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The Gadd45 family consists of three genes that are evolutionarily wellconserved; the molecular functions of their gene products are verysimilar when they are overexpressed in cell lines1. However, thesegenes are regulated differently and as a consequence their in vivofunctions are different. For example, Gadd45a is a direct target geneof the tumor suppressors p53 and BRCA1 (ref. 2). All Gadd45 familygenes are induced by various cellular stresses3,4, indicating involve-ment in the stress response. In addition to stress signals, Gadd45b isinduced by inflammatory cytokines such as interleukin 6 (IL-6), IL-18 and IL-12, tumor necrosis factor (TNF) and transforming growthfactor-β1 (TGF-β1)1,5–8. Gadd45g is also induced by cytokines such asIL-2 and IL-18 plus IL-12 (refs. 6,9,10). Gadd45b is induced by innatesignals triggered by lipopolysaccharide (LPS)11. All these observa-tions point to the idea that Gadd45β and Gadd45γ are important forinflammatory responses as well as mediating signals of the innate sys-tem. Further, they indicate that the biological functions of differentGadd45 family members are distinct.

Many studies using overexpression and deletion approaches havedemonstrated that Gadd45 family genes are involved in the elimina-tion of cells through cell cycle arrest and apoptosis4,6,10,12–18. Theantiproliferative activity of Gadd45 proteins is well established andinvolves direct interaction of Gadd45 family proteins with the cellcycle machinery, including PCNA, cdc2, cyclin B and p21 (refs.4,10,12,15,18,19). Gadd45 family genes are associated with the medi-ation of stress responses by stimulating p38-JNK mitogen-activatedprotein (MAP) kinase activation in various cell types including bothestablished cell lines and primary cells6,8,20,21. This was proposed as amechanism for Gadd45 family protein-induced apoptosis. In contrastto those last studies, a recent study showed that Gadd45β is antiapop-totic through its ability to downregulate JNK activation7. In contrast

to p38, which is activated by Gadd45 family proteins through MEKK4(MAP kinase (MAPK)-extracellular signal–regulated kinase (MEK)kinase 4), the regulation of JNK seems to be indirect and dependenton cell context5–8,16,20–23.

Studies have suggested that Gadd45β is important in mediating theeffects of the inflammatory cytokines IL-12 and IL-18 (ref. 5). Similarly,Gadd45g is also induced by IL-12 and potentiates TCR induced activa-tion of p38-JNK MAP kinases6. It is a matter of debate whether or inwhat circumstances Gadd45 family genes are required for TCR-inducedp38 activation. Here we studied the function of Gadd45β in mediatingboth cognate and inflammatory signals. Gadd45b was induced by thecognate signal, potentiated the activities of multiple MAP kinases andwas required for acquired immunity. Gadd45β is therefore a critical ele-ment within a positive feedback circuit that is responsible for perpetuat-ing signals by both the innate and acquired immune systems.

RESULTSGadd45β is induced after TCR challengeTo study the expression profile of Gadd45b during naive T cell activa-tion, we stimulated naive CD4+ T cells with plate-bound antibody toCD3 (anti-CD3) in the presence or absence of anti-CD28. TCR stimu-lation alone for 1 h was already sufficient to upregulate Gadd45bmRNA, which could be further augmented in the presence of CD28costimulation (Fig. 1a). We further studied the expression profile ofGadd45b and Gadd45g during early T cell activation and T helper type1 (TH1)-TH2 differentiation. We stimulated naive CD4+ T cells withanti-CD3 and anti-CD28 in TH0, TH1 and TH2 conditions for 1 h, 4 h,24 h and 48 h. We then withdrew cells from stimulation and culturedthem for another 2 d. We restimulated the cells at 96 h with anti-CD3for 2h. We examined Gadd45b and Gadd45g expression at all these time

1Section of Immunobiology and 2Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, Connecticut 06520-8011,USA. 3Present address: Department of Immunology, University of Pittsburgh School of Medicine, E1047 BST, 200 Lothrop Street, Pittsburgh, Pennsylvania 15261,USA. Correspondence should be addressed to R.A.F. (richard.flavell@yale.edu).

