealed that thymic cells produced a significant amount of IFN-␥ (1000 pg/ml) after IL-18 and IL-12 stimulation with concentrations as low as 1 ng/ml (Fig. 2D) and IL-13 (10 pg/ml) after addition of 1 ng/ml IL-18 plus 1 U/ml IL-2 (E). In- terestingly, levels of IL-12 that range from 0.4 to 15 ng/ml and 1 ng/ml IL-18 can be found in the sera of mice after infection with certain pathogens (14), thus indicating that the sensitivity of thymocytes to cytokine stimulation that we have observed is physio- logically relevant. Thymocytes from BCL-2 transgenic mice, which are more re- sistant to apoptotic death, produced equivalent amounts of IFN-␥ FIGURE 1. Cytokine mRNA expression following treatment in vivo or and IL-13 as control mice after IL-2-plus-IL-18 or IL-12-plus- in vitro with IL-18 in combination with IL-2 or IL-12. A, Mice were hy- IL-18 stimulation (data not shown). Thus, our results do not rep- drodynamically injected with control or IL-12-plus-IL-18, cDNA, and 24, resent a selection for thymocyte subsets that preferentially survive 48, 72, and 96 h later, thymuses were harvested and IFN-␥ mRNA expres- under these conditions. sion was evaluated by the RPA (BD Pharmingen multitemplate probe kit Downloaded from mck-1). Representative data from one of four independent experiments are Subpopulations of thymocytes have different and/or exclusive shown. C, Thymocytes, LN cells, and splenocytes were cultured with no capacities for producing IFN-␥ and IL-13 after IL-18-plus-IL-2 cytokines (NS), IL-2-plus-IL-18, or IL-12-plus-IL-18 for 48 h, and IL-5, or IL-18-plus-IL-12 stimulation IL-13, and IFN-␥ mRNA expression was evaluated by the RPA (BD Pharmingen multitemplate probe kit mck-1). Representative data from one To determine whether the production of IFN-␥ and IL-13 comes of two independent experiments are shown. B, D, and E, Densitometry was from thymocytes themselves or from thymic stromal cells (epithe- performed and the fold induction was measured by standardizing samples lial cells, dendritic cells, macrophages, fibroblast, etc.) present in http://www.jimmunol.org/ to the L32 signal (B) and then normalized to the NS sample for each tissue the bulk thymocytes cultures, we separated thymocytes into four (D and E). Spl, Spleen; Thy, thymus. groups based on their expression of CD4 and CD8 and investigated the cytokine expression after IL-2-plus-IL-18 or IL-12-plus-IL-18 stimulation. Fig. 3 shows that DN, CD4ϩ, and CD8ϩ thymocytes IL-18 rapidly and differentially synergizes with IL-2 and IL-12 were able to produce IFN-␥ to different extents (A). However, only for the production of Th1 and Th2 cytokines in thymocytes DN and CD4ϩ cells are able to produce IL-13 (Fig. 3B). Next, we evaluated how rapidly fresh thymocytes are able to ex- The synergy between IL-12 and IL-18 in IFN-␥ production is press Th1 and Th2 cytokines upon stimulation with IL-18 in com- partially explained by the mutual up-regulation of the cytokine by guest on September 23, 2021 bination with IL-2 or IL-12. As can be seen in Fig. 2, thymocytes receptors (12, 23). Thus, we evaluated the surface expression of produced increasing and significant amounts of IFN-␥ and IL-13 IL-2R, IL-12R, and IL-18R in the different subsets of thymocytes mRNA (A) and protein (B and C, respectively) up to 48 h post- and found that, in all cases, the expression of these receptors was stimulation. We did not continue the kinetic analysis beyond 48 h very low and did not change during any of the treatments used in because the viability of DP cells is significantly compromised at the experiments. However, at the RNA level, we detected up-reg- later time points. We also observed that IL-18 synergizes with both ulation of IL-12R␤1 and -␤2 by IL-18, and IL-18R by IL-12 (data IL-2 and IL-12 to produce both IL-13 and IFN-␥. IL-18-plus-IL-2 not shown), a result that has been reported previously in T treatment is a stronger stimulus for IL-13 and uniquely for IL-5 cells (11, 23).

