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The Journal of Immunology, 2006, 176: 4793-4803.
Copyright © 2006 by The American Association of Immunologists

Coordination of NF-{kappa}B and NFAT Antagonism by the Forkhead Transcription Factor Foxd11

Ling Lin* and Stanford L. Peng2,*,{dagger}

* Division of Rheumatology, Department of Internal Medicine, and {dagger} Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Forkhead transcription factors play critical roles in the maintenance of immune homeostasis. In this study, we demonstrate that this regulation most likely involves intricate interactions between the forkhead family members and inflammatory transcription factors: the forkhead member Foxd1 coordinates the regulation of the activity of two key inflammatory transcription factors, NF-AT and NF-{kappa}B, with Foxd1 deficiency resulting in multiorgan, systemic inflammation, exaggerated Th cell-derived cytokine production, and T cell proliferation in autologous MLRs. Foxd1-deficient T cells possess increased activity of both NF-AT and NF-{kappa}B: the former correlates with the ability of Foxd1 to regulate casein kinase 1, an NF-AT inhibitory kinase; the latter with the ability of Foxd1 to regulate Foxj1, which regulates the NF-{kappa}B inhibitory subunit I{kappa}Bbeta. Thus, Foxd1 modulates inflammatory reactions and prevents autoimmunity by directly regulating anti-inflammatory regulators of the NF-AT pathway, and by coordinating the suppression of the NF-{kappa}B pathway via Foxj1. These findings indicate the presence of a general network of forkhead proteins that enforce T cell quiescence.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many inflammatory reactions, such as autoimmune diseases or transplant rejection, are coordinated by Th cells, whose immediate-early activation is regulated in large part by members of the NF-{kappa}B and NF-AT transcription factor families (1, 2). Although the complex roles of these transcription factors in inflammation continue to be elucidated, clinical observations have repeatedly indicated their importance to pathological states: NF-AT inhibitors like cyclosporin A and FK506, as well as NF-{kappa}B inhibitors like glucocorticoids, sulfasalazine, leflunomide, aspirin, and salycylates have been long demonstrated to be efficacious in inflammatory conditions from infections to autoimmunity (3). Still, the cellular mechanisms that distinguish and/or coordinate such inflammatory activities of such transcription factors remain largely unclear.

The forkhead (Fox) family includes a diverse and growing array of helix-loop-helix transcription factors that play critical roles in immune homeostasis (4, 5). Whereas Foxp3 regulates the development of the regulatory T cell subset, Foxo3a and Foxj1 both intrinsically regulate Th cell activation, at least in part by antagonizing the NF-{kappa}B pathway. Both Foxo3a and Foxj1 deficiencies result in a spontaneous, systemic autoimmune syndrome that has been attributed to hyperactivated T cells that result from deficiencies in the inhibitory I{kappa}B proteins of the NF-{kappa}B pathway (6, 7). A similar, albeit less dramatic, role for Foxj1 appears to participate in B cell homeostasis (8). Because several inbred mouse strains prone to lupus-like disease possess reduced levels of both Foxj1 and Foxo activity, loss of function and/or activity of such forkhead genes may underlie the predisposition to systemic autoimmune disease (6, 7).

As such, we have been interested to understand the mechanisms that regulate the expression of such forkhead genes in T cells, particularly Foxj1 because of the dramatic phenotype of its deficiency (7), to gain further insight into the means by which inflammatory Th reactions are regulated. In this study, we identify Foxd1 as a regulator of Foxj1; however, Foxd1 appears to exert additional immunoregulatory functions via the NF-AT transcription factor pathway, suggesting the presence of a general network of forkhead activities that coordinately regulates lymphocyte activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and fetal liver chimerizations

C57BL/6 Foxd1-deficient (9), Foxj1-deficient (10), Foxo3a-deficient (6), C57BL/6, and C57BL/6 Rag-1-deficient (The Jackson Laboratory) mice were maintained under specific pathogen-free conditions at the Washington University School of Medicine. Fetal liver chimerization was performed in a manner similar to prior studies (7). Briefly, fetal livers of embryos of ages E12.5–14.5 (from Foxd1+/– x Foxd1+/– or Foxj1+/– x Foxj1+/– matings) were genotyped by PCR, and +/+ and/or –/– livers were used to reconstitute 6 Gy-irradiated Rag-deficient animals. Recipients were then maintained on trimethoprim/sulfamethoxazole-supplemented water for ~28 days before analyses ~4–6 wk (or at the times indicated) postchimerization. All experiments were performed in compliance with the relevant laws and institutional guidelines, as overseen by the Animal Studies Committee of the Washington University School of Medicine.