Published online 14 December 2003; doi:10.1038/ni1020

Gadd45β is important for perpetuating cognate andinflammatory signals in T cellsBinfeng Lu1,3, Anthony F Ferrandino1 & Richard A Flavell1,2

Gadd45β (growth arrest and DNA damage–inducible, β) is involved in cell cycle arrest, apoptosis, signal transduction and cellsurvival. In T cells, Gadd45b was rapidly induced by T cell receptor (TCR) and inflammatory signals. Deficiency of Gadd45βin CD4+ T cells impaired their responses to TCR stimulation or inflammatory cytokines. ERK, p38 and JNK activation were allsubstantially suppressed in Gadd45β-deficient CD4+ T cells. Cytokine production by Gadd45β-deficient CD4+ T cells was alsoimpaired. Furthermore, Gadd45β mediated inflammatory cytokine production by dendritic cells, and Gadd45β-deficient miceshowed an impaired T helper type 1 response during Listeria monocytogenes infection. Gadd45β is therefore a critical feedbackregulator that perpetuates both cognate and inflammatory signals.

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points by real-time PCR. Gadd45b mRNA peaked rapidly after TCRstimulation and settled at a lower level at later time points (Fig. 1b). IL-4 further repressed the Gadd45b mRNA at 48 h. But cells cultured inTH1 conditions did not have higher expression of Gadd45b mRNAcompared with cells cultured in TH0 conditions. Therefore, IL-12 didnot affect Gadd45b expression during early T cell activation. Gadd45btranscripts disappeared after cessation of TCR signals for 2 d (Fig. 1b,96 h), but were quickly induced after restimulation. Therefore, duringearly T cell activation, Gadd45b was regulated directly by TCR signalingbut not IL-12. In contrast, Gadd45g expression was later and inducedby both IL-12 and IL-4, consistent with published data6.

Generation of Gadd45β-deficient miceTo determine the in vivo function of Gadd45β in CD4+ T cells, we gen-erated Gadd45β-deficient mice. Gadd45b is composed of four codingexons (Fig. 2a). We designed a targeting construct to delete exons 3and 4. Homologous recombination between the targeting constructand the endogenous Gadd45b locus resulted in deletion of sequencesthat encode amino acids 42–161, which include the most crucialdomains of the protein16,24–28. We generated chimeric mice that trans-mitted the disrupted allele through the germline from two indepen-dent embryonic stem cell clones. Southern blot analysis showed thatoffspring of heterozygous parents included the three expected geno-types (Fig. 2b). Both male and female Gadd45b–/– mice were fertile.We intercrossed Gadd45b–/– mice and Gadd45g–/– mice to generateGadd45b+/–Gadd45g+/– mice, and intercrossed Gadd45b+/–Gadd45g+/–

mice to generate Gadd45b–/–Gadd45g–/– mice. Gadd45b–/–Gadd45g–/–

mice were born without any obvious developmental abnormality.To determine if Gadd45β deficiency affected lymphoid develop-

ment in general, we examined thymocytes, splenocytes and bonemarrow cells from 6- to 8-week-old mice of both wild-type andGadd45b–/– genotypes by flow cytometry. We examined thymocytesfor CD4, CD8, CD44 and CD25 expression. We assessed the expres-sion of surface markers such as CD4, CD8, CD44, CD62L, CD25,B220, IgM, Mac-1 and Gr-1 on splenocytes. We found no substantialchanges in any of these parameters (data not shown). These data indi-cate that Gadd45β was not essential for the development of lymphoidas well as some of the myeloid lineages.

Gadd45β mediates naive CD4+ T cell activationThe delivery of cognate signals through TCR stimulation results in MAPkinase and T cell activation. Because Gadd45b mRNA was rapidlyinduced by TCR stimulation, we examined whether it was required forMAP kinase activation in naive CD4+ T cells. We stimulated naive CD4+

T cells from wild-type and Gadd45b–/– mice with plate-bound anti-CD3for different amounts of time and assessed the activation of MAP

kinases by immunoblotting with antibodies that recognize the activeforms of these kinases (Fig. 3). We found a defect in the activation of theERK and p38 pathways in Gadd45b–/– CD4 T cells in response to TCRsignal. ERK activation after 10 min of TCR crosslinking was reduced andwas more rapidly downregulated in Gadd45b–/– naive CD4+ T cells atlater times than in wild-type cells (Fig. 3a). Similarly, p38 activation wasgreatly reduced in Gadd45b–/– naive CD4+ T cells compared with wild-type cells (Fig. 3a). We found low JNK activation in both wild-type andknockout naive T cells, consistent with previous observations29.