FIGURE 2. Kinetics of cytokine mRNA and protein expression in thymocytes from C57BL/6 mice. A, Thy- mocytes were cultured with no cytokines (NS), IL-2- plus-IL-18, or IL-12-plus-IL-18; samples were taken at speciﬁc time points and evaluated for IL-5, IL-13, and IFN-␥ mRNA expression by the RPA (BD Pharmingen multitemplate probe kit mck-1). Representative data from one of three independent experiments are shown. B and C, Supernatants were collected at each time point, and the levels of IFN-␥ (B) and IL-13 (C) were determined by ELISA. Representative data from one of three independent experiments are shown. D and E, Thymocytes were cultured for 48 h at different cytokine concentrations and assayed for IFN-␥ and IL-13, respectively, by ELISA. Representative data from one of two independent experiments are shown. The Journal of Immunology 2799

Table II. Frequency of IFN-␥-producing cells in the thymus: DN subsets

DN1 DN2 DN3 DN4 (CD44ϩCD25Ϫ) (CD44ϩCD25ϩ) (CD44ϪCD25ϩ) (CD44ϪCD25Ϫ)

IFN-␥ϩ 23% 40% 30% 22% cells

LNs, and spleen cells. The expression of these chemokine receptors is not due to the presence of IL-13 or IFN-␥ in the cultures because thymocytes from IFN-␥Ϫ/Ϫ mice or the addition of IL-13 neutralizing Ab does not change the chemokine receptors pattern seen in C57BL/6 mice after stimulation with the cytokine combinations (Fig. 4B and data not shown). Moreover, although CCR4 can be expressed by DN, CD4ϩ, and CD8ϩ cells (Fig. 4C), CCR5 can be predominantly expressed in DN and CD4ϩ cells and weakly FIGURE 3. Cytokine mRNA and protein expression in different thymo- in CD8ϩ cells (C). cyte populations. Thymocytes were stained with PE-CD4 and FITC-CD8, ϩ ϩ Downloaded from and DN, DP, CD4 , and CD8 cells were sorted. Cells were then cultured IL-12-plus-IL-18 stimulation induces signiﬁcant thymocyte death with no cytokines (NS), IL-2-plus-IL-18, or IL-12-plus-IL-18 for 48 h, and in vivo and in vitro: protective role of IFN-␥ supernatants were collected and the levels of IFN-␥ (A) and IL-13 (B) were determined by ELISA. Representative data from one of three independent It has been described that, in the presence of IFN-␥, TCR engage- experiments are shown. ment can cause apoptosis in CD4ϩ and CD8ϩ thymocytes (25). To evaluate whether the large amounts of IFN-␥ produced in the IL-

12-plus-IL-18 thymic cultures correlates with increased cell death, http://www.jimmunol.org/ Differential capacity for IFN-␥ production during T cell we generated thymic suspension cultures and ATOC where the development architecture of the organ is disrupted or intact, respectively, and The high expression of IL-13 and IFN-␥ observed in the DN subset analyzed apoptosis by annexin V/7-aminoactinomycin D (7AAD) (Fig. 3, A and B) could be due to the presence of a small population staining. of NKT cells present in the thymus. Therefore, we evaluated the Fig. 5A shows that no significant differences in cell death were percentage and type of IFN-␥-producing cells in the different sub- observed when we compared thymocytes stimulated with individ- sets of thymocytes after IL-18-plus-IL-12 stimulation (Tables I ual or combined cytokines in suspension cultures with the control and II). A high percentage of NKT cells (68%) was responsible for group. However, when apoptosis was assayed in ATOC, we found by guest on September 23, 2021 the production of IFN-␥ in the DN subset; however, 34% of the that IL-12-plus-IL-18 treatment results in significantly increased remaining DN thymocytes could still produce IFN-␥ (Table I). cell death compared with control cultures (75 vs 55%, respec- Ͻ Interestingly, six-color flow cytometry analysis revealed that, in tively; p 0.05) (Fig. 5A). Analysis of apoptosis in the different the DN subset, cells in the DN2 and DN3 stages show higher numbers of IFN-␥ϩ cells by intracellular staining than in the DN1 and DN4 stages (Table II). Moreover, the number of thymocytes with the capacity to produce IFN-␥ decreases from DN (34%) to a very low number when they become DP (5%), and then recovers to some extent when they mature to CD8ϩ (22%) and CD4ϩ (10%) (Table I).

