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Leishmania majorCell Development and Resistance to

Th1+B1 Is Required for Optimal CD4κNF-

Phillip ScottGoldschmidt, Jorge Caamaño, Christopher A. Hunter and David Artis, Kendra Speirs, Karen Joyce, Michael

http://www.jimmunol.org/content/170/4/1995doi: 10.4049/jimmunol.170.4.1995

2003; 170:1995-2003; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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NF-�B1 Is Required for Optimal CD4� Th1 Cell Developmentand Resistance toLeishmania major1

David Artis,* Kendra Speirs,* Karen Joyce,* Michael Goldschmidt,* Jorge Caamano,†

Christopher A. Hunter,* and Phillip Scott 2*

The NF-�B family of transcription factors regulates the expression of a wide range of immune response genes involved inimmunity to pathogens. However, the need for individual family members in regulating innate and adaptive immune responsesin vivo has yet to be clearly defined. We investigated the role of NF-�B1 in the induction of protective IL-12-dependent Th1 cellresponses following infection with the intracellular protozoan parasiteLeishmania major. Whereas wild-type C57BL/6 micecontrolled parasite replication, NF-�B1 knockout (KO) mice were susceptible to infection, developing chronic unresolving lesionsassociated with persistent parasites. There was a profound defect in Ag-specific CD4� T cell proliferation and IFN- � productionin infected KO mice, although innate responses—including IL-12 production and control of intracellular parasite replication bymacrophages—were intact. In vitro polyclonal stimulation of purified naive KO T cells revealed an intrinsic defect in CD4� T cellproliferation associated with reduced IL-2 receptor expression, but operating independently of APC function and IL-2 production.Critically, the frequency of proliferating KO CD4 � T cells secreting IFN-� matched that of wild-type cells, suggesting that NF-�B1was not required for efficient transcription of the IFN- � gene. Taken together, these results identify a novel role for NF-�B1 inCD4� T cell proliferation and the development of Th1 cell responses required for protective immunity against intracellularpathogens. The Journal of Immunology, 2003, 170: 1995–2003.

T he mammalian NF-�B family of transcription factors,composed of NF-�B1 (p50), NF-�B2 (p52), RelA, RelB,and c-Rel, plays a critical role in regulating expression of

immune response genes associated with innate and adaptive im-munity to pathogens (1, 2). In resting cells, NF-�B is maintainedin an inactive form in the cytoplasm bound to a family of proteinstermed I�B. After cells are exposed to a broad range of infectiousand inflammatory stimuli, these different signals converge at thelevel of I�B kinase, a complex of three proteins that initiates acascade of signaling events culminating in the phosphorylation,polyubiquitination, and subsequent degradation of I�B (3–5). Dis-sociation of NF-�B from I�B unmasks the nuclear localizationsequence, allowing NF-�B to translocate into the nucleus and ini-tiate gene transcription (6).

The complex role of NF-�B and the specific functions of indi-vidual family members in the development and regulation of im-mune responses following exposure to pathogens have becomeincreasingly apparent as mice with targeted deletions in individualNF-�B family members have become available (reviewed in Refs.7 and 8). For example, the failure of RelB-deficient mice to controlinfection with lymphocytic choriomeningitis virus and Toxo-

plasma gondii (an intracellular protozoan parasite) was associatedwith defective Th1 cell responses (9, 10). In contrast, IFN-� re-sponses and the ability to control viral infections were intact inNF-�B2 knockout (KO)3 and c-Rel KO mice (11–13). Resistanceto intestinal helminth infection was also independent of NF-�B2and c-Rel, but required NF-�B1 (14). Studies have also begun toexamine the roles of specific NF-�B family members in the de-velopment of protective immunity against the intracellular proto-zoan parasite Leishmania major. Control of infection requires theproduction of IL-12 and subsequent expansion of CD4� Th1 cellssecreting IFN-� (15–17). Mice deficient in NF-�B2 exhibited de-fective CD40-induced IL-12 responses after L. major infection,resulting in chronic disease, although Th1 cell responses and NO-mediated parasite killing were intact (13). In contrast, susceptibil-ity to L. major infection in c-Rel KO mice was associated withdefective NO production and parasite killing (18).

Although the role of NF-�B1 in immunity to L. major has notbeen examined, previous studies suggest a role for this familymember in Th2 cell development. NF-�B1-deficient CD4� T cellsfailed to up-regulate GATA-3—a transcription factor critical inTh2 cell development (19, 20)—and exhibited significant defectsin secretion of IL-4 and IL-13 following in vitro stimulation (21).KO mice were also defective in the expression of Th2 cytokines inmurine models of airway hyperresponsiveness and helminth infec-tion (14, 22). However, the role of NF-�B1 in Th1 cell polarizationand IFN-� production has remained controversial (23). Whereas invitro studies have reported that expression of T-bet—a criticaltranscription factor in Th1 cell development (24, 25)—and IFN-�production were normal in NF-�B1-deficient T cells (21), impairedIFN-� responses and less severe Th1-mediated pathology have

*Department of Pathobiology, School of Veterinary Medicine, University of Penn-sylvania, Philadelphia, PA 19104; and †Medical Research Council Center for ImmuneRegulation, School of Medicine, University of Birmingham, Birmingham, UnitedKingdom

Received for publication September 13, 2002. Accepted for publication December6, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant 059967/B/99/Z from The Wellcome Trust (toD.A.) and by Grants AI.35914 (to P.S.) and AI.46288 (to C.A.H.) from the NationalInstitutes of Health.2 Address correspondence and reprint requests to Dr. Phillip Scott, Department ofPathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800Spruce Street, Philadelphia, PA 19104.

