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Cutting Edge: Differential Expression of in Th1 and Th2 Cells Is Dependent on Stat6 But Not Stat41

Shangming Zhang,* Nicholas W. Lukacs,† Victoria A. Lawless,* Steven L. Kunkel,† and Mark H. Kaplan2*

Although the exact nature of the STAT activated genetic The in vivo function of Th cell subsets is largely dependent on ؉ program is unknown, it has been demonstrated that other the ability of differentiated CD4 T cells to be recruited to than are differentially expressed between Th1 and Th2 specific sites and secrete restricted sets of cytokines. In this cells. Several of these are receptors including CXCR3, paper we demonstrate that Th1 and Th2 cells secrete discrete CCR5, and CCR7, preferentially expressed in Th1 cells (9–11), patterns of chemokines, small m.w. cytokines that function as and CCR3, CCR4, and CCR8, which are preferentially expressed chemoattractants in inflammatory reactions. Th2 cells secrete in Th2 cells (9, 10, 12, 13). This provides a mechanism by which -derived chemokine and activation 3, Th subsets can be selectively recruited to sites of inflammation. and acquisition of this pattern of expression is dependent on Indeed, Th1 cells selectively undergo chemotaxis to IFN-␥-induc- Stat6. In contrast, Th1 cells secrete lymphotactin and RAN- ible protein (IP-10), a for CXCR3, and RANTES, macro- TES, though unlike IFN-␥, expression of these chemokines is phage inflammatory protein 1␣ (MIP-1␣)3 and MIP-1␤, ligands independent of Stat4. We further show that supernatants from for CCR5 (9, 14). Similarly, Th2 cells selectively undergo chemo- activated Th2 cells preferentially induce the chemotaxis of Th2 taxis to macrophage-derived chemokine (MDC), T cell activation over Th1 cells, corresponding with Stat6-dependent expression gene 3 (TCA3), and - and activation-regulated chemokine of CCR4 and CCR8 in Th2 cells. These data provide the basis (TARC), chemokines that are ligands for CCR4 and CCR8 (9, 13, for restricted and direct T cell-mediated cellular recruitment 15–17). to sites of inflammation. The Journal of Immunology, 2000, Because Th subsets are important in regulating inflammatory 165: 10–14. processes, it seemed likely that they would also secrete discrete patterns of chemokines to recruit restricted types of cells to sites of inflammation. Although chemokine expression in Th sub- ifferentiated CD4ϩ Th cells can be divided into Th1 and sets has been extensively described, restricted expression of che- Th2 subsets based on their production follow- mokines between Th subsets has not been carefully examined. We D ing Ag stimulation (1, 2). Th1 cells produce IFN-␥ and have determined the expression patterns of 18 chemokines and mediate delayed-type hypersensitivity and protection against in- shown that there is restricted expression of chemokines between tracellular pathogens. The Th2 subset produces IL-4, IL-5, IL-10, Th1 and Th2 cells that is dependent on the function of Stat6 but not and IL-13 and is implicated in humoral and allergic responses. The Stat4. We further demonstrate that Th2 supernatants preferentially differentiation of these Th subsets is dependent on cytokine-stim- recruit Th2 rather than Th1 cells. ulated genetic programs. IL-12-activated Stat4 is required for the development of fully functional Th1 cells (3–5). Similarly, IL-4- Materials and Methods activated Stat6 is essential for the differentiation of Th2 cells Mice (6–8). The generation of Stat4- and Stat6-deficient mice has been previously de- scribed (3, 6). Both strains have been backcrossed 10 generations to the BALB/c genetic background and were bred as homozygotes in the Indiana University Laboratory Animal Resource Center. Wild-type BALB/c mice *Department of Microbiology and Immunology, Walther Oncology Center, Indiana were purchased from Harlan Bioproducts (Indianapolis, IN). University School of Medicine, and Walther Cancer Institute, Indianapolis, IN 46208; † and Department of Pathology, University of Michigan Medical School, Ann Arbor, T cell differentiation MI 48109 ϩ ϩ ϩ ϩ Received for publication March 1, 2000. Accepted for publication May 1, 2000. Total spleen cells were depleted of CD8 , B220 , and FcR cells. CD4 cells were positively selected from the resulting populations using mag- The costs of publication of this article were defrayed in part by the payment of page netic beads from Miltenyi Biotec (Auburn, CA) according to the manu- charges. This article must therefore be hereby marked advertisement in accordance ϩ Ͼ ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. facturer’s instructions. Final CD4 populations were 97% CD4 as de- termined by FACS analysis. CD4ϩ T cells cultured at 106 cells/ml were 1 M.H.K. is a Special Fellow of the Leukemia and Lymphoma Society. This work was then differentiated with 2 ␮g/ml plate-bound anti-CD3 (145-2C11) plus 1 supported by a grant from the National Institutes of Health (AI45515) to M.H.K. 2 Address correspondence and reprint requests to Dr. Mark H. Kaplan, Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School 3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; MDC, of Medicine, 1044 West Walnut Street, Room 302, Indianapolis, IN 46202. E-mail macrophage-derived chemokine; TARC, thymus- and activation-regulated chemo- address: [email protected] kine; MIG, monokine induced by IFN-␥; RPA, RNase protection assay.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