Flow cytometry

Flow cytometric analyses were performed on a FACSCalibur System (BD Biosciences) using splenocytes cleared of RBC by osmotic lysis or lymph node cells. Abs used included: FITC-16A (anti-CD45RB), allophycocyanin-53-7.3 (anti-CD5), FITC-7D4 (anti-CD25), PE-IM7 (anti-CD44), PE-MEL-14 (anti-CD62L), CyChrome-RA3-6B2 (anti-CD45R/B220), PE-R6-60.2 (anti-IgM), and CyChrome-RM4-4 (anti-CD4; BD Pharmingen).

Lymphocyte cultures and in vitro differentiation

For bulk CD4+ Th cell analyses, splenocytes were cleared of erythrocytes by osmotic lysis, and CD4+ cells were purified by positive magnetic bead selection (Miltenyi Biotec). For naive-enriched CD4+ Th cell analyses, lymph node cells from cervical, axillary, brachial, inguinal, and popliteal nodes were first cleared of CD8+, MHC II+, and CD44+ cells by negative magnetic bead selection (Miltenyi Biotec), followed by positive CD4+ magnetic selection. In general, cells were then incubated in complete RPMI 1640 medium supplemented with 10% FCS (BioWhittaker), 10 mM HEPES, 1 mM sodium pyruvate, 2 mM glutamine, 50 mM 2-ME, and 100 U of penicillin/streptomycin (Sigma-Aldrich) in 96-well U-bottom plates precoated with anti-CD3 (145-2C11; BD Pharmingen) at the concentrations indicated at 5 x 104 cells/well. Where indicated, 1 µg/ml soluble anti-CD28 (37.51; BD Pharmingen) and/or 100 U/ml human rIL-2 (PeproTech) were added. Cells and/or supernatants were harvested at the indicated times. For Th1/Th2 skewing, Th cells were incubated in 12-well tissue culture plates at 0.5 x 106 cells/ml with 1 µg/ml plate-bound anti-CD3 and 1 µg/ml soluble anti-CD28. For Th1 conditions, cultures were supplemented with 10 ng/ml murine rIL-12 (PeproTech) and 10 µg/ml anti-IL-4 (11B11; BD Pharmingen); for Th2 conditions, cultures were supplemented with 10 ng/ml murine rIL-4 (PeproTech), 10 µg/ml anti-IFN-{gamma} (XMG1.2), and 10 µg/ml anti-IL-12 (C17.8; BD Pharmingen). On day 3–4, T cells were expanded in complete medium containing 100 U/ml IL-2, and restimulated on day 6 with 1 µg/ml plate-bound anti-CD3. At the times stated, culture supernatants were assayed for IL-2, IL-4, IL-5, and IFN-{gamma} by ELISA (BD Pharmingen).

Proliferation was assessed by BrdU incorporation (BrdU Labeling and Detection Kit III; Roche Molecular Biochemicals). For autologous MLRs, CD4 cells were purified and, where indicated, stimulated as above. APCs were prepared from Rag-deficient chimera splenocytes, irradiated by 30 Gy, and combined with T cells at a 1:1 ratio in complete medium, 5 x 104 cells/well in a 96-well flat-bottom plate. Where indicated, phosphorothioate decoy- or control decoy-annealed NF-{kappa}B oligonucleotides, which inhibit the activity of all NF-{kappa}B subunits, were added at 10 µM (11); cyclosporin A, rapamycin, the casein kinase 1 (CK1)3 inhibitor 4-(4-(2,3-dihyrdobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide D4476 (Calbiochem; dissolved in ethanol), and FK506 (Biomol) were added at the concentrations indicated. Proliferation was assessed by BrdU incorporation, as above.

RNA transcript analysis

For RNA analyses, RNA was prepared from cells at the times indicated in the text with the RNeasy Mini Kit (Qiagen), and first-strand cDNA was synthesized using oligo(dT) primers and SuperScript II reverse transcriptase (Invitrogen Life Technologies). Samples were then subjected to real-time PCR analysis on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) under standard conditions, with specificity reinforced via the dissociation protocol. Gene-specific primers have been described previously (7, 12) and/or were obtained from PrimerBank (13). Relative mRNA abundance of each transcript was normalized against tubulin (14), calculated as 2(Ct[tubulin] – Ct[gene]), where Ct represents the threshold cycle for each transcript.