3 Abbreviations used in this paper: KO, knockout; WT, wild type; SLA, solubleleishmanial Ag; LN, lymph node; BMDM, bone marrow-derived macrophages; DC,dendritic cells; MFI, mean fluorescence intensity; CpG, cytosine-phosphorothioate-guanine.

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been reported in KO mice in vivo following induction of autoim-munity (26).

Here we investigated the role of NF-�B1 in the development ofprotective Th1 responses after L. major infection. Whereas wild-type (WT) C57BL/6 mice effectively controlled parasite replica-tion, NF-�B1 KO mice developed chronic lesions associated withpersistent parasites. Susceptibility to infection was not associatedwith defective innate immune responses, as IL-12 production andparasite killing by macrophages were intact. In contrast, CD4� Tcell functions were dramatically impaired in the absence of NF-�B1, with infected KO mice exhibiting low Ag-specific prolifer-ation and IFN-� responses. In vitro polyclonal stimulation of pu-rified KO T cells demonstrated a proliferation defect in NF-�B1KO CD4� T cells associated with defective IL-2R expression.Furthermore, analysis of proliferating KO CD4� T cells suggestedthat defects in IFN-� production were due to defective T cell pro-liferation, rather than a direct effect on cytokine gene transcription.Taken together, these results suggest that in addition to influencingTh2 cell development, NF-�B1 activation is a critical upstreamsignaling event required for optimal entry of CD4� T cells into theproliferative cycle.

Materials and MethodsAnimals

Female C57BL/6 mice were purchased from The Jackson Laboratory (BarHarbor, ME). NF-�B1-deficient mice were generated by Dr. W. Sha andDr. D. Baltimore (Berkeley, CA) (27) and bred at the University of Penn-sylvania on a B6 � 129 background or backcrossed at least eight times toC57BL/6. In most experiments, groups of four to six backcrossed female(5–8 wk old) mice were used. No differences were observed between KOmice on a B6 or B6 � 129 background. Animals were maintained in aspecific-pathogen-free environment and tested negative for pathogens inroutine screening. All experiments were conducted following the guide-lines of the University of Pennsylvania institutional animal care and usecommittee.

Parasite, Ag, and infection

L. major parasites (MHOM/IL/80/Friedlin) were grown in Grace’s insectculture medium (Life Technologies, Gaithersburg, MD) supplemented with20% heat-inactivated FBS (HyClone Laboratories, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 �g/ml streptomycin (Sigma-Al-drich, St. Louis, MO). Amastigote parasites were isolated from lesions ofinfected recombination-activating gene KO mice. Soluble leishmanial Ag(SLA) was prepared as previously described (28). Mice were injected in thehind left footpad with 5 � 106 stationary phase promastigote parasites.Footpad swelling was measured weekly using digital calipers (Mitutoyo,Kanagawa-Ken, Japan), and lesion size was determined by subtracting thesize of the uninfected contralateral footpad from the size of the infectedfootpad. To quantify parasites in tissues, single-cell suspensions of lesionswere prepared and plated in 10-fold serial dilutions (initial dilution of1:100) in Grace’s insect culture medium. Each sample was plated in qua-druplicate, and the mean of the negative log parasite titer was determinedafter 7 days of culture at 26°C.

EMSAs

Popliteal lymph node (LN) cells were isolated from naive and infectedmice, and nuclear extracts were prepared from B cell-depleted LN culturesas previously described (29). In brief, double-stranded oligodeoxynucle-otides corresponding to the palindromic B site (5�-GGGAATTCCC-3�)were labeled by filling the overlapping ends with the Klenow fragment ofDNA polymerase I and [32P]dCTP. Unincorporated nucleotides were re-moved, and 10 fmol of labeled oligonucleotide (50,000 cpm) were incu-bated with 10 �g of protein extracts and 2 �g of poly(dI-dC) in a buffercontaining 20 mM HEPES (pH 7.9), 100 mM NaCl, 5 mM MgCl2, 1 mMDTT, 0.7 mM PMSF, and 17% glycerol in a final volume of 22 �l for 15min at 20°C. Equivalent free probe was detected in all wells, confirmingthat probe was added in excess to all samples. Antisera against p50 (SC114-G) (Santa Cruz Biotechnology, Santa Cruz, CA) were used in super-shift assays. Complexes were separated on 5.5% polyacrylamide gels runon 0.25% Tris-borate-EDTA buffer, dried, and exposed to Kodak X-OmatAR film (Rochester, NY) at �70°C.

Histological analysis

Footpad tissues were fixed in 10% neutral buffered formalin, decalcified,routinely processed, and embedded in paraffin. Five micrometer sectionswere stained with H&E before microscopic evaluation.