Both CCR4 and CCR5 are expressed after IL-2-plus-IL-18 stimulation but only CCR5 after IL-12-plus-IL-18 stimulation in DN, CD4ϩ, and CD8ϩ cells It has been reported that Th1 cells preferentially express the chemokine receptor CCR5 (24), whereas Th2 cells express CCR4 (24). To evaluate whether the treatment with the combined cytokines results not only in the ability to produce IFN-␥ and IL-13 but also a chemokine receptor pattern that correlates with the Th1 and/or Th2 proﬁles, we performed a RPA to evaluate the expression of CCR4 and CCR5 mRNA. As shown in Fig. 4A, IL-2-plus- IL-18 induces the expression of both CCR4 and CCR5, whereas IL-12-plus-IL-18 induces the expression of only CCR5 in thymus, FIGURE 4. Chemokine receptor mRNA expression following stimulation with IL-18 in combination with IL-2 or IL-12. Thymocytes, LNs cells and splenocytes from C57BL/6 (A) or thymocytes from C57BL/6 and IFN- Table I. Frequency of IFN-␥-producing cells in the thymus: major ␥Ϫ/Ϫ mice (B) or thymocytes stained with PE-CD4 and FITC-CD8 (C) subsets were sorted for DN, DP, CD4ϩ, and CD8ϩ cells, and then cultured in vitro for 48 h with the cytokines indicated in the ﬁgure. CCR4 and CCR5 mRNA DN DP CD4ϩ CD8ϩ NKT expression was evaluated by RPA (BD Pharmingen multitemplate probe kit mcr-5). Representative data from one of three independent experiments ␥ϩ IFN- cells 34% 5% 10% 22% 68% are shown. 2800 CYTOKINES AND THYMOCYTE APOPTOSIS AND Th1 AND Th2 POLARIZATION Downloaded from

FIGURE 5. IL-12-plus-IL-18 induce in vitro (ATOC) and in vivo significant cell death in thymocytes. Protective role for IFN-␥. A, Thymocytes from ATOC or suspension cultures were stimulated for 24 h with the designated cytokines. Then cells were stained with CD4, CD8, annexin V, and 7AAD, and the percentage of cell death was calculated as follow: 100 Ϫ % of annexin VϪ7AADϪ cells of a total of 150,000 events acquired. B, DN, DP, CD4ϩ, and CD8ϩ were gated out, and the percentage of annexin Vϩ cells in the different thymocyte subsets was analyzed. C–E, C57BL/6 and IFN-␥-deficient mice were hydrodynamically injected with IL-12-plus-IL-18 cDNA, and 96 h later, thymocytes were harvested. Cells from C57BL/6 mice were counted, and the absolute cell number (C) and percentage of annexin Vϩ cells (D) were determined in the bulk thymocyte population of C57BL/6 vs GKO mice. Then, cells from C57BL/6 mice were stained with CD4 and CD8 Abs, and the percentage of annexin Vϩ cells were determined in the DN, DP, CD4ϩ, and CD8ϩ http://www.jimmunol.org/ subsets (E). The data are taken from one representative experiment of four performed with three to five mice per group. subset of thymocytes in the ATOC demonstrated that IL-12-plus- cocultured with thymocytes in the presence of IL-12-plus-IL-18, IL-18 induces cell death preferentially in DP cells with 22% more up-regulate IA-IE expression; moreover, this effect does not need annexin Vϩ cells than in the control ATOC (Fig. 5B). This effect cell-cell interaction because supernatants from IL-12-plus-IL-18- can also be seen in vivo because we observed a marked reduction stimulated thymocytes induced IA-IE expression in both ANV in the absolute cell number in the thymuses of mice injected with IL-12-plus-IL-18 cDNA compared with control cDNA-injected by guest on September 23, 2021 mice (Fig. 5C). Moreover, the loss of cells in the IL-12-plus-IL- 18-injected mice correlated with an increase in the percentage of annexin Vϩ cell in the bulk population (Fig. 5D) that corresponds to a selective apoptosis in the DN and DP subsets (E). To evaluate the role of IFN-␥ in cell death induction in vivo after IL-12-plus-IL-18 cDNA injection and in vitro in ATOC, we performed our studies in IFN-␥-deficient mice. Fig. 5C shows a significant reduction in the absolute cell numbers in thymocytes coming from either C56BL/6 or IFN-␥Ϫ/Ϫ mice after cDNA injection. Accordingly, an increased percentage of apoptotic cells can be seen in both strains of mice after IL-12-plus-IL-18 stimulation in ATOC (data not shown) or after IL-12-plus-IL-18 cDNA injection (Fig. 5D). Finally, IL-12-plus-IL-18 stimulation induces apoptosis preferentially in DP and DN cells in vitro and in vivo in the presence (Fig. 5, B and E, respectively) or absence of IFN-␥ (data not shown). ⌴oreover, IFN-␥ seems to play some type of overall protective role because only ϳ60% of IFN-␥Ϫ/Ϫ mice sur- vived 5 days post-IL-12-plus-IL-18 cDNA injection compared with 100% survival in the cDNA-injected control mice (data not FIGURE 6. IA-IE up-regulation in cortical and medullary TEC by IL- 12-plus-IL-18-stimulated thymocytes is IFN-␥ dependent. A, The ANV shown). 