Th1 and Th2 Cell Differentiation Polarizing Cytokine Determines The

Responsiveness of Naive CD4 T Cells to Polarizing Cytokine Determines the Ratio of Th1 and Th2 Cell Differentiation

This information is current as Natallia Mikhalkevich, Brian Becknell, Michael A. Caligiuri, of October 2, 2021. Michael D. Bates, Richard Harvey and Wei-ping Zheng J Immunol 2006; 176:1553-1560; ; doi: 10.4049/jimmunol.176.3.1553 http://www.jimmunol.org/content/176/3/1553 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Responsiveness of Naive CD4 T Cells to Polarizing Cytokine Determines the Ratio of Th1 and Th2 Cell Differentiation1

Natallia Mikhalkevich,* Brian Becknell,† Michael A. Caligiuri,† Michael D. Bates,‡ Richard Harvey,§¶ and Wei-ping Zheng2*

The intrinsic features of naive CD4 T cells that affect their ability to respond to polarizing signals for Th cell differentiation are not well understood. In this study, we show that naive CD4 T cells from mice transgenic for the Hlx gene expressed lower levels of IL-4R␣. The down-regulation of IL-4R␣ diminished IL-4 signaling and the Th2 response and enhanced the Th1 response under suboptimal polarizing conditions. In nontransgenic CD4 T cells, blocking IL-4R␣ with Abs had the same effect in an Ab dose- dependent manner. Conversely, Hlx haploinsufficiency caused higher expression of IL-4R␣ to favor Th2 cell differentiation. Thus, the IL-4R␣ level on naive CD4 T cells is genetically controlled by Hlx and determines the ratio of Th1 and Th2 cell differentiation. The Journal of Immunology, 2006, 176: 1553–1560. Downloaded from he dichotomy of type 1 and type 2 Th cells stands as a GATA-3 is up-regulated during Th2 cell differentiation (4). These central paradigm in cellular immunology that provides the two transcription factors, respectively, function as master regula- T framework to understand the nature of an immune re- tors of the differentiation of Th1 and Th2 cells (3–9). Additionally, sponse that is either beneficial or detrimental to the host (1, 2). Th1 Th1 cells specifically express the transcription factor Hlx and Erm and Th2 cells are characterized by their mutually exclusive ex- (10–12), and Th2 cells specifically express c-Maf (13), to enhance ␥ pression patterns of cytokines. Th1 cells produce IFN- , whereas differentiation and effector cytokine expression. http://www.jimmunol.org/ Th2 cells produce IL-4, IL-5, and IL-13 (2). Functionally, Th1 Cell surface molecular interaction is an important means of de- responses are required for the clearance of intracellular infections, livering the initial polarizing signals. Early studies have shown that and Th2 responses are required for the clearance of helminth in- different strengths of TCR ligation and duration could dictate Th1 fection. The failure to generate the appropriate Th cell responses is or Th2 cell differentiation (14). Numerous studies have also doc- often the cause of chronic infectious diseases. Under autoimmune umented costimulatory and accessory interactions of CD28/B7, conditions, polarized Th1 and Th2 responses are associated with ICOS/B7h, OX40/OX40 ligand, and LFA-1/ICAM to regulate Th organ-specific autoimmune diseases and allergies, respectively. cell differentiation. More recently, the Notch ligands Delta and Therefore, the mechanisms for controlling the differentiation of Jagged expressed on APC are found to induce Th1 and Th2 cell Th1 and Th2 cells from their naive precursors are crucial to our by guest on October 2, 2021 differentiation, respectively (15). understanding of the complex regulation of immune responses under these pathological conditions. Apart from the surface molecules, cytokines in the environment Studies in the past have established an instruction model of Th in which naive CD4 T cells encounter their specific Ags provide cell differentiation. In this model, surface molecules on the APC or potent polarizing signals for Th subset differentiation (16). Al- cytokines in the surrounding tissue environment deliver polarizing though other cytokines have been reported to regulate Th cell dif- signals to naive CD4 T cells upon Ag stimulation. Such polarizing ferentiation, the best understood and perhaps the most important signals induce up-regulation of transcription factors that drive the polarizing cytokines are IFN-␥, IL-4, and IL-12. Interestingly, differentiation of Th1 and Th2 cells. During Th1 cell differentia- IFN-␥ and IL-4 are both the effector cytokines and the inducers of tion, the transcription factor T-bet is up-regulated (3), whereas the differentiation of their own producers (17–21). IFN-␥, through Stat1 activation, induces T-bet expression (18, 22, 23), whereas IL-4, through Stat6 activation, up-regulates GATA-3 expression *David H. Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical Sciences, Department of Microbiology and Immunology, University of (4, 6, 24–26). IL-12 activates Stat4, which plays an important role Rochester School of Medicine and Dentistry, Rochester, NY 14642; †Ohio State in Th1 cell differentiation because Th1 response in mice deficient ‡ University Comprehensive Cancer Center, Columbus, OH 43210; Division of Gas- in Stat4 or IL-12 is severely diminished (27, 28). In fact, IL-12 and troenterology, Hepatology, and Nutrition, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229; §Victor IL-4 are the polarizing cytokines typically used for in vitro differ- Chang Cardiac Research Institute, St. Vincent’s Hospital, Darlinghurst, Australia; and entiation of Th1 and Th2 cells, respectively. ¶Faculties of Medicine and Life Sciences, University of New South Wales, Kensing- ton, Australia The polarizing cytokines are derived from multiple sources. NK Received for publication August 17, 2005. Accepted for publication November cells (29, 30) and a special population of acute responding CD8 T 10, 2005. cells (31) could provide the initial IFN-␥ for Th1 cell differenti- The costs of publication of this article were defrayed in part by the payment of page ation. An important source of early IL-4 is the naive CD4 T cells charges. This article must therefore be hereby marked advertisement in accordance themselves, which can produce IL-4 immediately upon TCR stim- with 18 U.S.C. Section 1734 solely to indicate this fact. 1 ulation to induce their own differentiation to Th2 cells (17, 21, 32). This work is supported by National Institutes of Health Grants AI47263 and ϩ AI53745 (to W.Z.) and CA95426 and CA68458 (to M.A.C.). Other potential sources of the early IL-4 include the NK1.1 CD4 Ϫ 2 Address correspondence and reprint requests to Dr. Wei-ping Zheng, David H. T cells (33), a special population of NK1.1 CD4 T cells (34, 35), Smith Center for Vaccine Biology and Immunology, Aab Institute of Biomedical mast cells (36), and eosinophils (37, 38). IL-12 is produced by Sciences, Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Box 609, 601 Elmwood Avenue, Rochester, NY APC, such as macrophages and dendritic cells (39–43). Studies 14642. E-mail address: [email protected] using infectious disease models have shown that at least some