Bone marrow cultures, NO production, and in vitro killingassays

Bone marrow-derived macrophages (BMDM) and dendritic cells (DC)were cultured as previously described (30–32). In brief, macrophages weregrown in RPMI containing 10% FCS, 100 U/ml penicillin, 100 �g/mlstreptomycin, 2 mM glutamine, and 25 mM HEPES and supplementedwith 30% L929 cell-conditioned medium for 7 days. DC were cultured for10 days in medium supplemented with rmGM-CSF (PeproTech, RockyHill, NJ). Cells were resuspended at 1 � 106 cells/ml in 96-well plates andcultured either in medium alone, in LPS (100 ng/ml) (Sigma-Aldrich), inanti-CD40 mAb (20 �g/ml) (IC10 clone; R&D Systems, Minneapolis,MN), or in cytosine-phosphorothioate-guanine (CpG)-containing oligonu-cleotide (5�-TTCATGACGTTCCTGACGTT-3�) (1 �g/ml) (synthesized atthe University of Pennsylvania) (33, 34) for 48 h. Nitrite production wasdetermined by the Greiss reaction (35). For in vitro killing assays, adherentmacrophages were harvested, resuspended at 1 � 106/ml in polypropylenetubes, and primed with 100 U/ml rmIFN-� (R&D Systems) for 4 h. Fol-lowing incubation of suspension cultures with L. major amastigotes (2:1parasite:cell ratio) for 2 h, cells were washed to remove extracellular par-asites, resuspended in medium containing rmIFN-� (100 IU/ml), and cul-tured for 72 h at 34°C as previously described (36). Cytospins were pre-pared and stained with Hema 3 (Biochemical Sciences, Swedesboro, NJ),and the number of intracellular parasites was determined under lightmicroscopy.

Cell culture and analysis of cytokine secretion

Spleen cells were harvested and single-cell suspensions prepared inDMEM supplemented with 10% heat-inactivated FBS, 2 mM glutamine,100 U/ml penicillin, 100 �g/ml streptomycin, 25 mM HEPES, and 5 �10�5 M 2-ME. Cells were plated at 4 � 106/ml in 24-well culture plates inmedium or SLA (50 �g/ml). In addition, 5 �g/ml anti-IL-4R mAb (M1;a gift from Dr. F. Finkelman, University of Cincinnati, and Dr. C.Maliswjeski, Immunex, Seattle, WA) was added to cultures to enhancedetection of IL-4. Supernatants were harvested after 72 h and assayed forcytokine production by sandwich ELISA using paired mAb to detect IL-4(11B11 and BVD6-24G2.3), IFN-� (R46A2 and polyclonal rabbit anti-IFN-�), and IL-12 (C17.8 and C15.6).

CFSE labeling, T cell proliferation, and intracellular cytokineanalysis

Cells were harvested from naive and infected mice, and single-cell sus-pensions prepared. In some experiments, CD3� T cells were purified usingT cell enrichment columns (R&D Systems) following manufacturer’s in-structions. Purities obtained using this technique were determined byFACS analysis (see below) and were routinely between 85 and 90%. Pro-liferation was analyzed by labeling cells with CFSE (Molecular Probes,Eugene, OR), a fluorescent dye that makes cell division visible after stim-ulation (37). Cells were washed and suspended in PBS at 4 � 106/ml andlabeled with CFSE (1.25 �M) for 5 min at 37°C. Labeling was quenchedwith FBS, and cells were washed twice and resuspended in DMEM sup-plemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mlpenicillin, 100 �g/ml streptomycin, 25 mM HEPES, and 5 � 10�5 M2-ME. Cells were cultured at 2 � 105 cells/well in 96-well U-bottom platesor at 4 � 106/ml in 24-well culture plates (Costar, Cambridge, MA) inmedium alone, in SLA (50 �g/ml), or in anti-CD3/anti-CD28 (each at 1�g/ml) (BD PharMingen, San Diego, CA) for 3–5 days. In some experi-ments cells were cultured under Th1 polarizing conditions (rmIL-12, 10ng/ml (a gift from Dr. S. Wolf and Dr. J. Sypek, Genetics Institute, Cam-bridge, MA); anti-IL-4 mAb (11B11), 10 �g/ml) or in the presence ofexogenous IL-2 (500 IU/ml; Chiron, Emeryville, CA). The reduction inintensity of CFSE labeling, concomitant with cell division, was comparedwith cells cultured in medium alone. Before harvesting, supernatants werecollected for ELISA analysis, and cells were pulsed with PMA (50 ng/ml),ionomycin (500 ng/ml), and brefeldin A (10 �g/ml) (Sigma-Aldrich) for4–5 h. Cells were washed; incubated with Fc block (anti-FcR�II/III mAb(2.4G2) (10 �g/ml) and rat IgG (10 �g/ml)) for 10 min; surface-stainedwith anti-CD4, anti-CD25, or isotype control mAb (BD PharMingen); andfixed overnight in 1% paraformaldehyde (Electron Microscopy Sciences,Fort Washington, PA). Cells were then permeabilized with 0.2% saponin(Sigma-Aldrich) and intracellularly stained with anti-IL-2, anti-IFN-�, orisotype control mAb (BD PharMingen). Cells were washed, and 1 � 104

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live CD4� T cells were acquired on a FACSCalibur cytometer (BD Bio-sciences, San Jose, CA). Acquisition and analysis were conducted usingCellQuest software (BD Biosciences). All dot plots shown have a log axisof 100–104.