41.2 (cortical) and TE-71 (medullary) TEC were cocultured with thymocytes from C57BL/6 mice in the absence (empty histograms) or presence IL-12-plus-IL-18-stimulated thymocytes induce IA-IE expression ␥ (black histograms) of IL-12-plus-IL-18. After 24-h culture, TEC were re- in cortical and medullary epithelial cell lines in an IFN- - covered, and IA-IE expression was evaluated by flow cytometric analysis. dependent manner B, Supernatants from C57BL/6 thymocytes culture for 24 h in the absence In our model, we evaluated whether thymocytes that have been (empty histograms) or presence (black histograms) of IL-12-plus-IL-18 exposed to the different combinations of cytokines have the po- were transferred to ANV 41.2 and TE-71 cells cultures, and 24 h after, IA-IE expression was evaluated by flow cytometric analysis. C, TE-71 tential to affect cells from the thymic microenvironment by mod- cells were cocultured with thymocytes from C57BL/6 or IFN-␥Ϫ/Ϫ mice in ulating MHC class II expression in different TEC as has been pre- the absence (empty histograms) or presence (black histograms) of IL-12- viously described for human TEC (26). Fig. 6A shows that a plus-IL-18. After 24-h culture, TE-71 cells were recovered, and IA-IE ex- cortical (ANV 41.2) and a medullary (TE-71) TEC lines, when pression was evaluated by flow cytometric analysis. The Journal of Immunology 2801 Downloaded from

FIGURE 7. IFN-␥ and IL-13 mRNA and protein expression in Stat4Ϫ/Ϫ mice. A, Thymocytes from C57BL/6 and Stat4Ϫ/Ϫ mice were stimulated in Ϫ Ϫ http://www.jimmunol.org/ vitro for 48 h with the cytokines indicated in the ﬁgure, and IFN-␥ and FIGURE 8. IFN-␥ and IL-13 mRNA and protein expression in p50 / Ϫ Ϫ IL-13 mRNA expression was evaluated by the RPA (BD Pharmingen mul- mice. A, Thymocytes from C57BL/6 and p50 / mice were stimulated in titemplate probe kit mck-1). B and C, Supernatants were collected and the vitro for 48 h with the cytokines indicated in the ﬁgure, and IFN-␥ and levels of IFN-␥ (B) and IL-13 (C) were determined by ELISA. Represen- IL-13 mRNA expression was evaluated by the RPA (BD Pharmingen mul- tative data from one of two independent experiments are shown. titemplate probe kit mck-1). B and C, Supernatants were collected, and the levels of IFN-␥ (B) and IL-13 (C) were determined by ELISA. Represen- tative data from one of two independent experiments are shown.

41.2 or TE-71 cells (Fig. 6B). Finally, IA-IE up-regulation seems by guest on September 23, 2021 to be mediated by IFN-␥ produced by thymocytes after IL-12-plus- NF-␬B but not NFAT1 or NFAT4 is required for both IL-13 and IL-18 stimulation because ANV 41.2 and TE-71 do not express IFN-␥ production IL-12 or IL-18 receptors on their cell surface (data not shown) and It has been reported that NF-␬B and NFAT have an important role IFN-␥-deficient thymocytes do not have the capacity to induce in regulating IFN-␥ and IL-13 gene expression (11, 27, 30–32). To IA-IE in these TEC lines (Fig. 6C). evaluate whether these transcription factors are involved in IFN-␥, IL-13, and IL-5 expression after IL-2-plus-IL-18 or IL-12-plus- ␥ IFN- and IL-13 but not IL-5 expression is Stat4 dependent and IL-18 stimulation, we cultured fresh thymocytes from NF-␬B Stat6/T-bet independent in response to IL-18-plus-IL-2 or IL-18- p50Ϫ/Ϫ, NFAT1Ϫ/Ϫ, NFAT4Ϫ/Ϫ, and control littermate mice for plus-IL-12 stimulation 48 h with the combined cytokines. As shown in Fig. 8, whereas To gain insight into the transcription factors that participate in the IFN-␥ (B) and IL-13 (C) protein expression is considerably Ϫ Ϫ expression of IFN-␥, IL-13, and IL-5 in the thymus, we stimulated blocked in thymocytes from p50 / mice after both IL-2-plus- thymocytes with IL-18-plus-IL-2 or IL-18-plus-IL-12 in Stat4, IL-18 or IL-12-plus-IL-18 stimulation, the mRNA levels (A) are Stat6, and T-bet knockout (KO) mice. As previously reported, only partially affected. However, IL-5 mRNA expression (Fig. 8A) Stat4 activation correlates with the capacity to promote IFN-␥ pro- is completely absent in IL-2-plus-IL-18 cultures. In contrast, thy- Ϫ Ϫ duction in response to IL-12 signaling (12, 27). Stat6 is essential mocytes from NFAT1 or NFAT4 / mice expressed similar levels for chromatin remodeling