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 1554 GENETIC CONTROL OF IL-4R␣ EXPRESSION IN NAIVE CD4 T CELLS infectious agents can induce acute production of polarizing cyto- Technologies) according to the manufacturer’s instructions. Forty-eight kines to skew the Th response toward Th1 or Th2. For example, hours after transfection, culture supernatants were harvested and centri- ϫ Th2 responses to Leishmania major and Schistosoma mansoni in- fuged at 6000 g to concentrate the viruses. For T cell infection, naive CD4 T cells were stimulated for 12 h. Culture supernatants were removed fection could be significantly attributed to an early burst of IL-4 and kept at 4°C. Cells were then mixed with freshly prepared viruses plus secretion from a population of CD4 T cells expressing the 8 ␮g/ml polybene (American Bioanalytical) 0.5 ng/ml IL-4, and 50 U/ml V␤4V␣8 TCR and eosinophils, respectively (34, 44). In contrast, IL-2, then centrifuged at 1800 rpm for 45 min at room temperature. After intracellular infection of Listeria or Toxoplasma stimulates IL-12 12 h of incubation at 37°C, viruses were removed, and the saved culture supernatants with fresh IL-4 were added back to the cells. expression in macrophages and IFN-␥ expression in NK cells to instruct Th1 cell differentiation (39, 40, 45). Flow cytometry As supported by these studies, the current instruction model of For intracellular cytokine staining, differentiated CD4 T cells were acti- Th cell differentiation emphasizes the creation of a strong polar- vated with PMA (50 ng/ml) and ionomycin (1 ␮M), and stained for IL-4, izing cytokine environment to induce a dominant Th1 or Th2 re- IFN-␥, and CD4 or other surface Ags as previously described (12). For sponse. Less attention has been devoted to the contribution of the analysis of cell surface expression of IL-4R␣, total CD4 T cells prepared intrinsic features of the responding naive CD4 T cells to the dif- by negative selection with magnetic beads were first incubated with Fc block (BD Pharmingen), followed by incubation with biotinylated anti- ferentiation outcome. However, in many immune responses, po- mouse IL-4R␣ Ab. After washing, the cells were incubated with strepta- larizing cytokines for one subset may not always dominate over vidin-PE (BD Pharmingen), anti-CD4-allophycocyanin, anti-CD44- those for the other subset. Under such weak polarizing conditions, PE-Cy5, and anti-CD62L-FITC (eBioscience). All stained cells were polarizing cytokines for Th1 or Th2 cell differentiation probably analyzed on a FACSCalibur. coexist and compete to influence Th cell differentiation. In previ-

Real-time RT-PCR Downloaded from ous studies (10, 12), we and others found that the homeobox transcription factor Hlx is speciﬁcally expressed in Th1 cells and not Total RNA were isolated from FACS-sorted naive CD4 T cells using an Ultraspec-II RNA isolation system (Biotecx) and reverse transcribed with in Th2 cells. Consistent with its speciﬁc expression in Th1 cells, SuperScript II reverse transcriptase (Invitrogen Life Technologies) accord- overexpression of Hlx in CD4 T cells promoted Th1 cell differ- ing to the manufacturer’s instructions. Primers were purchased from Inte- entiation. However, Hlx is also expressed in naive CD4 T cells at low grated DNA Technologies. Sequences of the primers are as follows: gac levels. In this study we show that Hlx in naive CD4 T cells genetically cga atc tct gtg tac tat aat g and cca gac tga ccg aaa acg aag t for ␤-tubulin down-regulates IL-4R␣ expression in naive CD4 T cells. The genet- V, gtt act tca gca tta cca aga tta ag and agt ttt cca gtg cgg gta ctt g for http://www.jimmunol.org/ IL-4Ra, tgc cat ggt gcc acc tta tg and gtg agg ctc ctg aaa aga tg for Stat6, ically predetermined IL-4R␣ level dictates the ratio of Th1 and Th2 cat cgg cat ttt gaa cga ggt ca and gct acg gac cta agt agc tat tc for IL-4, and cell differentiation under weak polarizing conditions. gta cta caa gct tca taa tat taa c and ggt tga agg agc tgc tct tg for GATA-3. PCR were set up with the 2ϫ SYBR Green real-time PCR Master Mix Materials and Methods (Applied Biosystems). The reactions were run on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Data were analyzed Mice with the SDS2.0 software. Transcripts of each gene relative to the ␤-tu- Ϫ⌬Ct CD4-Hlx transgenic mice were described previously (12) and maintained bulin V gene were determined with the 2 method (47). as heterozygous by crossing to C57BL/6 mice. FVB Hlxϩ/Ϫ mice were ϩ/Ϫ Western blot