ResultsNF-�B1 is activated following L. major infection of resistantB6 mice

Previous studies have shown that NF-�B is activated in vitro or invivo after exposure to a range of pathogens including viruses, bac-teria, and parasites (7, 8). To determine whether infection of re-sistant B6 mice with L. major resulted in the activation of NF-�B,nuclear extracts were prepared from B cell-depleted lymphocytesuspensions isolated from naive and infected mice, and �B bindingactivity was analyzed by EMSA. As shown in Fig. 1, NF-�B bind-ing was not detected in nuclear extracts isolated from naive mice(samples from naive mice were collected on day 0 and day 42 p.i.;only those from day zero are included, because equivalent resultswere obtained with both). In contrast, NF-�B binding was detectedin the draining LN of infected mice as early as day 3 p.i. andremained at day 42. A 100-fold molar excess of unlabeled com-petitor oligonucleotide was routinely used in competition assaysand efficiently blocked detection of NF-�B binding (data notshown). Addition of anti-p50 mAb resulted in a shift in NF-�Bcomplexes at both time points, demonstrating that NF-�B1-con-taining complexes were activated after L. major infection (Fig. 1).The presence of two distinct bands implies that at least two dif-ferent NF-�B complexes were activated following infection.

NF-�B1 KO mice fail to control L. major infection

We investigated whether the deletion of NF-�B1 would affect theoutcome of L. major infection using gene-targeted mice. In WTmice, lesion development peaked between wk 3 and 4 and resolvedby wk 12 p.i. (Fig. 2A). In contrast, NF-�B1 KO mice developedchronic lesions that failed to resolve by wk 18 p.i. Interestingly,lesion development in infected KO mice was significantly delayedin the first 4 wk p.i. (Fig. 2A). Because infection of KO mice isassociated with defective CD4� T cell proliferation (see below),this observation is consistent with previous reports demonstratingthat early lesion development after Leishmania infection is aCD4� T cell dependent phenomenon (13, 38–40). At wk 10 p.i.,cutaneous lesions in WT mice were resolving (Fig. 2A). Histolog-ical evaluation demonstrated the presence of few viable parasitesand a small number of inflammatory cells in the dermis, typical ofresistant strains at this time point (Fig. 2B). In contrast, foci of

inflammatory cells (composed of granulocytes, lymphocytes, andmacrophages), tissue necrosis, and fibrin deposition were presentin the dermal layer of lesions isolated from infected KO mice (Fig.2C). The epidermis was also thickened, with evidence of micro-abscesses, focal necrosis, and spongiosis. Numerous viable intra-cellular parasites were observed within epidermal keratinocytes(Fig. 2D, arrows), suggesting that a reservoir of infection existedin the epidermal layer of infected KO mice.

Consistent with the development of chronic lesions (Fig. 2A)and the presence of parasites in histological sections from KOmice (Fig. 2, C and D), quantification of parasite numbers in thefootpad by limiting dilution demonstrated that KO mice had sig-nificantly more parasites than WT mice had beyond wk 4 p.i. (Fig.2E). By wk 10 p.i., parasite numbers in WT mice had fallen belowthe limit of detection, while viable promastigotes were detected at10�8 dilutions in lesions isolated from KO mice (Fig. 2E). Inter-estingly, the number of parasites did not become progressivelygreater in the absence of NF-�B1 (as is the case in susceptibleBALB/c or recombination-activating gene KO mice), demonstrat-ing that NF-�B1 KO mice were able to control parasite replicationto some extent—although the response was not sufficient to me-diate disease resolution.

Intact macrophage function in NF-�B1 KO mice

Previous studies demonstrated that disruption of NF-�B activationcan impair the production of NO (41), an effector molecule critical

FIGURE 1. NF-�B1 is activated in the draining popliteal LN followingL. major infection. B6 mice were infected with 5 � 106 L. major promas-tigotes in the left hind footpad, and nuclear extracts were prepared from Bcell-depleted draining popliteal LN cell suspensions at days 3 and 42 p.i.Extracts were incubated with palindromic �B oligonucleotides and ana-lyzed by EMSA. Supershift analysis following addition of anti-p50 mAbdemonstrated that NF-�B1 was activated in both homo- and heterodimericforms. Results are representative of three experiments.

FIGURE 2. NF-�B1-deficient mice develop chronic lesions and fail tocontrol parasite replication following L. major infection. KO and WT micewere infected with 5 � 106 L. major promastigotes in the left hind footpad.A, Lesion size was determined by subtracting the size of the uninfectedcontralateral footpad from the size of the infected footpad. Histologicalanalysis at wk 10 p.i. demonstrated that while lesions had resolved in WTmice (B), KO mice exhibited chronic lesions composed of foci of inflam-matory cells (�), fibrin deposition (�), and persistent parasites (arrows) indermal and epidermal tissues (C and D). E, Mice were sacrificed at varioustimes p.i., and the parasite load in the footpad was quantified by limitingdilution analysis. Results are expressed as the mean � SD for four or fivemice per group and are representative of three experiments. �, Significantdifference between KO and WT (p � 0.05). Bar, 40 �m (B, C) or 5 �m (D).

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to the control of L. major infection (42, 43). Therefore, we inves-tigated whether NF-�B1-deficient macrophages were defective inNO production and the control of L. major replication in vitro.Following stimulation of IFN-�-activated BMDM with either LPSor CpG, nitrite production was greater in cultures of KO macro-phages than in WT (Fig. 3A). To investigate whether the absenceof NF-�B1 altered the control of L. major replication in vitro,IFN-�-activated KO and WT macrophages were infected with L.major and cultured for 72 h. Consistent with intact NO responses(Fig. 3A), no significant differences were observed in the numberof parasites per 100 KO or WT macrophages examined, suggestingequivalent control of parasite replication by KO and WT cells (Fig.3B). In addition, equivalent control of parasite replication was ob-served in infected KO and WT macrophages treated with LPS(data not shown). Together, these results suggested that activatedNF-�B1-deficient macrophages were able to produce NO and tocontrol growth of intracellular L. major.