at the Th2 cytokine loci and for the of IFN-␥ and IL-13 RNA and protein when compared with control production of IL-4, IL-5, and IL-13 (27–29). T-bet, a member of mice (data not shown), indicating that NFAT1 and -4 do not play the T-box family transcription factors, has been shown to be re- a role in the expression of these cytokines in response to IL-12- quired for the induction of IFN-␥ by CD4ϩ cells (12, 27). How- plus-IL-18 or IL-2-plus-IL-18. ever, our analysis demonstrated that neither Stat6 nor T-bet is required for the expression of IL-13, IL-5, or IFN-␥ after IL-18- Discussion plus-IL-2 or IL-18-plus-IL-12 stimulation because levels of Recently, it has been demonstrated that the proinflammatory cy- expression in the thymocytes from these KO mice were compara- tokines IL-12 and IL-18 are expressed in the thymus and may play ble with those observed in the wild-type mice (data not shown). a role during thymic development (4–8, 13). Our initial experi- However, thymocytes from Stat4Ϫ/Ϫ mice showed a profound de- ments using injections of cytokine-inducing cDNAs demonstrated crease in IFN-␥ and IL-13 at the mRNA (Fig. 7A) and protein level that thymocytes in vivo respond to circulating levels of IL-12 and (B and C) in response to IL-18-plus-IL-12. Interestingly, when the IL-18 by expressing IFN-␥ (Fig. 1〈). These findings are quite cells were stimulated with IL-18-plus-IL-2, IFN-␥ was consider- interesting considering the thymus is a closed organ, and the en- ably reduced, whereas IL-13 was only partially affected in the trance of macromolecules including cytokines into the thymus is Stat4Ϫ/Ϫ mice (Fig. 7). limited (3). 2802 CYTOKINES AND THYMOCYTE APOPTOSIS AND Th1 AND Th2 POLARIZATION

In vitro, we found that thymocytes rapidly and strongly respond can induce apoptosis in the absence of IFN-␥ (Fig. 5C). IFN-␥ to IL-18 in combination with IL-12 or IL-2 to produce the Th1 seems to have a protective effect considering that the 60% of cytokine IFN-␥ and the Th2 cytokines IL-5 and IL-13. Interest- thymocytes lost after IL-12-plus-IL-18 treatment in C57BL/6 ingly, the levels of IFN-␥ produced by thymocytes in vitro under mice (NS vs IL-12-plus-IL-18, p ϭ 0.001) increases to 80% in these conditions are comparable with those produced by spleen IFN-␥Ϫ/Ϫ mice (NS vs IL-12-plus-IL-18, p ϭ 0.0001) (Fig. and LN cells in vitro (data not shown). Moreover, the Th2 cyto- 5C). This phenomenon might be explained based on previous kines, IL-5 and IL-13, are more highly expressed in the thymus reports where it has been proposed that IFN-␥ can induce IL-15 than in spleen or LNs. These findings surprised us because thy- which in turn mediates an antiapoptotic effect (41). This regu- mocytes have been reported to be poor cytokine producers them- latory mechanism exerted by IL-15 may be more systemic, be- selves, unless they receive activation signals (e.g., PMA, ionomy- cause ϳ40% of IFN-␥Ϫ/Ϫ mice died after 5 days post-IL-12- cin, Con A, anti-CD3 Abs) (3, 13, 33). plus-IL-18 injection compared with 100% survival in control The production of IFN-␥ by thymocytes in response to IL-2- C57BL/6 mice (data not shown). plus-IL-18 or IL-12-plus-IL-18 is relevant because IL-2, IL-12, Over the past years, important progress has been made in un- and IL-18 are present in the thymus (4–8) and because IFN-␥ derstanding the molecular events resulting in strong IFN-␥ expres- plays multiple roles in thymic development (34–36). IFN-␥ can sion in mature T cells in response to IL-12 and IL-18. Considering modulate TEC interaction with thymocytes and induce Ag presen- that the transcription factors that participate in cytokine expression ␥ tation in TEC (34, 36). Moreover, as an effector molecule, IFN- by mature T cells could be differentially expressed in thymocyte induces NO in thymic stromal cells restricting thymocyte devel- subsets, we considered it important to evaluate how this synergy opment by inducing apoptosis (35). In our model, we found that works in immature T cells. Downloaded from ␥ thymocytes exposed to IL-12-plus-IL-18 produce IFN- that in IL-12 induces phosphorylation of specific proteins known as turn can mediate IA-IE expression in cortical and medullary TEC. Stats, and it has been demonstrated that the Stat4 is linked to IL- This is important considering that MHC class II expression by 12R signaling and is critical for IFN-␥ expression in T cells me- TEC is essential during negative and positive selection (37, 38). ϩ ϩ diated by IL-12 alone or in synergy with IL-18 (42). In thymo- Separation of DN, DP, CD4 , CD8 cells provided a clearer cytes, we confirmed that Stat4 is an important mediator in the insight regarding the ability of these subsets to express cytokines expression of IFN-␥ and surprisingly found that Stat4 is required http://www.jimmunol.org/ following specific stimulation. Our data revealed that DN cells are for IL-13 expression by IL-12-plus-IL-18 stimulation as well as in the main source of IFN-␥ and IL-13 after IL-2-plus-IL-18 or IL- IL-2-plus-IL-18-treated cultures, in the absence of IL-12. 