derived from the originally reported Hlx mice (46) by crossing to the by guest on October 2, 2021 wild-type FVB mice for Ͼ10 generations. Inbred mice were purchased Cells were lysed in lysis buffer (300 mM NaCl, 10 mM Tris-HCl (pH 7.5), from the National Institutes of Health. All animal studies were conducted 1 mM EDTA, 1 mM EGTA, 0.2 mM Na3VO4, 1 mM sodium fluoride, 50 in accordance with procedures approved by the University of Rochester mM ␤-glycerol phosphate, 1% Triton X-100, and 0.5% Nonidet P40). The committee for animal research. cell lysates were directly used for Western blot analyses. Primary Abs are goat anti-mouse actin (Santa Cruz Biotechnology), rabbit anti-mouse Stat6 Preparation of CD4 T cells and APC Ab (Cell Signaling Technology), and rabbit anti-Hlx Abs. The rabbit poly- Total CD4 T cells were isolated by negative selection with magnetic beads clonal anti-Hlx Abs were raised against a synthetic peptide of Hlx and as previously described (12). APC were prepared by depleting T and NK cross-reacted to the human Hlx homologue HB24. After the primary Ab cells in spleen and lymph node cells and were irradiated with 3000 rad. As incubation, the protein blots were incubated with HRP-conjugated rabbit specified in the experiments, naive CD4 T cells were isolated from total anti-goat IgG (Sigma-Aldrich) or goat anti-rabbit IgG (Santa Cruz Bio- CD4 T cells either by MACS sorting of the CD62Lhigh population (12) or technology). The proteins were then detected using Western Lightening FACS sorting of the CD4ϩCD44lowCD62Lhigh population from the total Chemiluminescence Reagent Plus (PerkinElmer). CD4 T cell preparations. Immunoprecipitation Th cell differentiation Cell lysates were diluted with an equal volume of dilution buffer (10 mM ␤ Cultures for Th cell differentiation were set up essentially as previously Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 50 mM -glycerol phos- described (4). Briefly, equal numbers of naive CD4 T cells and APC (5 ϫ phate, and 20% glycerol) plus proteinase inhibitors. The diluted lysates 105/ml) were stimulated with Con A (2.5 ␮g/ml; Roche) in RPMI 1640 were incubated with anti-Stat6 Ab. The protein-Ab complexes were pulled plus 10% FBS and 50 U/ml IL-2 (Chiron). As specified in each experiment, down with protein G beads (Amersham Biosciences) and washed with the anti-IL-4 Abs (11B11) or exogenous IL-4 were added to the cultures at the diluted lysis buffer. Protein blots were probed with HRP-conjugated anti- indicated concentrations. After 4 days of differentiation, the cells were phospotyrosine Ab 4G10 (Upstate Biotechnology). rested in fresh medium plus 50 U/ml IL-2 for 1–1.5 days before intracellular cytokine analysis. Results Effect of the Hlx transgene on Th cell differentiation in the Retroviral infection of T cells absence of exogenous polarizing cytokines The retroviral vector MigR1 is gift from Drs. W. Pear and S. Reiner (De- The transcription factor Hlx is expressed in naive CD4 T cells and partment of Pathology and Laboratory Medicine, University of Pennsyl- vania, Philadelphia, PA). The IL-4R␣-MigR1 for bicistronic expression of specifically up-regulated during Th1 cell differentiation (10, 12). In IL-4R␣ and GFP is a gift from Dr. M. Boothby (Department of Microbi- an earlier study, we found that transgenic mice expressing Hlx ology and Immunology, Vanderbilt University School of Medicine, Nash- under a CD4 promoter generated more Th1 cells than their wild- ville, TN). The packaging cell line Phoenix Eco was originally developed type littermates when immunized with the protein Ag keyhole lim- in Dr. G. Nolan’s laboratory (Department of Molecular Pharmacology, Stanford University Medical Center, Stanford, CA) and was purchased pet hemocyanin. However, when naive CD4 T cells were stimu- from American Type Culture Collection. The packaging cells were translated to differentiate in vitro under extreme polarizing conditions fected with retroviral vectors using LipofectaMINE 2000 (Invitrogen Life for Th1 cell differentiation, no differences in Th cell differentiation The Journal of Immunology 1555 were observed between wild-type and transgenic CD4 T cells (12). Western blot analysis of 293T cells transfected with Hlx expres- We reasoned that the extreme polarizing conditions used in the in sion vector. As shown in Fig. 1b (left), a strong band was detected vitro cultures might have masked the effect of Hlx on Th cell in the lysate of 293T cells transfected with the Hlx expression differentiation under less polarizing conditions, which was proba- vector. A weak band was also detected in 293T cells transfected bly the case for the in vivo environment. To better mimic the in with the empty vector due to endogenous expression of the human vivo conditions, we compared in vitro Th cell differentiation of wild- Hlx homologue HB24, which could also be detected by RT-PCR type and transgenic naive CD4 T cells in the absence of exogenous (data not shown). When the Abs were used to examine the expres- IL-4, IL-12, and neutralizing Abs. After differentiation, the cells were sion of Hlx in Hlx transgenic naive CD4 T cells, a relatively high restimulated for intracellular cytokine staining to detect Th1 and Th2 level of expression was detected. Consistent with previous analysis cells (Fig. 1a, upper panel). A markedly higher percentage of Th1 with RT-PCR (10, 12), a relatively low level of Hlx was also cells was detected in the Hlx transgenic CD4 T cells than in the wild- detected in the wild-type naive CD4 T cells (Fig. 1b, right). type CD4 T cells. Conversely, a significant number of Th2 cells were ␣ observed in the wild-type CD4 T cells, but only a minimum number Down-regulation of IL-4R in Hlx transgenic naive CD4 T cells of Th2 cells in the transgenic CD4 T cells. Thus, the Hlx transgene not Our data have indicated that endogenous IL-4 signaling is key to only enhanced Th1, but also diminished Th2 cell differentiation. This the difference in the ratio of Th1 and Th2 cells between transgenic effect was naive CD4 T cell intrinsic because the same wild-type APC and wild-type CD4 T cells differentiated under the unmanipulated, were used in the differentiation cultures of both wild-type and trans- weak, polarizing condition. To uncover the underlying mechanism, genic CD4 T cells. we examined whether the early expression of IL-4 would be dif- We suspected that the use of anti-IL-4-neutralizing Abs in the in ferent and responsible for the different outcomes. Naive CD4 T vitro cultures for extreme polarization of Th1 cell differentiation cells were stimulated in the absence of exogenous IL-4 and IL-12. Downloaded from might be responsible for the lack of difference between transgenic and Thirty-four hours later, CD4 T cells were sorted for RNA extrac- wild-type T cells after differentiation. To test this, we set up cultures tion and real-time RT-PCR. Albeit at low levels, IL-4 messages with anti-IL-4 neutralizing Abs, but without exogenous IL-12. Anti- were detected in both wild-type and transgenic CD4 T cells. The IL-4 Abs completely blocked the generation of IL-4 single-producer IL-4 message level of wild-type CD4 T cells was not higher, but and IL-4, IFN-␥ double-producer cells in both wild-type and trans- instead slightly lower than the transgenic CD4 T cells (Fig. 2a). genic cells (Fig. 1a, lower panel), so that only Th1 cells were de- Based on these data, we believed that the amount of early en- http://www.jimmunol.org/ tected. The percentages of Th1 cells were essentially the same be- dogenous IL-4 was unlikely to be responsible for the different out- tween wild-type and transgenic CD4 T cells differentiated in the comes of differentiation between wild-type and transgenic naive presence of anti-IL-4 Abs. Therefore, anti-IL-4 Ab is sufficient to CD4 T cells. We then suspected that signaling potentials of the abolish the differences between wild-type and transgenic CD4 T cells wild-type and transgenic naive CD4 T cells might be different. To differentiated under unmanipulated conditions. test this possibility, we analyzed the expression of IL-4R␣ and To verify the difference in Hlx expression between wild-type Stat6 in wild-type and Hlx transgenic naive CD4 T cells by real- and transgenic naive CD4 T cells, we performed Western blot time RT-PCR. Although no difference in Stat6 expression was de- analysis of Hlx expression. Rabbit polyclonal Abs were raised tected between wild-type and transgenic naive CD4 T cells, the by guest on October 2, 2021 against an Hlx peptide. The specificity of the Ab was confirmed by IL-4R␣ level in the transgenic naive CD4 T cells was ϳ4-fold