Defective Ag-specific IFN-�, but not IL-12 responses, in L.major infected NF-�B1 KO mice

Analysis of cytokine responses at wk 2 and 8 p.i. revealed signif-icantly lower levels of Ag-induced IFN-� in cultures of KO cellscompared with WT cells (Fig. 4, A and B). Flow cytometric anal-ysis of lymphocyte cultures also demonstrated that a significantlylower percentage of KO as opposed to WT CD4� T cells hadshifted in their forward scatter (KO, 10.8 � 1.5; WT, 48.6 � 7.4)(Fig. 4, C and D), an indication that the proportion of cells under-going proliferation was lower in KO cultures (see below). In ad-dition, intracellular cytokine staining showed a significantly lowerfrequency of KO CD4� T cells staining positive for IFN-� at wk8 p.i. (KO, 1.4 � 0.1%; WT, 23 � 5%) (Fig. 4, C and D). How-ever, the failure of KO mice to control infection was not due to thedevelopment of a nonprotective Th2 response, because the levelsof IL-4 were either lower (consistent with previous reports of de-fective Th2 response in NF-�B1 KO mice) (14, 21) or equivalentfollowing stimulation of KO and WT cells (Fig. 4, E and F).

The development of protective IFN-� responses following L.major infection is dependent on IL-12 production (44–46). Be-cause previous studies have shown that NF-�B family members,notably c-Rel and NF-�B2, can regulate IL-12 p40 production inresponse to certain stimuli (13, 47, 48), we analyzed infection-induced IL-12 p40 levels in NF-�B1 KO mice. No significant dif-ferences in the production of IL-12 were observed between KO and WT mice at wk 4, 8, and 12 p.i. (Fig. 5, A–C), suggesting that

NF-�B1 is not essential for IL-12 responses after L. major infec-tion. IL-12 responses were also normal following in vitro stimu-lation of NF-�B1 KO BMDM or DC with LPS or anti-CD40 mAb(Fig. 5, D and E), both potent inducers of IL-12. In addition, invivo serum IL-12 p40 responses following systemic exposure toLPS or anti-CD40 mAb were equivalent in KO and WT mice (LPSstimulation: KO, 157.0 � 7.1 ng/ml; WT, 105.3 � 1.8 ng/ml;anti-CD40 stimulation: KO, 143.0 � 8.5 ng/ml; WT, 159.0 � 13.5ng/ml). Taken together, these results suggested that the defectivedevelopment of Ag-specific CD4� T cells secreting IFN-� in L.major-infected NF-�B1 KO mice was not due to a deficiency inIL-12 production.

NF-�B1 KO CD4� T cells have an intrinsic defect inproliferation

Previous studies have shown that the expression of IFN-� isclosely linked to lymphocyte proliferation (49, 50). Therefore, weinvestigated the proliferative capability of NF-�B1-deficientCD4� T cells isolated from L. major-infected mice. Proliferationfollowing ex vivo exposure to Ag was determined by evaluatingCFSE dilution of CD4� T cells using flow cytometry. A clear shift

FIGURE 3. NO production and control of L. major replication in vitroare intact in NF-�B1-deficient macrophages. A, Macrophages were grownfrom KO and WT bone marrow in vitro and stimulated with LPS or CpGfor 48 h; nitrite levels were assessed by Greiss reaction. Macrophages werealso infected with L. major and cultured for 72 h in the presence ofrmIFN-�. B, Cytospins were prepared, and the number of infected cells wasanalyzed under light microscopy. Results are expressed as the mean of twoor three animals per group and are representative of two experiments. �,Significant difference between KO and WT (p � 0.05).

FIGURE 4. NF-�B1 KO mice develop poor Ag-specific Th1 responsesfollowing L. major infection. At wk 2 and 8 p.i., spleen cells were isolatedand cultured in medium alone (med) or with SLA for 72 h. Supernatantswere assayed for IFN-� (A and B) and IL-4 (E and F) production by sand-wich ELISA. Values represent the mean � SD for four or five mice pergroup. The frequency of WT (C) and KO (D) splenic CD4� T cells se-creting IFN-� after restimulation with SLA at wk 8 p.i. was analyzed byFACS. Numbers represent the percentage of CD4� T cells secreting IFN-�.�, Significant difference between KO and WT (p � 0.05).

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in CFSE profile was observed in CD4� T cells from infected WTmice as early as 2 wk p.i., and this shift grew in magnitude as theinfection progressed (Fig. 6, A and B). In contrast, a profounddefect in proliferation was detected in KO CD4� T cells through-out the course of infection (Fig. 6, A and B).