12-plus-IL-18 stimulation. However, it has been described that In contrast to Stat4, our studies in T-bet- and Stat6-deficient small populations in the thymus, including NKT cells and ␥-␦ mice demonstrate that IFN-␥ and IL-13 expression is independent thymocytes, can strongly express cytokines (39). As these cells are of T-bet and Stat6 in response to IL-2-plus-IL-18 or IL-12-plus- present in the thymus in an extremely low percentage, their puri- IL-18 (data not shown). Because the role of Stat6 in Th2 differ- fication is a significant technical challenge. To overcome this prob- entiation is well established (27–29), it is interesting to note that lem, we stimulated thymocytes with IL-12-plus-IL-18 and ana-

IL-13 and IL-5 expression after IL-2-plus-IL-18 treatment repre- by guest on September 23, 2021 lyzed IFN-␥ expression by intracellular staining. The data sents a novel way to induce these two Th2 cytokines in a Stat6- demonstrated that, in the DN subset, NKT cells may in fact highly ϩ independent manner. contribute to the production of IFN-␥, because 68% were IFN-␥ . Ϫ Our group and other laboratories have reported an important Remarkably, we found 34% of DN NK1.1 cells also produce IFN-␥, especially in the intermediate stage of DN T cell develop- role for the transcription factor NFAT family in the regulation of ␥ ment, when CD25 is expressed (DN2 and DN3, 40 and 30%, re- IFN- (30, 32) and IL-13 gene expression (31). NFAT1 has been ␥ spectively) compared with when it is absent (DN1 and DN4 stages, reported to be a major regulator of IFN- in vivo (30) and impor- 22 and 23%, respectively). The number of IFN-␥ϩ cells decreased tant during IL-13 transcription after CD3 stimulation in vitro (43). ␥ considerably in the DP stage (5%) as has been previously reported In contrast, NFAT4 has been proposed as an enhancer of IFN- (40). Interestingly, at the final stage of T cell maturation, IL-12- and suppressor of Th2 cytokines (44). plus-IL-18 induces more CD8ϩ cells to produce IFN-␥ than CD4ϩ Surprisingly, thymocytes from NFAT1- or NFAT4-deficient ␥ cells (20 vs 10%, respectively). mice expressed similar levels of IFN- and IL-13 in response to It has been proposed that IL-12 influences intrathymic T cell IL-2-plus-IL-18 or IL-12-plus-IL-18 compared with control mice development through induction of apoptosis in immature thymo- (data not shown). Although these results rule out a role for NFAT1 ␥ cytes (5). In our model, even when the baseline of apoptosis ob- and NFAT4 in regulating IFN- and IL-13 expression by thymo- served in ATOC in unstimulated cultures is very high but expected cytes, a potential role for other NFAT family members awaits fur- based on a previous report describing ATOC (18), the IL-12-in- ther analysis. duced apoptosis observed is significantly increased when IL-18 is NF-␬B is an important transcription factor induced following added (Fig. 5A). The apoptotic effect is more prominent after IL- IL-18 signaling and is known to be involved in the regulation of 12-plus-IL-18 in vivo expression where more than half of thymo- IFN-␥ gene expression (11, 27, 32). In our analysis, thymocytes cytes (especially DN and DP cells) are lost. Moreover, the nor- from p50 KO mice demonstrated a dramatic decrease in IFN-␥ and mal thymic environment seems to be essential, because when IL-13 expression especially at the protein level, indicating a role the tissue is disrupted and cells cultured in suspension, thymo- for the p50 subunit in regulating cytokine gene expression. cytes still produce large amounts of IFN-␥ but the apoptotic Due in part of the physical proximity of the il-4, il-13, and il-5 effect is completely lost. genes, chromatin remodeling during T cell differentiation may per- Because IFN-␥ produced after CD3 stimulation has been asso- mit