Naive CD4+ T cells respond to TCR stimulation by makingcytokines such as IL-2. Consistent with the hyporesponsiveness toTCR triggering, IL-2 production by naive CD4+ T cells was also com-promised when Gadd45b was deleted (Fig. 3b). Therefore, Gadd45βis important for cytokine production by naive CD4+ T cells.

Gadd45β sustains p38 activationEffector CD4+ T cells, both TH1 and TH2, maintained low amounts ofGadd45b mRNA, which could be rapidly upregulated after TCR res-timulation (Fig. 1b). In contrast, Gadd45g mRNA expression was highin effector T cells but not induced by TCR stimulation (Fig. 1b).Therefore, Gadd45g is regulated mostly by cytokines, whereasGadd45b is regulated by TCR signaling. In addition, Gadd45b wasexpressed earlier during T cell activation than Gadd45g (Fig. 1b),which is consistent with involvement of Gadd45β in naive CD4+ T cellactivation (Fig. 1b). To study the function of Gadd45β in mediatingMAP kinase activation by TCR signaling in effector CD4+ T cells, weused both wild-type and Gadd45b–/– TH1 cells derived in vitro. We cul-tured naive CD4+ T cells from both wild-type and Gadd45b–/– mice inTH1-polarizing conditions for 4 d. We then collected these cells andallowed them to ‘rest’ in culture media free of cytokines for 1 h. Wethen stimulated them with plate-bound anti-CD3 at high and low doses

Figure 1 Gadd45b message is induced by TCR stimulation and enhancedby costimulation. (a) Naive CD4+ T cells were stimulated with plate-bound anti-CD3 (α-CD3) or α-CD3 plus α-CD28 for 1 h. The Gadd45bmessage was detected with RT-PCR. (b) Naive CD4+ T cells werestimulated with plate-bound α-CD3 and α-CD28 for 1 h (1,2,3), 4 h (4,5,6), 24 h (7,8,9), 48 h (10,11,12) or 96 h (13,14,15) andrestimulated (16,17,18). Cells were cultured in TH0 (1,4,7,10,13,16),TH1 (2,5,8,11,14,17) and TH2 (3,6,9,12,15,18) conditions. Gadd45band Gadd45g mRNA was assayed with real-time RT-PCR. Data representfold changes versus naive CD4+ T cells.

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for 20 min (Fig. 4a) or at a high dose for 60 and 240 min (Fig. 4b). Weexamined extracts for the activation of MAP kinases withimmunoblotting. TCR stimulation resulted in sustained p38 activa-tion that peaked around 20 min and receded to a sustained but loweramount at 4 h after stimulation (Fig. 4a,b). Gadd45β affected the mag-nitude of the peak activation (Fig. 4a) and was also required for sus-tained p38 activation (Fig. 4b). TCR stimulation of these cells alsoresulted in sustained ERK activation (Fig. 4a,b). Deletion of Gadd45breduced the magnitude of peak ERK activation (Fig. 4a) but did notaffect the level of sustained activation (Fig. 4b). JNK activation wastransient; it peaked around 20 min and then decreased (data notshown). Gadd45β seemed to affect the magnitude of the peak but notthe duration of JNK activities (Fig. 4a). Therefore, the influence ofGadd45β on the activation of MAP kinases are twofold: it enhancesTCR-stimulated transient activation of all MAP kinases, and it isspecifically required for sustained p38 activation.

Gadd45β mediates effector T cell functionsBecause our study demonstrated that Gadd45β was required for bothtransient and sustained activation of MAP kinases by TCR stimulation,we examined whether it was important for effector cytokine productionby effector CD4+ T cells. We generated TH1 cells in vitro and stimulatedthem with plate-bound anti-CD3, and assessed cytokine production byenzyme-linked immunosorbent assay (ELISA). Consistent with itsfunction in mediating MAP kinase activation, Gadd45β seems to beimportant for production of IL-2 and interferon-γ(IFN-γ) by TH1 cells.