To further investigate the defect in CD4� T cell proliferation,spleen cells were isolated from naive NF-�B1 KO and WT mice,labeled with CFSE, and stimulated with soluble anti-CD3/anti-CD28 under neutral and Th1 polarizing conditions. As shown inFig. 7, the dilution of intracellular CFSE staining distinguishedindividual generations of dividing cells 3 days after stimulation. Atleast four generations of dividing CD4� T cells were visible in WTcultures, while the majority of NF-�B1 KO CD4� T cells had onlyprogressed as far as the second proliferative generation (Fig. 7A).We observed similar results using anti-CD3 stimulation alone(data not shown). To determine whether the reduced proliferationobserved in NF-�B1-deficient CD4� T cells was related to defec-tive growth or survival signals from APC or from accessory cells,CD3� T cells were purified from KO and WT mice and stimulatedwith plate-bound anti-CD3/anti-CD28 for 3 days. Consistent withour results with whole spleen cell cultures, four generations ofproliferating CD4� T cells were visible in cultures of purified Tcells from WT mice, while only two generations were discernablein KO cultures (Fig. 7B)—demonstrating that NF-�B1-deficientCD4� T cells had an intrinsic defect in their ability to proliferatefollowing stimulation through the TCR and CD28.

Production of IFN-� by proliferating NF-�B1 KO CD4� T cellsis intact following in vitro polyclonal stimulation

In addition to regulating T cell proliferation, NF-�B can directlyregulate expression of IFN-� through �B binding sites in the

IFN-� promoter (51). We investigated whether the defects inIFN-� production after stimulation were due to a direct effecton IFN-� gene transcription or were an indirect effect of defectiveT cell proliferation. Spleen cells were labeled with CFSE and stim-ulated with anti-CD3/anti-CD28; the frequency of proliferatingCD4� T cells expressing IFN-� was determined by flow cytom-etry. Confirming our previous results, the frequency of NF-�B1KO CD4� T cells undergoing proliferation was lower than thatobserved in cultures of WT cells (Fig. 8, A and B). However, thefrequency of proliferating CD4� T cells that produced IFN-� wassimilar in KO and WT cultures under both neutral and Th1 polar-izing conditions (Fig. 8, A and B).

To further examine whether IFN-� responses were equivalent inproliferating KO and WT CD4� T cells, we analyzed the fluores-cence intensity of the IFN-�-positive cells from KO and WT T cellcultures. Despite equivalent frequencies of IFN-�-positive prolif-erating cells, the mean fluorescence intensity (MFI) of the IFN-�signal was lower in proliferating KO than in WT cultures (KO, 41;WT, 51). This difference may have been the result of reducedtranscription of the IFN-� gene in KO T cells or because IFN-�bright T cells (i.e., those with the highest MFI) were found pre-dominantly in the fourth generation in WT cultures, a populationthat was absent in cultures of KO T cells. Therefore, the reducedMFI of IFN-� staining in KO T cells may have been the result ofimpaired proliferation. To test this, we conducted T cell genera-tion-specific analysis of the fluorescence intensity of IFN-� stain-ing. We found that the geometric mean of IFN-� staining was

FIGURE 5. IL-12 production is normal in NF-�B1 KO mice. KO andWT mice were infected with 5 � 106 L. major promastigotes in the lefthind footpad. At wk 4 (A), 8 (B), and 12 (C) p.i., spleen cells were isolatedand cultured in medium (med) or SLA for 72 h. BMDM (D) and DC (E)were isolated from naive KO and WT mice and cultured in medium, LPS,or �CD40 mAb for 48 h. Supernatants were assayed for IL-12 p40 pro-duction by sandwich ELISA. Values represent the mean � SD for four orfive mice per group.

FIGURE 6. Ag-specific CD4� T cell proliferation is impaired in NF-�B1 KO mice following L. major infection. Spleen cells were isolated frominfected KO and WT mice, labeled with CFSE, and cultured in medium orSLA for 5 days. Cells were stained for CD4, and Ag-specific proliferationwas analyzed by flow cytometry at wk 2 (A) and 8 (B) p.i. Gray linedenotes the proliferation of labeled cells cultured in medium alone. Resultsare representative of three or four mice per group at each time point.

FIGURE 7. Naive NF-�B1 KO CD4� T cells are defective in prolifer-ation following in vitro polyclonal stimulation. Whole spleens (A) or pu-rified CD3� T cells (B) were labeled with CFSE and cultured for 3 dayswith anti-CD3 and anti-CD28 under neutral or Th1 polarizing conditions.Cells were surface-stained for CD4, and proliferation was analyzed by flowcytometry. Results are representative of three experiments.

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equivalent in the first, second, and third generations of KO and WTcultures (first generation: KO, 39.7; WT, 34.3; second generation:KO, 40.9; WT, 42.5; third generation: KO, 47.5; WT, 56.5). Theintensity of IFN-� staining increased markedly in the fourth gen-eration of proliferating WT T cells (83.9), suggesting that the re-duced overall MFI of IFN-� staining observed in KO T cells wasthe result of reduced T cell proliferation. Together, the similar

frequency of proliferating T cells secreting IFN-� and the equiv-alent MFI of the IFN-� signal between KO and WT CD4� T cellsin early generations provide compelling evidence that defectiveIFN-� production in NF-�B1 KO mice was a result of defectiveCD4� T cell proliferation and was not related to direct transcrip-tional regulation of the IFN-� gene. Furthermore, these results in-dicated that IL-12R expression and responsiveness were intact inKO CD4� T cells.