transcription of all these genes simultaneously (45). However, ciated with an increased apoptotic effect in human medullary thy- it is intriguing to note that, following IL-2-plus-IL-18 stimulation, mocytes (25), we questioned whether the IL-12-plus-IL-18 effect we observed expression of IL-5 and IL-13 but not IL-4. The con- on increased cell death correlates with augmented levels of IFN-␥. trol of the il-4 loci is tightly regulated at the chromatin level (45). The in vitro (ATOC) and in vivo (cDNA injections) experi- To address this question, we evaluated the levels of histone acet- ments using IFN-␥-deficient mice suggest that IL-12-plus-IL-18 ylation at sites shown previously to correlate with IL-13 and IL-4 The Journal of Immunology 2803 expression, respectively (46), in either nontreated or IL-2-plus-IL- References ϩ 18-treated bulk thymocyte cultures or in purified CD4 cells. The 1. Galy, A. H., E. M. Hadden, J. L. Touraine, and J. W. Hadden. 1989. Effects of results indicated equivalent levels of histone acetylation that were cytokines on human thymic epithelial cells in culture: IL1 induces thymic epithelial cell proliferation and change in morphology. Cell. Immunol. 124:13. not affected by the cytokine treatments (data not shown). It re- 2. Wolf, S. S., and A. Cohen. 1992. Expression of cytokines and their receptors by mains to be determined whether other mechanisms, such as DNA human thymocytes and thymic stromal cells. Immunology 77:362. methylation, are responsible for the differential gene expression 3. Yarilin, A. A., and I. M. Belyakov. 2004. Cytokines in the thymus: production and biological effects. Curr. Med. Chem. 11:447. observed in our experiments. 4. Foss, D. L., M. J. Zilliox, and M. P. Murtaugh. 2001. Bacterially induced acti- An essential event during T cell development is the continuous vation of interleukin-18 in porcine intestinal mucosa. Vet. Immunol. Immuno- migration of cells through the thymus and to the peripheral com- pathol. 78:263. 5. Godfrey, D. I., J. Kennedy, M. K. Gately, J. Hakimi, B. R. Hubbard, and partments of the immune system (16). This process is mediated in A. Zlotnik. 1994. IL-12 influences intrathymic T cell development. J. Immunol. part by the interaction of chemokines with their receptors (15, 16, 152:2729. 6. Li, L., H. C. Hsu, C. R. Stockard, P. Yang, J. Zhou, Q. Wu, W. E. Grizzle, and 47). In this study, we found that stimulation of thymocytes with J. D. Mountz. 2004. IL-12 inhibits thymic involution by enhancing IL-7- and IL-2-plus-IL-18 induces the expression of CCR4 and CCR5, IL-2-induced thymocyte proliferation. J. Immunol. 172:2909. whereas IL-12-plus-IL-18 triggers the expression of only CCR5. 7. Ludviksson, B. R., R. O. Ehrhardt, and W. Strober. 1999. Role of IL-12 in intrathymic negative selection. J. Immunol. 163:4349. Although CCR5 has been described to be weakly expressed in a 8. Whalen, B. J., J. Marounek, J. P. Mordes, A. A. Rossini, and D. L. Greiner. 2003. minor fraction of thymocytes and is nonfunctional (47), the up- Type 1 cytokines polarize thymocytes during T cell development in adult thymus regulation of CCR4 expression by IL-2-plus-IL-18 could be of organ cultures. J. Autoimmun. 20:27. 9. Yoshimoto, T., H. Mizutani, H. Tsutsui, N. Noben-Trauth, K. Yamanaka, importance considering that CCR4 is responsible for the retention M. Tanaka, S. Izumi, H. Okamura, W. E. Paul, and K. Nakanishi. 2000. IL-18 ϩ of mature cells in the thymic medulla (15). In addition, CCR4 induction of IgE: dependence on CD4 T cells, IL-4 and STAT6. Nat. Immunol. Downloaded from 1:132. coexpression with CD30 has been implicated in the process of 10. Hoshino, T., R. T. Winkler-Pickett, A. T. Mason, J. R. Ortaldo, and H. A. Young. negative selection (16). In the peripheral immune tissues, CCR4 is 1999. IL-13 production by NK cells: IL-13-producing NK and T cells are present the major chemokine receptor functionally expressed on in vitro- in vivo in the absence of IFN-␥. J. Immunol. 162:51. 11. Nakanishi, K., T. Yoshimoto, H. Tsutsui, and H. Okamura. 2001. Interleukin-18 polarized Th2 T cells (24), whereas CCR5 is preferentially ex- is a unique cytokine that stimulates both Th1 and Th2 responses depending on its pressed on cells with a Th1 polarization (48). Overall, the predom- cytokine milieu. Cytokine Growth Factor Rev. 12:53. ϩ 12. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and inant expression of CCR5 and CCR4 in DN and CD4 cells http://www.jimmunol.org/ adaptive immunity. Nat. Rev. Immunol. 