In addition, deletion of Gadd45b reduced the frequency of effector cellsable to produce IL-2 and IFN-γ (Fig. 5a,b). To examine whether thereduction in IFN-γ production was a result of TH2 skewing inGadd45β-deficient T cells, we cultured naive CD4+ T cells in nonpolar-izing conditions. In these conditions, IL-2 production by T cells wasreduced (Fig. 5a). Moreover, no IL-4 was produced by either wild-typeor Gadd45b–/– T cells (data not shown). Therefore, deletion of Gadd45bdoes not skew naive CD4+ T cells to a TH2 response. In addition, whenwe examined cells cultured in TH2 conditions for their cytokine pro-duction, we found a modest reduction in IL-4 production in Gadd45β-deficient T cells (Fig. 5a). Gadd45β was therefore not required forTH1-TH2 differentiation but was required for the function of all effec-tor T cells. In addition to stimulating cells with plate-bound anti-CD3plus anti-CD28, we also cultured T cells with soluble anti-CD3 plusantigen-presenting cells (splenocyte populations depleted of T cells)and obtained similar results (data not shown).

Gadd45β mediates IL-12 and IL-18 functionsGadd45β overexpression indicates it is involved in the IL-12–IL-18-induced activation of p38 and IFN-γ production5. The mechanismof p38 activation was proposed to occur through MEKK4, based on

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data generated with a dominant negative form of this kinase. Wetherefore decided to test this hypothesis with Gadd45β-deficientTH1 cells. Unexpectedly, Gadd45b deletion did not result in reducedtransient activation of p38 by IL-12 and IL-18 (Fig. 6a). However, itdid affect sustained activation of p38 in TH1 cells (Fig. 6a).Consistent with its function in sustaining p38 activation, deletionof Gadd45b also resulted in drastically reduced IFN-γ activation inTH1 cells (Fig. 6b). Therefore, Gadd45β is an important signalingmodulator that enhances and sustains p38 activation. The sus-tained activation of p38 seems to be important for IFN-γ produc-tion, which is in agreement with studies showing that p38 isrequired for TH1 IFN-γ production30.

Gadd45β regulates in vivo TH1 responsesTo extend our study of in vitro cultured T cells to physiological condi-tions, we used two established methods to examine whether Gadd45βis required for TH1 function in vivo. We first immunized both wild-type and Gadd45β-deficient mice with keyhole limpet hemocyanin(KLH) plus complete Freund’s adjuvant (CFA). We isolated cells fromdraining lymph nodes 7 d later and restimulated them ex vivo withKLH. Cytokines are produced mainly by CD4+ T cells in this type ofassay. We did not find great differences in IL-2 and IL-4 production,indicating again that no TH2 skewing occurred (data not shown).However, we did note a large reduction in IFN-γ in cells fromGadd45β-deficient mice (Fig. 7a). Gadd45β is therefore important forthe generation of TH1 cells in vivo. A second well-characterized mousemodel used for studying cell-mediated immunity is infection withListeria monocytogenes. Infection of mice with this pathogen elicits astrong T cell response that is essential for bacterial clearance. Studieswith major histocompatibility complex–deficient mice have showedthat both CD4 and CD8 T cells contribute to protection against liste-riosis31. L. monocytogenes is able to induce CD4+ T cells that arerestricted to the TH1 phenotype32 and has been proposed as a vaccinevector for immunity induction against human immunodeficiencyvirus and tumors33,34. We therefore used this system to examinewhether Gadd45β is involved in the generation of TH1 cells in vivo. Weinfected both wild-type and Gadd45b–/– mice intravenously with

5 × 103 live L. monocytogenes. We isolated spleen cells 7 d later and stim-ulated them with a dominant epitope for CD4+ T cells. We thenrecorded the frequency of IFN-γ-producing T cells by flow cytometry.In wild-type mice, about 15% of the CD4+ T cells at this stage of infec-tion were IFN-γpositive after restimulation. In contrast, in Gadd45b–/–

mice, only about 5% of CD4+ T cells were able to make IFN-γ(Fig. 7b).Therefore, Gadd45β is important for the generation of TH1 cells duringinfection. Gadd45γ is also expressed in effector TH1 cells and is impor-tant for p38 activation and IFN-γ production by TH1 cells6,35. Becausewe found residual TH1 cells in the Gadd45b–/– mice, we examinedwhether Gadd45γ was responsible for this. Deletion of Gadd45gresulted in only a slightly reduced primary TH1 response (Fig. 7b).Compound deletion of Gadd45b and Gadd45g did result in a furtherdecrease in TH1 cell numbers (Fig. 7b). Gadd45β-deficient, Gadd45γ-deficient, and Gadd45β and Gadd45γ double-deficient mice were notsusceptible to listeria infection at the sublethal dose used despite thereduction in TH1 responses.