FIGURE 1. Comparison of Th cell differentiation in the absence of exogenous polarizing cytokines and Hlx expression between wild-type and Hlx transgenic naive CD4 T cells. a, MACS-sorted naive CD4 T cells (CD4ϩCD62Lhigh) from wild-type and Hlx transgenic mice were stimulated with Con A (2.5 ␮g/ml) plus T and NK cell-depleted APC and IL-2 (50 u/ml) either without (upper panel) or with anti-IL-4 Abs (lower panel)inthe absence of exogenous IL-4 or IL-12. After 4 days of differentiation, the cells were rested for 1 day and restimulated with PMA and ionomycin. Dot plots of intracellular staining of IL-4 and IFN-␥ on gated CD4 T cells are shown. Numbers in the plots are percentages of each cell populations. b, Detection of Hlx proteins in naive CD4 T cells. The speciﬁcity of rabbit anti-mouse Hlx polyclonal Abs was tested by Western blot using cell lysates of 293T transfected with MigR1 GFP, or MigR1 Hlx. Proteins were separated by PAGE. Actin and Hlx proteins were detected using the anti-Hlx and anti-actin Abs (left panel). Right panel, Naive CD4 T cells were isolated by FACS sorting of the CD4ϩCD44lowCD62Lhigh population. Cell lysates were analyzed as described in the left panel. 1556 GENETIC CONTROL OF IL-4R␣ EXPRESSION IN NAIVE CD4 T CELLS