Constructs, luciferase assays, and in vitro cell culture assays

A CK1{alpha} promoter-reporter construct was constructed by PCR from C57BL/6 genomic DNA, using primers 5'-GGGGTACCGCAAAAGGGGACGGCCCCAGAGTGACCTGG and 5'-GAAGATCTAGAAGCGCGGCGGGCAACCTAAACCCAAG, which produced a ~824-bp fragment corresponding to the putative CK1{alpha} promoter (–748 to +76, respective to the transcriptional start site), flanked by KpnI and BglII restriction sites. The amplicon was cloned into the KpnI-BglII sites of TK-luc (15) and then confirmed by routine sequencing. A Foxd1 promoter-reporter construct was created similarly, using 5'-GGGGTACCAACGCCAGCCTAACCGCATTTTTATTAACAG and 5'-GAAGATCTGGGCCGCTCCGCTCCGCTACTTGGCGAGCAGGGCT, which produced a ~824-bp fragment corresponding to –596/+228, respective to the transcriptional start site. To generate the expression plasmid for Foxd1, a ~1469-bp KpnI-XbaI fragment containing the cDNA of human Foxd1 was removed from a pEVRF0-Foxd1 plasmid (16) and cloned into the KpnI-XbaI sites of pcDNA3 (Invitrogen Life Technologies). The Foxj1 promoter-luciferase construct containing ~4.2 kb of the murine Foxj1 promoter was provided by S. Brody (Washington University School of Medicine, St. Louis, MO) (17). A pEGFP-C1 (BD Clontech)-based vector expressing GFP fused to the N terminus of human NF-ATc2 was provided by R. Lieberson and L. Glimcher (Harvard School of Public Health, Boston, MA). A pCMV-SPORT6-based expression vector for CK1{alpha} (Csnk1a1) was obtained from the American Type Culture Collection (MGC-30571); an antisense CK1{alpha} construct was generated by cutting this construct with SalI and NotI (liberating the Csnk1a1 cDNA), blunt ending with Klenow, religating, and selecting for a clone in the antisense orientation.

Reporter assays also used pNF-AT-luc (a 3x NF-AT reporter; Stratagene), 2x NF-{kappa}B-luc, Foxo-luc (6x DBE-luc, a Foxo reporter) (18), and pRL-CMV (Renilla luciferase control reporter; Promega). For studies in Jurkat T cells, 107 cells in 400 µl of complete RPMI 1640 medium were electroporated in a 0.4-cm cuvette at 280 mV, 975 µF in the presence of 10 µg of luciferase reporter, 40 ng of pRL-CMV, and 10 µg of pcDNA3 (Invitrogen Life Technologies) or pcDNA3-Foxd1 expression plasmid, and then returned to cell culture medium. Where indicated, PMA and ionomycin were added at 20 ng/ml and 1 nM, respectively. For primary T cells, electroporation-based assays were performed as described (19), except that we used 2 x 107 purified CD4+ cells, 20 µg of NF-{kappa}B- or NF-AT-luc, and 0.4 µg of pRL-CMV. Where indicated, primary T cells were alternatively nucleofected using 2 µg of NF-{kappa}B- or NF-AT-luc, 2 µg of expression plasmid vector, and 1 ng of pRL-CMV, according to the manufacturer’s instructions (Mouse T Cell Nucleofector Kit I; Amaxa Biosystems). At the indicated times, reporter activity was determined by the Dual-Luciferase Reporter Assay System (Promega), and relative activity was determined after normalization for Renilla luciferase. For reconstitution studies, primary T cells were nucleofected with 2 µg of pcDNA3, pcDNA3-Foxj1, pcDNA3-Foxd1, pCMV-SPORT6, or CK1{alpha} sense or antisense expression vector, as well as 0.2 µg of pIRESpuro3 (BD Clontech), and, where indicated, 2 µg of NF-{kappa}B- or NF-AT-luc, followed by selection in culture medium containing 400 U/ml human IL-2 and 2 µg/ml puromycin (Sigma-Aldrich) for 2–5 days before processing in T cell proliferation, cytokine, and/or luciferase assays, as indicated. Immunohistochemical staining for NF-AT in T cells was performed using 7A6 (mouse anti-NF-ATc1) and G1-D10 (anti-mouse NF-ATc2; Santa Cruz Biotechnology), followed by FITC anti-mouse IgG (Pierce), as directed by the manufacturer.

Chromatin immunoprecipitations were performed, as described (20). PCR primers for Foxj1 were 5'-TGACTGCCCTACCTGCTTCT and 5'-GGCCTGCAAGATTCTCAAAG (promoter, –917/–716), and 5'-GAGTGAGGGCAAGAGACTGG and 5'-TCAAGTCAGGCTGGAAGGTT (exon 3); for CK1 were 5'-CTTCAAACGCGGAAAGAAAG and 5'-GTGGCTCTCTTCGTGCTACC (promoter, –688/–526), and 5'-AGCCAGGGCTACACAGAGAA and 5'-GGCTCCTAGAAAGGGCTGTT (intron 3).