● The Journal of Immunology 11

␮g/ml anti-CD28 (PharMingen, San Diego CA) to Th1 (1 ng/ml IL-12 (Peprotech, Rocky Hill, NJ) and 10 ␮g/ml anti-IL-4 (11B11)) or Th2 (10 ng/ml IL-4 (Peprotech) plus 10 ␮g/ml anti-IFN-␥ (R4/6A2)) populations. After 6 days, cells were restimulated with 2 ␮g/ml plate-bound anti-CD3 for the indicated times for RNA analysis or for 24 h to recover supernatants. Chemokine mRNA analysis Total RNA samples were isolated from cell populations indicated using Trizol (Life Technologies/BRL, Bethesda, MD). RNase protection assays (RPA) were done according to the manufacturer’s instructions (PharMin- gen). cDNA probes for monokine induced by IFN-␥ (MIG), TARC, and CCR8 were generated by PCR, subcloned, and sequenced to confirm the identity of the probes. The cDNA probes for MDC (a gift of Dr. Uli Schin- dler, Tularik, South San Francisco, CA), Breast And Kidney, Exodus-1, -2 and -3 (gifts of Dr. Rob Hromas, Indiana University, Indianapolis, IN), and CCR4 (a gift of Dr. Byung Youn, Indiana University, Indianapolis, IN), and PCR probes were labeled using random decamers (Ambion, Austin, TX) and hybridized overnight in a formamide hybridization buffer. ELISAs ELISAs for IL-4 and IFN-␥ were performed as described previously (5, 6). Rabbit anti-murine MDC Abs were prepared by multiple-site immuniza- tion of New Zealand White rabbits with recombinant murine MDC (R&D Systems, Rochester, MN) in CFA. Polyclonal Abs were titered by direct ELISA and specifically verified by the failure to cross-react to other che- mokines. The IgG portion of the serum was purified over a protein A column and used in a sandwich ELISA. Goat anti-murine RANTES was made as previously described (18). The levels of chemokines in cell-free supernatants were measured by specific ELISA using a modification of a double-ligand method as previously described (19). Chemotaxis assay Chemotaxis assays were performed in a Costar (Cambridge, MA) Trans- well with a 3-␮m pore filter. Assays were performed in chemotaxis buffer (RPMI 1640, 1% BSA, and 20 mM HEPES). Supernatants from the indi- cated Th population were diluted 1:2 with chemotaxis buffer in the lower chamber and 5 ϫ 105 Th2 cells in the upper chamber. After 4 h, cells in the FIGURE 1. Expression of chemokines in Th1 and Th2 populations. A, ␥ ϩ ϩ lower chamber were counted using trypan blue exclusion. Rabbit anti- ELISA of IFN- and IL-4 expression in differentiated CD4 cells. CD4 MDC serum was purified using protein G and used in the assay at 10 cells were purified from wild-type, Stat4-deficient, and Stat6-deficient ␮g/ml. Anti-TCA3 was purchased from PharMingen. Control Ig was pu- spleens and differentiated to Th1 or Th2 populations as indicated. After 6 rified by protein G from normal rabbit serum. Supernatants were incubated days in culture, T cells were restimulated with anti-CD3 for 24 h and with anti-MDC for1honicebefore adding to Transwell culture. supernatants were recovered for analysis by ELISA. B, RPA of RNA from differentiated CD4ϩ cells. CD4ϩ cells, differentiated as in A, were acti- Results and Discussion vated with