in Th1 cells (Fig. 3a), demonstrating the Th2-promoting and Th1- suppressing activities of IL-4. However, at each concentration of exogenous IL-4, a lower percentage of Th2 and a higher percentage of Th1 cells were detected in transgenic cells than in wild-type cells. Therefore, Hlx transgenic naive CD4 T cells showed weaker responses to IL-4 in terms of both promoting Th2 and suppressing Th1 cell differentiation. Similar results were obtained when the cells were stimulated with anti-CD3 and anti-CD28 (Fig. 3b). In subsequent experiments we will use only Con A and APC for T cell stimulation. When the differentiated cells were analyzed for Hlx expression by Western blot, we found only a weak signal in nontransgenic CD4 T cells, which was further diminished in the presence of IL-4 (Fig. 3c, left). As expected, much stronger signals were detected in Hlx transgenic CD4 T cells (Fig. 3c, right). The difference between Hlx transgenic CD4 T cells differentiated in the absence or the presence of IL-4 was somewhat masked by the constitutive expression of Hlx driven by the CD4 promoter. Stat6 activation in wild-type and Hlx transgenic CD4 T cells We also measured the strength of IL-4 signaling in Hlx transgenic Downloaded from and wild-type CD4 T cells by detecting Stat6 activation. Trans- genic and wild-type naive CD4 T cells were activated with Con A and APC in the presence of suboptimal concentration of IL-4. Cells were harvested after a relatively short culture time of 20 h. Cell lysates were immunoprecipitated with anti-Stat6 Abs. The precipitated proteins were analyzed with anti-phospho-tyrosine http://www.jimmunol.org/ Abs in Western blot to detect phosphorylated Stat6. When equal FIGURE 2. Analyses of IL-4, IL-4R␣, and Stat6 gene expression. a, MACS-sorted naive CD4 T cells from Hlx transgenic and wild-type naive amounts of immunoprecipitated Stat6 were loaded, as determined CD4 T cells were set up for differentiation as described in Fig. 1a (upper by Western blot analysis with anti-Stat6 Abs, less phosphorylated panel). After 1.5 days of T cell activation, CD4 T cells were isolated by Stat6 was detected in Hlx transgenic cells than in wild-type cells FACS sorting. RNA was extracted from the sorted cells and analyzed for (Fig. 4a, left). We also examined the difference in IL-4 signaling be- gene expression by real-time RT-PCR. Expression of IL-4 is shown as tween Hlx transgenic and nontransgenic naive CD4 T cells (Fig. 4a, transcripts relative to Tubb5. b, Naive CD4 T cells were isolated by FACS right). In this case, fresh isolated naive CD4 T cells were stimulated ϩ sorting of the CD4 CD44lowCD62Lhigh population. RNA was extracted with IL-4. Cell lysates were analyzed in the same way as before. As ␣ by guest on October 2, 2021 from the isolated cells and reverse transcribed. Expression of IL-4R , expected, less Stat6 activation was detected in Hlx transgenic naive ␤ Stat6, and the housekeeping gene -tubulin V (Tubb5) was analyzed by CD4 T cells than in nontransgenic naive CD4 T cells. To determine real-time RT-PCR. Levels of IL-4R␣ and Stat6 are presented as transcripts whether IL-4R␣ expression could enhance Th2 cell differentiation in relative to Tubb5. c, Left panel, Total CD4 T cells were incubated with biotinylated anti-IL4R␣ Ab or isotype-matched nonspecific Ab. The cells Hlx transgenic CD4 T cells, Hlx transgenic CD4 T cells were stimu- were then stained with streptavidin-PE, Abs against CD4 (allophycocya- lated to differentiate in the presence of IL-4. After 12 h of T cell nin), CD44 (PE-Cy5), and CD62L (FITC). An overlay histogram of PE stimulation, the cells were infected with retrovirus expressing a bicis- staining (IL-4R␣) on gated the CD4ϩCD44lowCD62Lhigh population is tronic IL-4R␣-GFP or GFP alone. After infection, the cells were re- shown. Right panel, Cell lysates of sorted naive CD4 T cells turned to differentiation in the presence of IL-4. The differentiated ϩ (CD4 CD44lowCD62Lhigh) from wild-type or Hlx transgenic mice were cells were analyzed by intracellular cytokine staining. As shown in analyzed by Western blot using anti-Stat6 or anti-actin Abs. Fig. 4b, more Th2 and fewer Th1 cells were detected in cells infected with retrovirus expressing IL-4R␣. These results directly demon- strated that a lower IL-4R␣ level in Hlx transgenic CD4 T cells not lower than in wild-type naive CD4 T cells (Fig. 2b). We also only led to less IL-4 signaling, but also to less Th2 cell differentiation analyzed the surface expression of IL-4R␣ by flow cytometry. and more Th1 cell differentiation. Consistent with the real-time RT-PCR results, the surface IL-4R␣ ␣ level on the Hlx transgenic naive CD4 T cells was dramatically Effect of anti-IL-4R Ab on Th cell differentiation lower than in wild-type naive CD4 T cells (Fig. 2c, left). In con- The study of Hlx transgenic mice clearly showed that the IL-4R␣ trast, Western blot analysis confirmed that similar protein levels of level is a key factor that controls the ratio of Th1 and Th2 cell Stat6 in wild-type and transgenic CD4 T cells (Fig. 2c, right). differentiation under suboptimal polarizing conditions. However, an alternative explanation, such as changes in T cell development IL-4 dose response of Hlx transgenic and wild-type naive CD4 in the transgenic mice, could be responsible for the effect. To dem- T cells onstrate that the IL-4R␣ level indeed determines the ratio of Th1 The gene expression data suggested that the responsiveness of Hlx and Th2 cell differentiation under suboptimal conditions, we used transgenic naive CD4 T cells to IL-4 might be impaired. To function- anti-IL-4R␣ neutralizing Ab to titrate down the IL-4R␣ level on ally analyze IL-4 responsiveness, we compared IL-4 dose-response normal CD4 T cells (Fig. 5). Thus, different concentrations of curves of transgenic and wild-type naive CD4 T cells. Despite the low anti-IL-4R␣ Abs were added to Th cell differentiation cultures level of IL-4R␣, Hlx transgenic naive CD4 T cells remained respon- either without exogenous IL-4 or with a suboptimal concentration sive to IL-4. In the differentiation cultures of both transgenic and of exogenous IL-4. As expected, exogenous IL-4 increased Th2 wild-type naive CD4 T cells stimulated with Con A and APC, we and decreased Th1 cell differentiation compared with cultures observed an IL-4 dose-dependent increase in Th2 cells and a decrease without exogenous IL-4. In both cases, the addition of anti-IL4R␣ The Journal of Immunology 1557