Western blotting

In general, Western blots consisted of total cell lysates resolved by 7.5% SDS-PAGE electrophoresis and blotted to nitrocellulose. Membranes were blocked with 5% nonfat dried milk (Sigma-Aldrich), incubated with primary Ab at 1/200 dilution for 1 h, washed thrice with PBS containing 0.05% Tween 20 (Sigma-Aldrich), incubated with HRP-conjugated mouse anti-goat or donkey anti-rabbit IgG (Pierce) at 1/5000 dilution for 1 h, washed thrice with PBS-Tween, and then developed using ECL Western Blotting Detection Reagents (Amersham Biosciences) and BioMax MR film (Eastman Kodak). Primary Abs included 4G6-G5 (mouse IgG2a anti-NF-ATc2), FL (rabbit anti-GFP), C-19 (goat anti-CK1{alpha}), C-18 (goat anti-CK1{delta}), H-60 (goat anti-CK1{epsilon}), FL (rabbit anti-I{kappa}B{alpha}), C-20 (rabbit anti-I{kappa}Bbeta) and M-364 (rabbit anti-I{kappa}B{epsilon}; Santa Cruz Biotechnology), anti-Foxd1 (Chemicon International), as well as rabbit polyclonal Abs against Foxj1 (7), glycogen synthase kinase-3beta (GSK-3beta) (Cell Signaling Technology), and phosphoserine (Zymed Laboratories).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of Foxd1 as a candidate regulator of Foxj1

Because some studies have suggested that forkhead genes are primarily controlled in an autoregulatory fashion (e.g., 21), we wondered whether Foxj1 might be regulated by itself or other forkhead genes. While assessing the expression pattern of various Fox family members in Th cells, we noted that one member of a separate forkhead subfamily, Foxd1, was most prominently expressed in naive Th cells, and was rapidly down-regulated upon stimulation, particularly with anti-CD3 or IL-2 (Fig. 1, A and B); this pattern was highly reminiscent of the expression pattern in T cells of Foxj1 (7), raising the possibility that Foxd1 is a transcriptional regulator of Foxj1. Indeed, whereas Foxj1 was unable to affect a reporter construct including 4.2 kb of its own promoter (17), Foxd1 reproducibly trans-activated the same Foxj1 reporter, at least in Jurkat T cells (Fig. 1C and our unpublished observations), and chromatin immunoprecipitation studies in CD4 T cells revealed that Foxd1 binds directly to the Foxj1 promoter (Fig. 1D).


Figure 1
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  		FIGURE 1. Regulation of Foxj1 by Foxd1. A and B, Expression of Foxd1 was assessed by real-time PCR (A) or Western blot (B) on C57BL/6 naive Th cells, 24 h after incubation with the stimuli indicated, in the presence ({blacksquare}) or absence ({square}) of anti-CD3. C, The activity of a Foxj1 promoter-luciferase construct was assessed in Jurkat T cells in the presence (pcDNA-Foxd1) or absence (pcDNA) of Foxd1. D, Chromatin immunoprecipitation of Foxj1 by an anti-Foxd1 Ab was assessed using C57BL/6 CD4 cells, followed by PCR with primers corresponding to the promoter or exon 3 of Foxj1. SDs reflect triplicate samples; data represent at least three experiments.

 
Foxd1 deficiency recapitulates the autoinflammatory phenotype of Foxj1 deficiency

Foxd1-deficient mice die within 24 h of birth with defects in renal morphogenesis (9). We therefore pursued fetal liver chimerization (FLC) of Rag–/– mice, to generate animals possessing either a Foxd1+/+ or Foxd1–/– lymphoid system. Foxd1-deficient FLCs developed a clinical syndrome highly reminiscent of Foxj1 deficiency: by 20–28 wk postreconstitution, a large proportion of animals that had received Foxd1–/– fetal livers appeared moribund, with ruffled fur and a hunched posture (18 of 24 vs 0 of 20 of their Foxd1+/+ counterparts; p < 0.0001). Histopathological examination as early as 12–16 wk after reconstitution revealed systemic autoimmune inflammation, including moderate to severe infiltrates of the salivary gland and kidney, but particularly of the lung, all of which were absent in Foxd1+/+ counterparts (Fig. 2). Although Foxd1–/– FLCs demonstrated essentially normal T and B cell development, with cellularities of spleens and lymph nodes comparable to their Foxd1+/+ counterparts (Fig. 3 and our unpublished data), CD4+ cells in Foxd1–/– FLCs displayed surface phenotypes consistent with increased in vivo activation, including modestly higher proportions of cells bearing a CD44highCD45RBlow activated phenotype, suggesting that T cell hyperactivation underlies the pathogenesis of this inflammatory syndrome, again similar to Foxj1 deficiency (7).