IL-2R expression is impaired in NF-�B1 KO CD4� T cellfollowing in vitro polyclonal stimulation

NF-�B family members play a critical role in regulating the ex-pression of IL-2 and IL-2R� (CD25) genes after T cell activation(52–54). Therefore, we investigated whether a deficiency in theexpression of IL-2 or its receptor may have been responsible forthe defects in T cell proliferation observed in NF-�B1 KO mice.Following stimulation of whole spleen cultures, the frequency ofCD4� T cells staining positive for intracellular IL-2 was higher inKO cells than in WT cells (66% vs 41%) (Fig. 9A). Elevated levelsof secreted IL-2 were also detected by ELISA in culture superna-tants from KO cells after polyclonal stimulation for 3 days (KO,3180 pg/ml; WT, 520 pg/ml). These results suggested that defectsin IL-2 production were not the basis for defective CD4� T cellproliferation in KO T cells. Supporting this hypothesis, adminis-tration of exogenous IL-2 enhanced proliferation in WT T cellcultures but failed to reverse the proliferation defect in NF-�B1KO T cell cultures (Fig. 9B). Because IL-2 production is an earlyevent in T cell activation, occurring in the G0-S phase (55), theseresults also seemed to show that early T cell activation was normalin KO mice.

In contrast to the intact IL-2 responses in NF-�B1 KO T cells,there was a significant defect in the ability of KO CD4� T cells to

FIGURE 8. Production of IFN-� by proliferating NF-�B1 KO CD4� Tcells is intact after in vitro polyclonal stimulation. Spleen cells were iso-lated from naive KO and WT mice, labeled with CFSE, and cultured for 3days with anti-CD3 and anti-CD28 under neutral or Th1 polarizing con-ditions. Cells were stained for surface CD4 and intracellular IFN-�. Pro-liferation and IFN-� production in WT (A) and KO (B) CD4� T cells wereanalyzed by flow cytometry. Generation-specific analysis was conducted todetermine the geometric MFI of IFN-�-positive cells in individual prolif-erating generations. Numbers represent the frequency of proliferatingCD4� T cells staining positive for IFN-�. Results are representative ofthree experiments. ND, Not done.

FIGURE 9. IL-2R expression is impaired in NF-�B1 KO CD4� T cells following in vitro polyclonalstimulation. Spleen cells were isolated from naive KOand WT mice and cultured for 3 days in medium or inanti-CD3 and anti-CD28. Cells were stained for CD4,intracellular IL-2 (A), and surface CD25 (C). B, Ex-ogenous IL-2 was administered to polyclonally stim-ulated KO and WT T cells, and proliferation was an-alyzed by dilution of CFSE. Numbers in A and Cdenote the frequency of positively stained CD4� Tcells and are representative of two experiments.

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up-regulate CD25 following stimulation. Whereas 53% of prolif-erating WT CD4� T cells expressed high levels of CD25, only25% did so in KO cultures, with significantly more KO CD4� Tcells expressing only intermediate levels of CD25 (63% in KOcultures compared with 38% in WT cultures) (Fig. 9C). Histogramanalysis also demonstrated that the MFI of IL-2R staining overbackground was 39% lower on stimulated KO CD4� T cells thanon WT CD4� T cells (KO, 401.0 � 17.1; WT, 655.1 � 118.7).This observation provides a possible explanation as to why KO Tcells failed to respond to exogenous IL-2 (Fig. 9B). Collectively,these results identified an intrinsic proliferative defect in NF-�B1-deficient CD4� T cells, operating independently of APC functionand IL-2 production. Furthermore, while early events in T cellactivation remained intact, NF-�B1 KO CD4� T cells exhibited adefect in their ability to up-regulate expression of CD25.

DiscussionThere is increasing evidence that NF-�B family members playunique roles in regulating innate and adaptive immune responsesto a range of pathogens. In the case of NF-�B1, previous studieshave demonstrated its importance in innate responses to viral andbacterial infection and in the expression of Th2 cytokines follow-ing helminth infection or induction of airway hyperresponsiveness(14, 22, 27). In this report we extend these observations, identify-ing a novel role for NF-�B1 in the development of polarized Th1cytokine responses and resistance to the intracellular protozoanparasite Leishmania major. Mice deficient in NF-�B1 developedchronic infections associated with a failure to control parasite rep-lication. Although IL-12 responses and parasite killing were inde-pendent of NF-�B1, KO mice exhibited a profound defect in thedevelopment of Ag-specific CD4� T cells secreting IFN-�. Poly-clonal activation of naive KO T cells demonstrated a specific re-quirement for NF-�B1 in regulating CD4� T cell proliferation inresponse to stimulation through the TCR and CD28. These resultssuggest that NF-�B1 may be critical to the upstream signalingevents involved in optimal T cell responsiveness to IL-2 and entryinto the proliferative cycle, which is necessary for the subsequentdevelopment of polarized Th cell responses.

Susceptibility to L. major infection has also been reported inmice deficient in NF-�B2 or c-Rel. Chronic infection in NF-�B2KO mice was associated with defective CD40-induced IL-12 re-sponses, while susceptibility to infection in c-Rel KO mice wasattributed to defective NO production and parasite killing (13, 18).Therefore, while innate responses operating against L. major, in-cluding IL-12 production and parasite killing, were partially de-pendent on NF-�B2 and c-Rel, respectively, they were indepen-dent of NF-�B1. In contrast, the results presented here identify anessential role for NF-�B1 in adaptive immune responses to L. ma-jor, because susceptibility to infection in NF-�B1 KO mice wasassociated with a marked defect in the expansion of Ag-specificCD4� T cells secreting IFN-�. Together, these studies highlightthe complex regulatory role that different NF-�B family membersplay in controlling innate and adaptive responses after infectionwith intracellular pathogens.