FIGURE 3. IL-4 dose response of wild-type and Hlx transgenic naive CD4 T cells. Th cell differentiation cultures were set up with MACS-sorted naive CD4 T cells in the presence of different concentrations of exogenous IL-4 and the absence of any neutralizing Abs. The T cells were stimulated with wild-type APC and Con A (a) or plate- bound anti-CD3 (1 ␮g/ml) and anti-CD28 (5 ␮g/ml) Abs (b). Differentiated cells were stained for CD4 and intracellular IL-4 and IFN-␥. The percentages of Th1 and Th2 cells on the gated CD4 T cells were plotted against the concentrations of the initial exogenous IL-4. c, Western blot analysis of Hlx expression in differentiated CD4 T cells. Naive

CD4 T cells from Hlx transgenic or nontransgenic mice Downloaded from (left and center panels) were differentiated in the absence or the presence of exogenous IL-4 (5 ng/ml). On day 3, cell lysates were analyzed for Hlx or actin expression by West- ern blot. http://www.jimmunol.org/

Abs effectively reduced Th2 cell differentiation and enhanced Th1 heterozygous naive CD4 T cells compared with wild-type naive cell differentiation proportionally to the Ab concentration. There- CD4 T cells. Consistently, real-time RT-PCR results showed a by guest on October 2, 2021 fore, modulating the IL-4R␣ level on the cell surface alone is higher mRNA level of IL-4R␣ in heterozygous than wild-type na- indeed sufficient to change the ratio of Th1 and Th2 cell differen- ive CD4 T cells. Real-time RT-PCR analysis also confirmed the tiation under weak or suboptimal polarizing conditions. expected lower expression of Hlx in heterozygous naive CD4 T cells (Fig. 7a, right). To functionally analyze the responsiveness to ϩ/Ϫ T cell development in Hlx heterozygous mice IL-4, we compared the IL-4 dose responses of heterozygous and We have presented evidence that overexpression of Hlx in trans- wild-type naive CD4 T cells (Fig. 7b). As expected, an inverse genic mice diminishes the responsiveness of naive CD4 T cells to relationship between Th1 cell differentiation and exogenous IL-4 IL-4. A logical prediction would be that the loss of Hlx would have concentration was observed, whereas a positive correlation existed the opposite effect on naive CD4 T cells. However, Hlx deficiency between Th2 cell differentiation and exogenous IL-4 concentra- is embryonically lethal and causes miniature fetal liver develop- tion. At each exogenous IL-4 concentration tested, heterozygous ment (46). We therefore opted to use Hlxϩ/Ϫ heterozygous mice to naive CD4 T cells gave rise to more Th2 cells and fewer Th1 cells study genetic regulation of IL-4 responsiveness of naive CD4 T than wild-type naive CD4 T cells. Real-time RT-PCR analysis cells by Hlx. We first examined whether there were any abnor- showed higher GATA-3 expression in Hlx heterozygous CD4 T malities in T cell development in Hlxϩ/Ϫ heterozygous mice. cells differentiated in the presence of IL-4 than wild-type CD4 T Staining of thymocytes for CD4 and CD8 showed essentially the cells (Fig. 7c), consistent with the fact that there were more Th2 same distribution of all four populations of thymocytes in het- cells in heterozygous CD4 T cells. Therefore, heterozygosity of erozygous and wild-type mice (Fig. 6a). Likewise, similar num- Hlx gene caused a heightened responsiveness of naive CD4 T cells bers of CD4 and CD8 single-positive cells were detected in the to IL-4, a phenotype opposite to that of the Hlx transgene. periphery in heterozygous and wild-type mice (Fig. 6b). The percentages of peripheral CD4 T cells with the naive phenotype Discussion (CD44lowCD62Lhigh) were also similar in heterozygous and Studies of Th1 and Th2 cell differentiation have greatly benefited ϩ Ϫ wild-type mice (Fig. 6c). Thus, T cell development in Hlx / from in vitro models that can provide a nearly homogeneous pop- heterozygous mice appeared to be normal. ulation of Th1 or Th2 cells. Using such in vitro differentiation models, much progress has been made toward understanding the ␣ ϩ/Ϫ IL-4R expression and IL-4 responsiveness of Hlx naive development of the transcription machineries responsible for ef- CD4 T cells fector cytokine expression in Th1 and Th2 cells. However, one of We compared IL-4R␣ expression on naive CD4 T cells of wild- the ultimate goals of studying Th cell differentiation is to under- type and heterozygous mice (Fig. 7a). Flow cytometric analysis stand how genetic and physiological factors influence the outcome showed a surprisingly clear up-regulation of IL-4R␣ on Hlxϩ/Ϫ of differentiation in vivo. Under in vivo conditions, neutralizing 1558 GENETIC CONTROL OF IL-4R␣ EXPRESSION IN NAIVE CD4 T CELLS