Figure 2
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  		FIGURE 2. Autoimmune inflammation in the absence of Foxd1. Note inflammation of the organs in Foxd1–/– chimeric animals, in contrast to normal-appearing tissues in Foxd1+/+ chimeras. Each panel is from a different animal, representative of 10 animals examined for each genotype, at 16 wk postreconstitution.

 

Figure 3
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  		FIGURE 3. Hyperactivated Th cell phenotypes in Foxd1–/– FLCs. Representative flow cytometric plots of spleens from Foxd1–/– vs Foxd1+/+ FLCs, 8 wk postreconstitution, are shown. Note the development of grossly normal CD4 vs CD8 T cell populations (top panels), as well as B cells, as judged by IgM vs IgD staining (bottom panels). Note also the mildly hyperactivated phenotype of CD4+ cells (middle panels), as demonstrated by increased populations of CD44highCD45RBlow cells. Plots are representative of at least five animals examined of each genotype.

 
Foxd1–/– Th cells exhibited significantly enhanced proliferative and cytokine-secreting capacities in vitro, and interestingly appeared only to require IL-2, in the absence of ligation of the TCR or CD28, to proliferate (Fig. 4, A–C). This ability to proliferate in response to IL-2 alone was a characteristic finding of Foxj1-deficient Th cells, which suggested the presence of activated autoreactive Th cells (7). Indeed, whereas Foxd1+/+ Th cells failed to proliferate when incubated in the presence of autologous APCs, Foxd1–/– Th cells proliferated vigorously (Fig. 4D), indicating an ability to activate in response to endogenous Ags. Thus, like Foxj1-deficient T cells, Foxd1–/– T cells are prone to autoreactivity and hyperactivation in response to autologous/self targets.


Figure 4
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  		FIGURE 4. T cell hyperactivation in the absence of Foxd1. A, Proliferative capacities of naive CD4+ T cells from Foxd1–/– ({blacksquare}) and Foxd1+/+ ({square}) chimeric animals were assessed after a 3-day incubation with (+) or without (–) IL-2 supplementation, in the absence of anti-CD3 or anti-CD28. B, Proliferative capacities of naive CD4+ T cells from Foxd1–/– (circles) and Foxd1+/+ (squares) chimeric animals, stimulated with the indicated amounts of plate-bound anti-CD3 in the presence (filled) or absence (open) of IL-2, with (right panel) or without (left panel) anti-CD28, were assessed on day 3 of incubation. Foxd1–/– Th cells were significantly different from Foxd1+/+ counterparts (p < 0.001) at all conditions tested, except for anti-CD3 doses ≤0.1 µg/ml when treated without IL-2. Note that A and B reflect the same set of data, with A shown separately to emphasize the difference between IL-2-treated Foxd1–/– and Foxd1+/+ samples. C, Cytokines secreted by Th1 (IL-2, IFN-{gamma})- or Th2 (IL-4)-differentiated Th cells from Foxd1–/– (circles) and Foxd1+/+ (squares) chimeric animals, stimulated with the indicated amounts of plate-bound anti-CD3, were assessed by ELISA on culture supernatants 16–20 h after stimulation. Note the use of a log-scale y-axis for IFN-{gamma}. For IL-2 secretion, responses were compared in the presence (filled) or absence (open) of exogenously added human IL-2. Foxd1–/– Th cells were significantly different from Foxd1+/+ counterparts (p < 0.001) at anti-CD3 doses ≥0.3 µg/ml for IFN-{gamma}, and ≥1.0 µg/ml for IL-2 and IL-4. D, AMLRs were performed on Foxd1–/– ({blacksquare}) and Foxd1+/+ ({square}) Th cells. Proliferation was assessed by BrdU incorporation on Th cells incubated alone, syngeneic APCs incubated alone, or Th cells incubated with APCs after 24 h of incubation. Foxd1–/– Th cells were significantly different from Foxd1+/+ counterparts (p < 0.0001) under both T cells only and T cells + APC conditions.