Gadd45β and Gadd45γ are required for innate responsesAcquired immunity is important for the recognition of foreign anti-gens and for mounting memory immune responses against foreignelements. The other important arm of immunity that is important forself–non-self discrimination is innate immunity. Gadd45b is inducedby LPS in both dendritic cells and macrophages, indicating thatGadd45β or Gadd45γalso mediates innate recognition signals11,36. Totest this hypothesis, we examined the response to LPS bymacrophages and dendritic cells from wild-type and Gadd45β- andGadd45γ-deficient mice. Although it has been reported that LPSinduces Gadd45b mRNA in macrophages11, we did not find any dif-ference in macrophage inflammatory cytokine production whenGadd45b was deleted. In contrast, when dendritic cells were stimu-lated with LPS at lower doses, we noted a very large reduction in IL-12and IL-6 production (Fig. 8a) in the Gadd45β-deficient background.We did not find any difference in TNFα production between wild-type and Gadd45β-deficient dendritic cells. In contrast, Gadd45γ didnot seem to affect inflammatory cytokine production in macrophagesand dendritic cells (data not shown).

Figure 6 Gadd45β mediates sustained p38 activation by IL-12 plus IL-18.(a) Effector TH1 cells were stimulated (times, above lanes) with IL-12 (10 ng/ml) and IL-18 (30 ng/ml). Cell extracts were analyzed by immunoblotwith anti-phospho-ERK (α-phospho-ERK), α-phospho-JNK, α-p38 and α-phospho-p38. Phospho-ERK and phospho-JNK were not detectable in thisassay. p-, phosphorylated. (b) Effector TH1 cells were stimulated with IL-12(10 ng/ml) and IL-18 (30 ng/ml) for 48 h. Supernatants were analyzed forIFN-γ by ELISA. Results are representative of three independentexperiments.

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Figure 7 Gadd45β is important for in vivogeneration of TH1 cells. (a) After KLHimmunization, lymph node cells were culturedwith KLH for 4 d (concentrations, horizontal axis) and IFN-γ production by CD4+ T cells was measured. Results are representative of three experiments. (b) A sublethal dose of L. monocytogenes was used to infect wild-type, Gadd45b–/–, Gadd45g–/– andGadd45b–/–Gadd45g–/– mice. Then, 7 d later,splenocytes were collected and stimulated ex vivowith a dominant epitope for CD4+ T cells. Cells producing IFN-γ were stained intracellularly and identified by flow cytometry. Data are representative of atleast three independent experiments. WT, wild-type; βKO, Gadd45b–/–; γKO, Gadd45g–/–. DKO, Gadd45b–/–Gadd45g–/–.

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In addition to recognizing pathogens, innate cells such as naturalkiller (NK) cells are very important effector cell types that are acti-vated by inflammatory cytokines such as IL-12 and IL-18. Because wefound that Gadd45β and Gadd45γmediated IL-12 and IL-18 functionin TH1 cells, we examined whether they have a similar function in NKcells. We isolated splenocytes from wild-type, Gadd45b–/– andGadd45g–/– mice and stimulated them with IL-12 and IL-18 for 48 h.This treatment activates NK cells to secrete IFN-γ. We then measuredIFN-γ production by ELISA. Deletion of Gadd45b did not affect IFN-γ production by NK cells (data not shown). In contrast, deletion ofGadd45g greatly reduced IFN-γ production by NK cells (Fig. 8b).Therefore, Gadd45γ, but not Gadd45β, mediates NK cell function.This is consistent with the idea that Gadd45γ is required to mediatethe function of inflammatory cytokines and Gadd45β is importantfor mediating cognate signaling for pathogenic molecular patterns inboth the innate and acquired immune systems.