Our results demonstrating that NF-�B1 is required for the de-velopment of polarized Th1 responses are in conflict with a pre-vious in vitro study that concluded that Th1 cell differentiation wasintact in NF-�B1 KO CD4� T cells (21). However, these differ-ences may reflect the genetic background of the mice, or the mag-nitude and duration of the stimulation used in the different studies.A role for NF-�B1 in regulating T cell proliferation and Th1 re-sponses is supported by studies in a murine model of collagen-induced arthritis. In the absence of NF-�B1, IFN-� production andthe severity of Th1-mediated pathology were significantly reduced

(26). In addition, the original description of NF-�B1 KO micereported that KO T cells exhibited a proliferation defect, althoughno data were included (27). To our knowledge, this is the firstdetailed analysis of proliferation and its effect on IFN-� productionin NF-�B1-deficient CD4� T cells.

The importance of other NF-�B family members in regulating Tcell proliferation has been documented in a number of studies.Analysis of I�B transgenic T cells that express a degradation-de-ficient form of I�B, which efficiently blocked c-Rel and RelA ac-tivation, demonstrated the importance of NF-�B for optimal T cellproliferation (56–58). Similar defects were observed in T cellsisolated from mice individually deficient in RelA or c-Rel (59–61). Here we extend these studies, identifying a critical role forNF-�B1 in optimal CD4� T cell proliferation. Although the mech-anisms underlying defective T cell proliferation once the NF-�Bpathway is disrupted are not fully understood, the presence of �Bbinding sites in the promoters of the IL-2 and CD25 genes (bothcrucial for optimal T cell activation and homeostasis) (62, 63)suggest that NF-�B may regulate the expression of these genesfollowing TCR ligation. However, IL-2 and CD25 levels were nor-mal in RelA-deficient T cells, indicating that this family memberwas not essential to the control of these genes (60). In c-Rel-de-ficient T cells, IL-2 production was reportedly defective, whileconclusions vary on the expression of CD25 and the responsive-ness to exogenous IL-2 (59, 61, 64). Unlike T cells deficient inc-Rel or RelA, the frequency of NF-�B1-deficient T cells secretingIL-2 in our experiments was greater than that observed in WTcultures following stimulation through the TCR and CD28. Thisresult is supported by previous studies that suggest that NF-�B1acts as a transcriptional repressor of cytokine genes under certaincircumstances (65–68). Furthermore, while expression of CD25has been reported to be normal in other NF-�B KO mice, ourresults show that NF-�B1 is critical for the optimal up-regulationof CD25. A defect in CD25 expression and IL-2 responsiveness isalso supported by our results, which demonstrated that prolifera-tion in KO CD4� T cell cultures did not improve after adminis-tration of exogenous IL-2. Taken together, these results identifydistinct mechanisms that underlie the defective proliferation ob-served in T cells deficient in individual NF-�B proteins, highlight-ing the elaborate role the NF-�B family plays in the regulation ofimmune response genes after T cell activation.

NF-�B has also been implicated in the regulation of signalingdownstream of the IL-2R (56, 58, 69); in addition to defectiveCD25 expression, NF-�B1 KO T cells may exhibit defects in thesubsequent signaling events that follow activation through CD25.Indeed, an attractive hypothesis is that NF-�B1 regulates STAT5aactivation, a transcription factor required for IL-2-mediated induc-tion of CD25 (70). In support of this hypothesis, it has been foundthat CD4� T cells deficient in STAT5a were also deficient inCD25 but not in IL-2 expression (70)—an effect similar to thephenotype we observed in NF-�B1 KO T cells. Alternatively, thedefects in T cell proliferation in the absence of NF-�B1 may in-volve pathways independent of CD25 expression and the activa-tion of STAT5a. For instance, NF-�B may directly or indirectlyregulate other early events in T cell activation, including control ofcyclin-dependent kinases. NF-�B1, in connection with other fam-ily members, has been shown to associate with cyclin E/CDK2, thenuclear abundance of which peaks in G1/S—concomitant with ir-reversible commitment to DNA synthesis and cell division (71,72). Other studies have demonstrated that NF-�B activation islinked to cell cycle progression through transcriptional activationof cyclin D, another cyclin required for passage through early cellcycle checkpoints (73, 74). However, the specific role of NF-�B1

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in regulating cyclin expression and other cell cycle machinery afterT cell activation remains to be defined.

The failure of NF-�B1 KO mice to mount protective Th1 cyto-kine responses sufficient to resolve L. major infection may alsohave implications for understanding the molecular mechanismsthat underlie chronic leishmaniasis. Murine models of cutaneousand visceral leishmaniasis posited a critical role for IL-2/IL-2Rinteractions in promoting optimal T cell responses, IFN-� produc-tion, and parasite killing (75–78). In addition, nonhealing diffusecutaneous lesions reported in human patients have been associatedwith reduced IL-2 and/or CD25 expression and defective T cellresponses (79, 80). Therefore, aberrant NF-�B1 activation mayprovide a molecular trigger for the development of anergic T cellresponses and chronic disease in humans. Taken together, theseresults suggest that targeting NF-�B1 levels may prove useful inmanipulating a range of Th cell-mediated inflammatory and patho-logical conditions associated with anergic responses, autoimmu-nity, and chronic inflammation.

AcknowledgmentsWe thank Dr. D. Baltimore and Dr. W. Sha for providing the originalNF-�B1 KO mice used in these experiments, and members of the Depart-ment of Pathobiology for useful discussions.

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