anti-CD3 and RNA was isolated at the indicated times. The Differential expression of chemokines in Th1 and Th2 cells protection assay was performed with the mCK-5 templates (PharMingen). Labels on the left indicate the probe for each chemokine. Labels on the Because chemokines are known to recruit cells to sites of inflam- right indicate the protected fragment following RNase digestion. The re- mation, and because Th subsets mediate distinct types of inflam- sults are representative of five experiments. mation, we examined whether Th1 and Th2 cells secreted distinct sets of chemokines. CD4ϩ T cells (Ͼ97% pure), purified from wild-type spleen cells, were differentiated to Th1 or Th2 pheno- types. After 1 wk in culture, cells were restimulated with anti-CD3. of Stat4-deficient lymphocytes to mediate reduced but significant Restimulation of Th1 and Th2 cultures resulted in secretion of tissue inflammation (5). IFN-␥ and IL-4, respectively (Fig. 1A). RPA were used to assess We also tested the differential expression of several other che- the expression of a panel of chemokines in cultures that were un- mokines by Northern blot analysis. Th cells were differentiated as stimulated or activated for the time periods indicated with anti- above and RNA was isolated at the time points indicated following CD3. Expression of RANTES and lymphotactin was induced by stimulation with anti-CD3. Of the chemokines tested, MDC (also anti-CD3 specifically in Th1 populations (Fig. 1B). By contrast, known as STCP-1 and ABCD-1) was inducibly expressed specif- Th2 cells selectively expressed TCA3 following stimulation with ically in Th2 cells and not in Th1 cells (Fig. 2A). Because IL-4 anti-CD3. To determine whether expression of these chemokines induces, and IFN-␥ represses, MDC expression from macro- in Th subsets was dependent on the genetic programs activated by phages, dendritic cells and B cells (16, 20, 21) we tested whether Stat4 and Stat6, we performed the differentiation described above anti-CD3 induced cytokines were responsible for Th2-restricted with CD4ϩ T cells from mice deficient in either Stat4 or Stat6. MDC expression. Wild-type Th2 cells restimulated with anti-CD3 These cultures secreted cytokines as predicted from previous re- in the presence of anti-IL-4 showed that the absence of IL-4 re- sults (Fig. 1A). TCA3 expression was dependent on Stat6, as Stat6- duced but did not abrogate MDC expression (Fig. 2B). Thus it is deficient CD4ϩ Th2 cultures did not express TCA3 (Fig. 1B). Sur- likely that both the anti-CD3 and IL-4 signals contribute to MDC prisingly, and in contrast to IFN-␥, RANTES and lymphotactin expression in Th2 cells. Th1 cells stimulated with anti-CD3 ex- were expressed in Stat4-deficient Th1 cultures (Fig. 1B). This sug- pressed barely detectable levels of MDC and even in the presence gests that Stat4 is not required for RANTES and lymphotactin of neutralizing anti-IFN-␥ were not inducible by IL-4 (Fig. 2B). expression in Th1 populations and also correlates with the ability The restricted expression of MDC was also dependent on Stat6, 12 CUTTING EDGE