DISCUSSIONIn this study we addressed the function of Gadd45β in sustainingboth cognate and innate signals. Gadd45b mRNA was specificallyinduced by TCR signaling in both naive and effector CD4+ T cells.This is probably mediated by TCR-induced activation of the tran-scription factor NF-κB37. Using naive CD4+ T cells derived fromGadd45β-deficient mice and comparing them with wild-typeCD4+ T cells, we found that Gadd45β regulates both the magnitudeand kinetics of ERK activation. In addition, Gadd45β was requiredfor p38 activation in naive CD4+ T cells triggered by TCR crosslink-ing. Moreover, in effector TH1 cells, Gadd45β was required forERK, p38 and JNK activation by TCR signaling. Gadd45β bothenhanced and prolonged p38 activation by TCR signals; it alsoenhanced ERK and JNK but did not sustain signaling by thesekinases in effector TH1 cells. In contrast, IL-12–IL-18-stimulatedtransient activation of p38 was not affected, but Gadd45β wasrequired for prolonged p38 activation by IL-12 plus IL-18. Finally,we provided evidence that Gadd45β was required for the genera-tion of TH1 cells in vivo.

There are probably at least two mechanisms leading to the activa-tion of MAP kinases by Gadd45 family proteins. The first mecha-nism is through direct activation of MAP3Ks such as MEKK4,which then turn on p38 through MAP2Ks such as MAPK kinase 3(MKK3) and MKK6. Ample evidence in many cellular contexts sup-port the idea of the existence of this pathway5,6,8,21. However, previ-ous work failed to prove Gadd45 family proteins as being upstreamof MAP kinases38,39 because many of the Gadd45 family genes arecoregulated, and therefore results obtained from experimental sys-tems using cells with only one gene deficiency should be interpretedcautiously. Compensation from other members of the same genefamily should be considered. In addition, most of the Gadd45 familygenes are not present constitutively and are instead induced by vari-ous stress signals. Transient activation of MAP kinases is thereforenot expected to be affected by deletion of these genes. Consistent

with this, studies have demonstrated that Gadd45 family genes arerequired for sustained action of p38 and JNK8,21. Therefore, theGadd45 family genes are not involved in the ‘trigger’ signal, but areinvolved in sustaining or amplifying the initial signals.

The other mode of action by Gadd45 family genes is likely throughsecondary mechanisms. JNK activation is either mediated by Gadd45family genes or antagonized by Gadd45 family genes7,16,20. Althoughthese results seem contradictory, the differences are probably due tothe different cellular contexts and different stimuli used. This is par-ticularly likely given that most of these MAP kinase pathways canantagonize each other40. Experimental systems with cell lines thathave been passaged for many generations after the initial geneticmanipulation can compound these secondary effects. In addition totheir direct involvement in activating MEKK4, a potential alternativemechanism for this type of Gadd45 involvement is the function ofGadd45 proteins in chromatin mobilization and their interactionswith cohorts of nuclear proteins including transcription factors26,27.These interactions are probably responsible for rapid changes of cel-lular context through the regulation of gene expression. Many phos-phatases are transcriptionally regulated and may be used by Gadd45family genes to regulate MAP kinases such as ERK and JNK. Ourresults also support the idea that ERK and JNK are not likely directtargets of Gadd45 family proteins such as Gadd45β and Gadd45γ.When we stimulated TH1 cells with IL-12 and IL-18, which inducedGadd45β and Gadd45γ, we were able to sustain p38 activation butfailed to activate JNK and ERK. However, in the same cellular context,TCR crosslinking was able to do so. Both types of stimuli increaseGadd45β expression. Therefore, the activity of Gadd45β on ERK andJNK is signal dependent. Gadd45β overexpression alone was not ableto activate ERK or JNK. All these data support the idea that ERK andJNK are probably regulated indirectly by Gadd45β or by otherGadd45 family members.

Gadd45β was proposed to be important for IFN-γ production dri-ven by IL-12 and IL-18 but not TCR-stimulated cytokine production5.Our data clearly favor the model that Gadd45β is crucial for IFN-γproduction driven by both TCR and IL-12 and IL-18, as it is indeedactivated by both TCR signaling and IL-12 plus IL-18. As we haveshown here, with both TCR and inflammatory stimuli, Gadd45β isimportant for the sustained activation of p38. In contrast to this, theexpression of Gadd45g is regulated mainly by cytokines such as IL-12.Gadd45γ is important for TCR-stimulated activation of JNK-p38MAP kinases. This is probably because Gadd45γ, which is present inlarge amounts in effector TH1 cells, sensitizes the p38-JNK MAPkinase pathways. Therefore, the function of Gadd45γ is to provide avenue for inflammatory cytokines to potentiate cognate signals.Gadd45β, in contrast, can be induced directly by either cognate orinflammatory signals and is therefore important for the mediation andamplification of both acquired and innate signals. In addition toresulting in TH1 deficiency, we found that Gadd45b mutation alsoresults in defects in the function of naive CD4+ T cells and dendriticcells. Therefore, Gadd45β is required not only for the function of TH1