3:133. correlates well with the higher expression of Th1 and Th2 cyto- 13. Carding, S. R., A. C. Hayday, and K. Bottomly. 1991. Cytokines in T-cell de- kines in these two subsets, indicating that DN and CD4ϩ thymo- velopment. Immunol. Today 12:239. 14. Mordue, D. G., F. Monroy, M. La Regina, C. A. Dinarello, and L. D. Sibley. cytes are more sensitive to express a Th1 or Th2-like phenotype 2001. Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines. after IL-12-plus-IL-18 or IL-2-plus-IL-18 stimulation, respectively J. Immunol. 167:4574. (Figs. 3 and 4). 15. Campbell, J. J., and E. C. Butcher. 2000. Chemokines in tissue-specific and microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 12:336. In this context, the thymic expression of IL-5, IL-13, and CCR4 16. Annunziato, F., P. Romagnani, L. Cosmi, E. Lazzeri, and S. Romagnani. 2001. (Th2 phenotype, preferentially mediated by IL-2-plus-IL-18) or Chemokines and lymphopoiesis in human thymus. Trends Immunol. 22:277. IFN-␥ and CCR5 (Th1 phenotype, mediated by IL-12-plus-IL-18) 17. Watanabe, M., K. L. McCormick, K. Volker, J. R. Ortaldo, J. M. Wigginton, M. J. Brunda, R. H. Wiltrout, and W. E. Fogler. 1997. Regulation of local host- by guest on September 23, 2021 could be important not only inside the thymus but also in releasing mediated anti-tumor mechanisms by cytokines: direct and indirect effects on leu- cells to the peripheral immune compartment with a Th1 or Th2 kocyte recruitment and angiogenesis. Am. J. Pathol. 150:1869. 18. Whalen, B. J., P. Weiser, J. Marounek, A. A. Rossini, J. P. Mordes, and pre-established bias. D. L. Greiner. 1999. Recapitulation of normal and abnormal BioBreeding rat T The data presented here address the possibility that endogenous cell development in adult thymus organ culture. J. Immunol. 162:4003. or exogenous production of IL-2, IL-12, and IL-18 during host 19. Farr, A. G., S. Hosier, S. C. Braddy, S. K. Anderson, D. J. Eisenhardt, Z. J. Yan, and C. P. Robles. 1989. Medullary epithelial cell lines from murine thymus con- defense and/or normal homeostasis could be responsible for the stitutively secrete IL-1 and hematopoietic growth factors and express class II production of cytokines and induction of phenotypic changes in T antigens in response to recombinant interferon-␥. Cell. Immunol. 119:427. cells during thymic maturation. This phenomenon is also im- 20. Wang, R., A. Nelson, K. Kimachi, H. M. Grey, and A. G. Farr. 1998. The role of peptides in thymic positive selection of class II major histocompatibility com- portant not only because of its potential effects on T cell de- plex-restricted T cells. Proc. Natl. Acad. Sci. USA 95:3804. velopment but also because it could represent an important 21. Hodge, D. L., W. B. Schill, J. M. Wang, I. Blanca, D. A. Reynolds, J. R. Ortaldo, and H. A. Young. 2002. IL-2 and IL-12 alter NK cell responsiveness to IFN-␥- mechanism of induction of Th phenotypes from naive precur- inducible protein 10 by down-regulating CXCR3 expression. J. Immunol. sors early in thymocyte development. Proof of this hypothesis 168:6090. awaits further experimentation. 22. Ortaldo, J. R., R. Winkler-Pickett, J. Willette-Brown, R. L. Wange, S. K. Anderson, G. J. Palumbo, L. H. Mason, and D. W. McVicar. 1999. Struc- ture/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK re- Acknowledgments ceptors. J. Immunol. 163:5269. 23. Tominaga, K., T. Yoshimoto, K. Torigoe, M. Kurimoto, K. Matsui, T. Hada, We thank Drs. John Ortaldo, Pablo Iribarren, Xia Zhang, Deborah Hodge, H. Okamura, and K. Nakanishi. 2000. IL-12 synergizes with IL-18 or IL-1␤ for and Scott Durum, and especially Dr. David M. Reynolds for helpful com- IFN-␥ production from human T cells. Int. Immunol. 12:151. ments and review of the manuscript. We also thank Della Reynolds for 24. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, expert technical assistance, Mike Sanford for performing ELISA and RPA S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, and F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness analysis, Mehrnoosh Abshari for flow cytometry sorting, and John Wine of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129. for his support in animal care and experimentation. The IL-2 neutralizing 25. Groux, H., D. Monte, B. Plouvier, A. Capron, and J. C. Ameisen. 1993. 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