 
Foxd1 is required for Foxj1 and I{kappa}Bbeta expression in Th cells

Foxd1–/– Th cells contained significantly diminished levels of Foxj1, as judged by both real-time PCR and Western blot (Fig. 5, A and B), supporting the concept that Foxd1 is a regulator of Foxj1 in vivo. Consistent with this, Foxd1–/– Th cells were deficient in the Foxj1 target gene I{kappa}Bbeta (Fig. 5, B and C), which inhibits spontaneous NF-{kappa}B activation, particularly of the RELA (p65) subunit, in Th cells (7), and they demonstrated enhanced NF-{kappa}B activity, as judged by an NF-{kappa}B-luciferase reporter (Fig. 5D). These findings of spontaneous autoimmunity, associated with Th cell and NF-{kappa}B hyperactivity, and I{kappa}Bbeta deficiency are reminiscent of Foxj1 deficiency (7), supporting a model in which Foxd1 suppresses spontaneous Th cell activation by promoting the expression of Foxj1, which in turn regulates the inhibitory I{kappa}B proteins of the NF-{kappa}B pathway. Indeed, nucleofection of Foxd1–/– Th cells with a Foxj1-expressing, but not control, plasmid largely normalized their NF-{kappa}B hyperactivity, at least as judged by luciferase reporter activity (Fig. 5E).


Figure 5
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  		FIGURE 5. Foxd1 antagonizes NF-{kappa}B via Foxj1. A, Expression of Foxj1 in Foxd1-deficient (knockout (KO)) and of Foxd1 in Foxj1-deficient (KO) naive Th cells was assessed by real-time PCR. B, Western blot of the indicated proteins was performed on lysates of naive Th cells derived from Foxd1+/+ (wild-type (WT)) vs Foxd1–/– (KO) FLCs. C, Real-time PCR was used to quantify the abundance of mRNAs corresponding to the indicated genes in Th cells derived from Foxd1 WT vs Foxd1 KO Th cells, after stimulation for 24 h with plate-bound anti-CD3 (1 µg/ml). D, Activities of NF-{kappa}B were assessed in Th cells derived from Foxd1 WT vs KO FLCs, using a luciferase reporter construct. E, The ability of Foxj1 vs Foxd1 to affect NF-{kappa}B vs NF-AT transcription factor activities was assessed by nucleofection of Foxd1 WT vs Foxd1 KO Th cells with pcDNA, pcDNA-Foxj1, or pcDNA-Foxd1. NF-{kappa}B vs NF-AT activities were assessed by luciferase reporter activity, normalized for cotransfected Renilla luciferase. SDs reflect at least triplicate samples, representative of at least three experiments.

 
Regulation of NF-AT by Foxd1

A characteristic observation in Foxj1–/– Th cells is the ability of antisense and/or decoy oligonucleotides that block NF-{kappa}B activity to abrogate a significant majority of their hyperactivated phenotype (Fig. 6A) (7). When we subjected Foxd1–/– Th cells to a similar assay, we were surprised to discover that decoy NF-{kappa}B oligonucleotides only partially inhibited the hyperproliferative phenotype (Fig. 6A), suggesting that Foxd1 affects an additional activating pathway in Th cells.


Figure 6
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  		FIGURE 6. Foxd1 regulates NF-AT activity. A, The roles of NF-{kappa}B vs NF-AT in the autoreactivity of Foxd1–/– vs Foxj1–/– Th cells were assessed by performing an AMLR with Foxd1–/– or Foxj1–/– Th cells + APCs, in the presence or absence of an NF-{kappa}B decoy oligonucleotide or control mismatch oligonucleotide, the NF-AT inhibitor cyclosporin A (CsA) or vehicle (ethanol) control. BrdU incorporation was assessed 24 h later. B, Real-time PCR was used to quantify the abundance of mRNAs corresponding to the indicated genes in Th cells derived from Foxd1 WT vs Foxd1 KO Th cells, after stimulation for 24 h with plate-bound anti-CD3 (1 µg/ml). C, Activities of NF-AT, and Foxo transcription factors were assessed in Th cells derived from Foxd1 WT vs KO FLCs, using luciferase reporter constructs. D, The ability of Foxd1 to regulate NF-AT activity was assessed using an NF-AT-luciferase reporter in Jurkat T cells in the presence (pcDNA-Foxd1) or absence (pcDNA) of Foxd1. After electroporation, cells were treated with or without PMA/ionomycin, and assessed 24 h later. E and F, Nuclear vs cytoplasmic localization of NF-AT was assessed in Foxd1 WT vs Foxd1 KO Th cells by immunohistochemical staining. One hundred cells were assessed visually for predominantly nuclear vs cytoplasmic localization, and tabulated in F. G, Levels of NF-AT phosphorylation were assessed in Foxd1 WT vs KO Th cells by immunoprecipitating NF-ATc2, followed by Western blot using NF-ATc2- or phosphoserine-specific Abs. Error bars indicate SDs of at least triplicate samples, representative of at least three experiments.