Figure 8 Gadd45β and Gadd45γ are important for innate immune responses.(a) Bone marrow derived–dendritic cells were stimulated for 24 h with LPS.IL-12 and IL-6 were measured by ELISA. Results are representative of threeindependent experiments. (b) NK cells were stimulated with IL-12 and IL-18 for 48 h and IFN-γ production was measured by ELISA. Results arerepresentative of three independent experiments.

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cells but also for the generation and differentiation of TH1 cells. Theconsequences of Gadd45b mutation are therefore more profound thanthat of Gadd45g mutation in terms of TH1 cell generation in vivo.

METHODSTargeting strategy. The bacteria artificial chromosome containing Gadd45bwas identified by searching the Celera Database, and the clone (RP23-70D7)was then obtained from P.J. de Jong (Children’s Hospital, Oakland,California). The targeting vector was constructed by subcloning the 5.5-kbBglII–ScaI gene fragment as the 5′ arm and the 2.1-kb HindIII–BglII genefragment as the 3′ arm into pEasyFlox (a gift from K. Rajewsky (HarvardUniversity, Boston, Massachusetts); Fig. 2a). This vector was designed toreplace the coding exons of Gadd45b with the neomycin-resistance (neor) cas-sette. The vector was electroporated into TC1, a gift from P. Leder (HarvardUniversity, Boston, Massachusetts). Drug-resistant mouse embryonic stemcell clones were screened by Southern blot analysis of genomic DNA digestedwith EcoRI, with a 200-bp genomic fragment, amplified by 5′-GGGGGATTTTGCAATCTTCT-3′ and 5′-ACGGTGAGGCGATCCTGA-3′, as a probe. Twoindependent correctly targeted embryonic stem cell clones were injected intoC57BL/6 blastocysts, and germline transmission was obtained from eachclone. Deletion of the region was confirmed by PCR with primer set 5′-ACTGCATCATGACCCTGGAAGAG-3′ and 5′-TTCGGTTGTGCCCAATGTCTCC-3′. Heterozygous mutant mice were screened by PCR with a primer setderived from the neor cassette: 5′-CTTGGGTGGAGAGGCTATTC-3′ and 5′-AGGTGAGATGACAGGAGATC-3′. Live mouse experiments approved byYale University Institutional Animal Care and Use Committee.

Expression analysis. Gadd45b was detected with a PCR primer set withsequences 5′-GGGGGATTTTGCAATCTTCT-3′ and 5′-ACGGTGAGGCGATCCTGA-3′ . Microarray data were retrieved from a published expressiondatabase41.

Cell culture. CD4+ T cells were prepared as described6. Naive (CD62L+CD44lo)cells were sorted on a Becton Dickinson Vantage cell sorter. Naive CD4+ T cellswere cultured on 24-well plates precoated with anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml). TH1 conditions were created with IL-12 (3.4 ng/ml), humanIL-2 (20 units/ml) and anti-IL4 (clone 11B11; 2 µg/ml). TH0 conditions werecreated with only human IL-2 (20 units/ml). TH2 conditions were created withIL-4 (3000 units/ml), human IL-2 (20 units/ml) and anti-IFNγ(clone XMG1.2;2 µg/ml). At 48 h after the culture was started, the cells were replated to anotherculture dish free of anti-CD3 with freshly added human IL-2 (5 units/ml). Cellswere cultured further for another 2 d and then were washed and stimulatedwith either plate-bound anti-CD3 or IL-12 (10 ng/ml) and IL-18 (30 ng/ml). Inanother culture procedure, naive CD4+ T cells were cultured with anti-CD3(clone 2C11) plus T cell–depleted cell cycle–arrested splenocyte populations asantigen-presenting cells. The same amounts of cytokines or antibodies wereadded in this type of culture.