FIGURE 5. Dose-dependent effect of anti-IL4R␣ Ab on Th cell differentiation. CD4 T cells from normal BALB/c mice were stimulated with Con A and APC either without exogenous IL-4 or with 0.5 ng/ml IL-4. Anti-IL-4R␣ neutralizing Abs (M1) were added at the beginning of the culture at the indicated concentrations. The differentiated cells were analyzed by intracellular cytokine staining of IL-4 and IFN-␥. The percentages FIGURE 4. a, Stat6 activation in wild-type and Hlx transgenic CD4 T of Th1 and Th2 cells in the gated CD4 T cell population are shown. cells. In the left panel, FACS-sorted naive CD4 T cells ϩ low high (CD4 CD44 CD62L ) were stimulated with Con A plus wild-type many studies have shown that this source of IL-4 is sufficient to APC and IL-2 in the presence of 0.5 ng/ml IL-4. Twenty hours later, the Downloaded from drive Th2 cell differentiation. In this study we found that the level cells were treated with Abs against MHC class II Ags plus rabbit comple- ␣ ment to remove residual APC in the cultures. The cells were then lysed and of IL-4R in Hlx transgenic naive CD4 T cells was markedly ␣ immunoprecipitated with anti-Stat6 Abs. The immunoprecipitated proteins reduced. The reduction of IL-4R expression led to less activation were analyzed by Western blot for Stat6 (lower panel). Eluants of immu- of Stat6 and ultimately less Th2 and more Th1 differentiation un- noprecipitation containing equal amounts of Stat6 were analyzed by West- der weak or suboptimal polarizing conditions. In contrast, Hlx hap- ern blot using the phosphotyrosine-specific Ab 4G10 (upper panel). In the loinsufficiency caused more IL-4R␣ expression and Th2 cell dif- right panel, fresh isolated naive CD4 T cells from Hlx transgenic and ferentiation and concomitantly less Th1 cell differentiation. http://www.jimmunol.org/ nontransgenic mice were stimulated with IL-4 (0.5 ng/ml) only for 20 min. Cell lysates were prepared and analyzed as described in the left panel. b, Effect of retroviral expression of IL-4R␣ on the outcome of Th cell differentiation in Hlx transgenic CD4 T cells. Naive CD4 T cells from Hlx transgenic mice were stimulated with Con A and APC in the presence of IL-4 (0.5 ng/ml). Twelve hours later, the cells were infected with retrovirus expression bicistronic IL-4R␣-GFP or GFP alone. After infection, cells continued to differentiate in the presence of IL-4. The differentiated cells were analyzed by intracellular cytokine staining. Dot plots of IL-4 and by guest on October 2, 2021 IFN-␥ staining on gated GFP-positive cells are shown. Numbers in the quadrants show the percentages of the cell populations.

Abs against polarizing cytokines used in the in vitro cultures to create extreme polarizing conditions do not exist, and polarizing cytokines for one subset may not always dominate over those for the other subset. Therefore, it is important to understand the factors that inﬂuence Th cell differentiation under such weak or compet- itive conditions. In this study we investigated an earlier observa- tion that Hlx transgenic mice generated elevated Th1 response to a protein Ag in vivo (12). This study led us to uncover a naive CD4 T cell intrinsic element that inﬂuences the outcome of Th cell differentiation. Our study demonstrates that Hlx modulates the responsiveness of naive CD4 T cells to the polarizing cytokine IL-4. The relative responsiveness of naive CD4 T cells to IL-4 is a crit- ical factor that determines the ratio of Th1 and Th2 cells generated from differentiation under weak polarizing conditions. The cytokine milieu in which naive CD4 T cells respond to Ag has long been considered to be the predominant factor that drives Th cell differentiation toward either a Th1- or Th2-dominant outcome (16). Some infectious agents are known to induce cytokine environment that overwhelmingly favors either Th1 or Th2 cell FIGURE 6. Thymic and peripheral T cell surface phenotypes of wild- ϩ/Ϫ differentiation. However, this mode of regulation may not be ap- type and Hlx heterozygous mice. a, Thymocytes from wild-type and Hlxϩ/Ϫ mice were stained for CD4 and CD8. The numbers in the plots plicable to many other immune responses, such as those against indicate the percentage of each cell population. b, Upper panel, Spleen autoantigens and environmental Ags, where no microbial products cells were stained for CD4 and CD8, and the percentages of single positive are present to stimulate innate immune responses to create a dom- cells are shown. Lower panel, Spleen cells were stained for CD4, CD44, inantly polarizing cytokine environment. Under these circum- and CD62L. Dot plots show CD44 and CD62L staining on gated CD4 T stances, the immediate production of IL-4 by the responding naive cells. The numbers indicate the percentages of naive populations CD4 T cells upon Ag stimulation is especially important because (CD44lowCD62Lhigh) on the gated CD4 T cells. The Journal of Immunology 1559 Downloaded from