 
Because NF-AT comprises one other major immediate-early pathway in TCR-induced Th cell differentiation, besides NF-{kappa}B (2), we wondered whether abnormalities in this transcription factor family might account for these findings. Foxd1–/– Th cells did in fact contain elevated levels of both NF-AT and NF-{kappa}B target genes, including IL-2 and IFN-{gamma} (Figs. 4C and 6B); consistent with this, Foxd1–/– Th cells also demonstrated significantly increased NF-AT activity, as judged by an NF-AT-luciferase reporter (Fig. 6C), and Foxd1 was capable of inhibiting NF-AT activation, as judged by the same luciferase reporter, in Jurkat T cells (Fig. 6D). Such differences did not appear to reflect a global effect on transcriptional activities, because Foxd1–/– Th cells contained Foxo activities comparable to their Foxd1+/+ counterparts, at least as judged by a luciferase reporter (Fig. 6C). Foxd1–/– Th cells also contained increased spontaneous nuclear localization of NF-AT, as judged by immunohistochemical staining (Fig. 6, E and F), as well as diminished serine phosphorylation, as judged by immunoprecipitation-Western blot analysis (Fig. 6G), both of which correspond with NF-AT activity (2). Finally, whereas individual inhibition of NF-{kappa}B by decoy oligonucleotides or of NF-AT by the inhibitor cyclosporin A only partially inhibited the hyperproliferative phenotype of Foxd1–/– Th cells, combined treatment significantly did (Fig. 6A). Similar results were obtained with FK506, which also inhibits NF-AT, but not with rapamycin, which affects pathways downstream of IL-2 (Fig. 7), and both cyclosporin A and FK506 could abrogate the NF-AT hyperactivity of Foxd1–/– Th cells (Fig. 8). Such findings suggest that combined excessive activity of both NF-AT and NF-{kappa}B accounted for our findings.


Figure 7
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  		FIGURE 7. Pharmacological manipulation of autoreactivity in Foxd1 KO Th cells. A, Foxd1 KO T cells were subjected to AMLRs in the presence or absence of control or decoy NF-{kappa}B oligonucleotides, as well as the indicated concentrations of the NF-AT inhibitors cyclosporin A (CsA) and FK506, as well as rapamycin. B, For comparison, Foxd1 WT Th cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of the indicated concentrations of compounds. SDs reflect at least triplicate samples, representative of at least two experiments.

 

Figure 8
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  		FIGURE 8. Dependence of Foxd1 KO NF-AT hyperactivity on NF-AT. Th cells from Foxd1 WT and KO FLCs were nucleofected with an NF-AT-luciferase and CMV-Renilla reporter, and incubated in the presence of the indicated compounds. Reporter activity, normalized for Renilla, was determined after 24 h. SDs reflect at least triplicate samples, representative of at least two experiments.

 
Foxd1 regulates the NF-AT regulator CK1

Given these results, we examined the ability of Foxd1 to affect NF-AT nuclear-cytoplasmic translocation. Whereas control-transfected Jurkat T cells demonstrated clear nuclear translocation of an NF-ATc2-GFP fusion protein in response to PMA/ionomycin stimulation, Foxd1-transfected cells were consistently impaired in this activity (Fig. 9, A and B). Because NF-AT activity is negatively regulated by kinases like GSK3, CK1, and MAPK kinase kinase 1 of the MAPK pathway, which mediate nuclear export and/or impair nuclear import of the NF-ATs (22, 23), and because Foxj1 regulates NF-{kappa}B activity via the inhibitory I{kappa}B proteins (7), we hypothesized that Foxd1 might regulate NF-AT in an analogous fashion by promoting the expression and/or activity of a gene with inhibitory activity. Although Foxd1 deficiency had essentially no effect on GSK3 or MAPK kinase kinase 1 expression (Figs. 5B and 9C), Foxd1–/– Th cells consistently demonstrated significantly reduced levels of CK1{alpha}, but not CK1{delta} or CK1{epsilon}, as judged by real-time PCR and/or Western blot (Figs. 5B and 9C). Consistent with this observation, transfection of Foxd1 induced or enhanced both CK1{alpha} expression and serine phosphorylation of NF-AT in Jurkat T cells (Fig. 9, D and E). Foxd1 was furthermore capable of trans-activating a putative CK1{alpha} promoter construct (Fig. 9F) and binds the CK1a promoter in vivo, as indicated by chromatin immunoprecipitation studies (Fig. 9G).