To obtain bone marrow–derived dendritic cells, we cultured 1 × 106 bonemarrow cells for 5 d in 1 ml RPMI medium plus 5% FCS supplemented withgranulocyte-monocyte colony-stimulating factor. Fresh media were addedevery other day. On day 5, cells were stimulated with LPS for 24 h. Cytokineproduction was analyzed by ELISA.

To stimulate NK cells, we collected splenocytes and plated these at a densityof 4 × 106 cells/well in a final volume of 1 ml. Cells were then cultured for 48 hat 37 °C with 10 ng/ml of IL-12 (Genetics Institute) plus 10 ng/ml of IL-18(R&D Systems) to stimulate NK cell production of IFN-γ. Supernatants from48-hour cultures were used to measure IFN-γprotein by ELISA.

RNA quantification. Quantitative RT-PCR used the ICycler (Bio-RadLaboratories). The double-stranded DNA–specific dye SYBR Green I (MolecularProbes) was used to quantify the PCR reaction. The cycling conditions were 95 °Cfor 20 s, 57 °C for 30 s and 72 °C for 45 s. Fluorescence was captured at 72 °C. Theresults were normalized by hypoxanthine guanine phosphoribosyl transferase(HPRT). The primers were: Gadd45β, 5′-GGGGGATTTTGCAATCTTCT-3′ and5′-CGGTGAGGCGATCCTGA-3′; Gadd45γ, 5′-CATTTCACGTTGATTCAGGCG-3′ and 5′-AACAAACTGAGCTTCTCCAAGGC-3′; and HPRT, 5′-GTTGGATACAGGCCAGACTTTGTTG-3′ and 5′-GAGGGTAGGCTGGCCTATAGGCT-3′.

Immunoblot analysis. For each condition, 25 µg extract was used. Anti-phosphorylated (phospho)-p38, anti-phospho-JNK and anti-phospho-ERKwere obtained from New England Biolabs (9211S, 9255S and 9101S, respec-tively). Immunoblotting followed the manufacturer’s instructions.

KLH. Three 6- to 8-week-old female Gadd45b–/– mice and three wild-type lit-termates were immunized by footpad injection of 50 µg/mouse of KLH inCFA. At day 8 after injection, cells were collected from the draining lymphnodes and stimulated in vitro with KLH at various concentration for 1 d and 4 d. The IL-2 concentration in supernatants collected from cells cultured for24 h and IFN-γ and IL-4 concentrations in supernatants collected from cellscultured for 96 h were measured by ELISA with antibody pairs purchased fromBD PharMingen.

Infection of mice and in vitro restimulation with a dominant peptide.Infection and ex vivo stimulation were done as described with minor modifi-cations42. L. monocytogenes were grown overnight in tryptic soy broth to anabsorbance at 600 nm of between 0.1 and 0.4. Mice were infected intra-venously with 5 × 103 bacteria. At day 7 after the infection, spleen cells werestimulated for 5 h with 1 µmol of the peptide LLO189–201 (WNEKYAQAYP-NVS; Keck HHMI Biopolymer, Yale University). During the final 3 h of cul-ture, Golgi Block (BD PharMingen) was added. Cultured cells were washedand incubated at 4 °C for 30 min with anti-CD16-CD32 to block nonspecificantibody binding. Subsequently, cells were stained with fluorescein isothio-cyanate–conjugated monoclonal anti-CD4 (BD PharMingen). Cells werewashed with PBS and fixed for 20 min at room temperature with 2%paraformaldehyde in PBS. Finally cells were washed with 0.1% BSA in PBS,permeabilized with 0.1% BSA and 0.5% saponin in PBS (Sigma-Aldrich) andstained with phycoerythrin-conjugated monoclonal anti-IFNγ (BDPharMingen). Cells were analyzed with a FACSCalibur (BD Biosciences). Weacquired 100,000 lymphocyte-gated cells from each sample.

ACKNOWLEDGMENTSWe thank E. Eynon for scientific interactions; E. Tran for suggestions and readingthe manuscript; L. Evangelisti, J. Miller and J. Stein for technical assistance; G. Chenell for secretarial assistance; and F. Manzo for assistance with manuscriptpreparation. We also thank Wyeth (formerly Genetics Institute) for the gift ofrecombinant IL-12. Supported by National Institutes of Health grants (1 P01AI36529 and 1 K01 AR048854) and the Howard Hughes Medical Institute.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 16 June; accepted 5 November 2003Published online at http://www.nature.com/natureimmunology/

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