FIGURE 7. IL-4R␣ expression and IL-4 dose response of wild-type and Hlxϩ/Ϫ heterozygous naive CD4 T cells. a, Staining for IL-4R␣ was performed as described in Fig. 2. Overlay histograms of IL-4R␣-PE staining and isotype IgG control of gated naive CD4 T cells (CD4ϩCD44lowCD62Lhigh)of wild-type and heterozygous mice are shown (left). FACS-sorted naive CD4 T cells (CD4ϩCD44lowCD62Lhigh) from wild-type and Hlxϩ/Ϫ heterozygous mice were analyzed for the expression of IL-4R␣ and Hlx by real-time RT-PCR. Transcripts relative to the Tubb5 gene are shown (right). b, MACS-sorted naive CD4 T cells were activated by Con A and wild-type APC in the presence of different concentrations of exogenous IL-4. After 4 days of differentiation, the cells were rested and reactivated by PMA and ionomycin. The cells were stained for CD4 and intracellular IL-4 and IFN-␥. The percentages of Th1 http://www.jimmunol.org/ or Th2 cells on gated CD4 T cells were plotted against the initial concentration of the exogenous IL-4. c, GATA-3 expression in differentiated CD4 T cells. Naive CD4 T cells from wild-type or Hlx heterozygous mice were differentiated in the presence of IL-4 (1 ng/ml). On day 4, RNA were extracted and analyzed for GATA-3 expression by real-time RT-PCR.

Therefore, we have established that IL-4R␣ expression on naive dual regulatory role in naive CD4 T cells and differentiating Th CD4 T cells is genetically controlled by Hlx. Furthermore, the cells. Our study is the first to unravel the transcription regulatory level of IL-4R␣ is a determining factor that controls the ratio of network in naive CD4 T cells that controls their potential to re- by guest on October 2, 2021 Th1 and Th2 cell differentiation under weak polarizing conditions. spond to polarizing cytokines. However, other transcription fac- Supporting the idea of IL-4R␣ as a key factor for regulating Th2 tors, such as GATA-3 and T-bet, are also expressed at low levels cell differentiation in vivo is the genetic lineage of atopic diseases to in naive CD4 T cells (4, 50). It would be interesting to know polymorphism of IL-4R␣ amino acid sequences. Atopic diseases are whether they are also part of the transcription regulatory system caused by biased Th2 responses to environmental Ags. Allelic poly- that maintains the balance of responsiveness to Th1- and Th2- morphism at certain amino acid positions of the IL-4R␣ correlates polarizing cytokines in naive CD4 T cells. with the risk level of developing atopic diseases (48, 49). Our study Transcriptional regulation of IL-4R␣ expression is poorly un- shows that not only the allelic amino acid sequence variation, but also derstood. Our data demonstrate that Hlx at least genetically con- the level of IL-4R␣, is a potential risk factor for atopy. Based on our trols IL-4R␣ expression at the transcription level. IL-4R␣ could be study, it can be speculated how a biased Th2 response might be gen- a direct target gene of Hlx, because Hlx itself is also expressed in erated against environmental Ags. We believe that individuals ex- naive CD4 T cells, albeit at a low level. The low expression of Hlx pressing higher levels of IL-4R␣ may generate more Th2 cells upon in naive CD4 T cells is not surprising, because high levels of Hlx initial exposure to allergens than normal individuals. Because Th cell promote IFN-␥ expression. Therefore, Hlx must be kept at low differentiation is a self-reinforcing process in which effector cytokines levels to avoid aberrant expression of IFN-␥ in naive CD4 T cells. promote the differentiation of their producers and concomitantly sup- Thus, the requirement for a threshold level of Hlx to regulate IL- press the opposite subset, CD4 T cell responses to allergens in these 4R␣ expression is much lower than that of Hlx to regulate IFN-␥ individuals may become increasingly biased and eventually become gene expression. Such dose-dependent regulation of gene expres- Th2 dominant. sion is reminiscent of that of GATA-3, for which different thresh- The role of Hlx in regulating IL-4R␣ expression in resting naive old levels seem to be required for activating different Th2 cytokine CD4 T cells must be distinguished from its role in promoting Th1 genes (4). The biochemical bases for such quantitative require- cell differentiation in differentiating Th1 cells. During Th1 cell ments are of great biological significance and remain a challenge differentiation, Hlx is up-regulated from its basal level in naive for future studies. CD4 T cells, whereas its expression is further down-regulated or In conclusion, our study has modified the instruction model of extinguished during Th2 cell differentiation (10, 12). Because the Th cell differentiation to include the intrinsic difference in the re- Hlx transgene prevents silencing of the IFN-␥ gene in CD4 T cells sponsiveness of naive CD4 T cells to polarizing cytokine as a key differentiated even under extreme Th2-polarizing conditions, Hlx factor in determining the outcome of differentiation. We provided appears to maintain a transcriptionally active chromatin structure definitive evidence that the responsiveness of naive CD4 T cells to at the IFN-␥ gene during Th1 cell differentiation, a role that syn- polarizing cytokine is genetically controlled. The genetically de- ergizes with that of T-bet (10, 12). To date, Hlx is the only Th cell termined variations of the responsiveness predispose naive CD4 T subset-specific transcription factor that has been shown to have a cells to generate quantitative differences in Th1 and Th2 responses. 1560 GENETIC CONTROL OF IL-4R␣ EXPRESSION IN NAIVE CD4 T CELLS

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