these chemokines than Th2 cells. Importantly, expression of both MIP-1␣ and MIP-1␤ was readily detectable in Th2 cells (Fig. 1B). This difference was also reflected in ELISAs where MIP-1␣ was readily detectable in supernatants of Th2 cells, but accumulated in Th1 supernatants at 2- to 5-fold higher levels than in Th2 super- natants (data not shown). Furthermore, the absence of either Stat4 or Stat6 had only minor affects on the expression of MIP-1␣ and MIP-1␤ (Fig. 1B) or the secretion of MIP-1␣ (data not shown). Other chemokines had undetectable expression in either Th1 or Th2 cells. These included chemokines that were analyzed by RPA in Fig. 1B (eotaxin, monocyte chemoattractant protein-1 (MCP-1), MIP-2, and IFN-␥-inducible protein (IP-10)), Northern blot anal- ysis (MIG, TARC, Exodus-1 (LARC/MIP-3␣), Exodus-2 (TCA4/ SLC/6Ckine), Exodus-3 (ELC/MIP-3␤/CK␤11) and BRAK; data not shown), and ELISA (C10 and MCP-3; data not shown). Th2 supernatants preferentially recruit Th2 cells The chemokines that are preferentially expressed in Th2 cells are also ligands for receptors that are differentially expressed between Th subsets. The Th2 chemokines MDC and TCA3 signal through FIGURE 2. Expression of MDC in Th1 and Th2 populations. A, North- CCR4 and CCR8, respectively, which are also expressed at greater ern blot analysis of MDC expression. CD4ϩ T cells were differentiated and levels in Th2 cells than in Th1 cells and are expressed in a Stat6- restimulated with anti-CD3 for the time periods indicated as in Fig. 1. RNA was isolated, run on formaldehyde agarose gels, transferred to nylon mem- dependent manner (Fig. 4A). This led us to the prediction that brane, and probed with a MDC cDNA. Filters were stripped and reprobed Th2-generated chemokines would be more effective at stimulating with a TCR␣ probe as a loading control. B, Wild-type or Stat6-deficient T the chemotaxis of Th2 cells. To test this, we used Th2 supernatants cells were differentiated as in A and were left unstimulated or stimulated generated as above, in a standard, two-chamber migration assay. with anti-CD3 for 24 h in the presence or absence of 10 ␮g/ml anti-IFN-␥, As shown in Fig. 4B, Th2 supernatants were significantly more 1or10␮g/ml anti-IL-4, or 10 ng/ml IL-4 as indicated. Northern blot effective in stimulating chemotaxis in Th2 cells than were Th1 analysis was performed as in A. The results shown are representative of supernatants or media alone ( p Ͻ 0.01). Th2-induced chemotaxis four experiments for A and two experiments for B. could be inhibited by coincubation of the supernatant with anti- MDC, demonstrating that Th2-derived MDC is an important me- diator of Th2 chemoattraction. Incubation with anti-TCA3 did not because Stat6-deficient Th2 cultures did not express MDC even significantly affect Th2 migration in response to Th2 supernatants. when supplemented with IL-4 (Fig. 2B). Thus, Th2-derived chemokines can differentially recruit Th To demonstrate that the differentially expressed chemokines subsets. were indeed being secreted, we also performed ELISAs on super- This report describes the differential expression of specific CC natants from Th1, Th2, Stat4-deficient Th1, and Stat6-deficient chemokines in Th1 and Th2 subsets. The issue of differential ex- Th2 cultures stimulated for 24 h with anti-CD3. Secretion of RAN- pression of chemokines in Th subsets has been addressed in sev- TES and MDC was detectable in supernatants (Fig. 3) with a sim- eral reports with differing results. MDC expression has been ob- ilar restricted pattern of expression as seen in RPA and Northern served in neither or both Th1 or Th2 populations/clones (16, 25, blot analysis (Figs. 1 and 2). 26). TCA3 was expressed in transgenic Th2 cell populations but was also seen expressed in both Th1 and Th2 clones (27, 28). Common expression of chemokines in Th1 and Th2 cells MIP-1␣, MIP-1␤, and RANTES have also been shown to be both MIP-1␣ and MIP-1␤ are often associated with Th1-mediated in- differentially and similarly expressed in Th1 and Th2 populations flammatory lesions (22–24). However, our RPA analysis demon- (25, 28). Some of these discrepancies may be simply explained by strated that Th1 cells express only about 2-fold more RNA for the use of methods that were not quantitative enough to distinguish preferential expression in Th subsets or by differences in cultured primary T cells vs long-term T cell clones. Our report describes the systematic and quantitative examination of the expression of 18 chemokines in primary, polyclonally activated, and purified CD4ϩ cells that have been polarized to the Th1 or Th2 phenotype. The differential dependence on STAT for the acquisi- tion of chemokine secreting phenotypes is distinct from that re- quired for cytokines. Although Stat6 appears to be necessary for the development of Th2 cells and secretion of IL-4, MDC, and TCA3, there is not the same requirement for Stat4. In the absence of Stat4, Th1 cells can still acquire a chemokine-secreting pheno- type that includes RANTES and lymphotactin. We have previously suggested that Th1-like populations can be generated in the ab- FIGURE 3. Secretion of RANTES and MDC by Th1 and Th2 popula- sence of Stat4 (5). However, this observation was complicated by ϩ ␥ tions. CD4 T cells of the indicated genotypes were differentiated and the direct effects of Stat4 on IFN- expression, which served as the restimulated with anti-CD3 for 24 h as in Fig. 1A. Supernatants were re- sole marker for Th1 cells (3, 5). Using RANTES and lymphotactin covered and tested by ELISA for levels of RANTES and MDC. Results are as representative chemokines of Th1 and not Th2 cells, we provide representative of three experiments. further evidence here that cells with some characteristics of Th1 The Journal of Immunology 13

photactin may act primarily to recruit CD8ϩ cytotoxic cells to inflamed sites. These observations provide the basis for under- standing specific T cell-mediated recruitment of leukocytes to sites of inflammation. They also suggest additional targets for modu- lating chemokine-mediated inflammation in vivo.

Acknowledgments We thank Ulrike Schindler, Byung Youn, and Rob Hromas for providing cDNA probes and Michael Grusby for providing Stat4Ϫ/Ϫ and Stat6Ϫ/Ϫ breeding pairs. We also thank Drs. Alexander Dent and Hal Broxmeyer for critical review of this manuscript.

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