Figure 9
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  		FIGURE 9. Casein kinase is a Foxd1 target. A and B, Jurkat T cells were cotransfected with a plasmid encoding an NF-ATc2-GFP fusion protein as well as either a Foxd1 (pcDNA-Foxd1) or control (pcDNA) expression vector. After 24 h, cells were treated with or without PMA/ionomycin for 15 min, and then visualized by fluorescence microscopy. The percentage of GFP-positive cells, of 100 counted, which contained nuclear NF-ATc2-GFP staining, were graphically represented in B. C, Real-time PCR was used to quantify the abundance of mRNAs corresponding to the indicated genes in Th cells derived from Foxd1 WT vs Foxd1 KO Th cells. D, Expression of both CK1{alpha} and GSK3 was assessed by Western blot in Jurkat T cells transfected 24 h previously with a Foxd1 (pcDNA-Foxd1) or control (pcDNA) expression vector. E, The effect of Foxd1 upon serine phosphorylation of NF-AT was assessed using Jurkat T cells cotransfected with a plasmid encoding an NF-ATc2-GFP fusion protein as well as either a Foxd1– (pcDNA-Foxd1) or control (pcDNA) expression vector. After 24 h, the fusion protein was immunoprecipitated by an anti-GFP Ab, and Western blotting was performed on the precipitate using phosphoserine- and NF-ATc2-specific Abs. F, The ability of Foxd1 to trans-activate the CK1{alpha} promoter was assessed in Jurkat T cells transfected with CK1{alpha}-luc along with a Foxd1 (pcDNA-Foxd1) or control (pcDNA) expression vector. After 24 h, luciferase activity was assessed. G, Chromatin immunoprecipitation of CK1 by an anti-Foxd1 Ab was assessed using C57BL/6 CD4 cells, followed by PCR with primers corresponding to the promoter or intron 3 of CK1. SDs reflect triplicate samples, representative of at least three separate experiments.

 
Nucleofection of Foxd1–/– Th cells with a CK1{alpha}– but not control expression vector normalized their NF-AT, but not NF-{kappa}B, hyperactivity (Fig. 10A). Conversely, nucleofection of Foxd1+/+ Th cells with an antisense CK1{alpha}– expression vector increased nuclear localization and activity of NF-AT (Fig. 10, B and C), as did treatment of Foxd1+/+ Th cells with the CK1-specific inhibitor D4476 (Fig. 11). In contrast, nucleofection of Foxd1–/– Th cells with a Foxj1 expression vector normalized their NF-{kappa}B, but not NF-AT, hyperactivity (Fig. 5E), correlating with a failure to affect nuclear NF-AT localization (Fig. 10D) and an incomplete suppression of proliferation and cytokine production in autologous MLRs (AMLRs) (Fig. 10, E and F). In contrast, nucleofection of Foxd1–/– Th cells with a Foxd1 expression vector normalized both NF-{kappa}B and NF-AT hyperactivity (Fig. 5E), NF-AT subcellular localization (Fig. 10D), as well as proliferation and cytokine production in AMLRs (Fig. 10, E and F). Thus, deficiency in Foxd1 leads to spontaneous activation of both NF-AT and NF-{kappa}B, the former via CK1{alpha} deficiency, the latter via Foxj1 deficiency, resulting in immune dysregulation.


Figure 10
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  		FIGURE 10. Normalization of the Foxd1 phenotype by Foxj1 vs casein kinase. A, The ability of CK1{alpha} to affect NF-AT vs NF-{kappa}B activities in Foxd1 WT vs Foxd1 KO Th cells was assessed by luciferase reporter activity in Foxd1 WT vs KO T cells, conucleofected with control vs CK1{alpha}-expressing plasmids. Normalized luciferase activity was assessed 5 h later. B and C, Foxd1 WT Th cells were nucleofected with a control vs CK1{alpha}-expressing plasmid, with or without an NF-AT luciferase reporter. After 24 h, NF-AT localization was assessed by immunohistochemistry (B), and NF-AT activity was determined by normalized luciferase activity (C). D–F, The ability of Foxj1 vs Foxd1 to complement the phenotype of Foxd1 KO Th cells was assessed by nucleofection of Foxd1 KO Th cells with a control, Foxj1, or Foxd1 expression plasmid. NF-AT localization was assessed after 24 h by immunohistochemistry (D). Transfected cells were also subjected to AMLR assays, including assessment of proliferation by BrdU incorporation (E) and cytokine production by ELISA (F) 1 day after exposure to stimulator cells. SDs reflect at least triplicate samples, representative of at least three separate experiments.

 

Figure 11
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  		FIGURE 11. Role of CK1 in NF-AT regulation. Th cells from Foxd1 WT FLCs were incubated in the presence of the indicated concentrations of the CK1 inhibitor D4476. Left, After 24 h, NF-AT nuclear-cytoplasmic localization was determined by immunohistochemistry. Right, Th cells were nucleofected with NF-AT-luciferase and CMV-Renilla reporters before incubation with D4476, with normalized reporter activity determined after 24 h. SDs reflect at least triplicate samples, representative of at least two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This ability of Foxd1 to regulate simultaneously both NF-AT activity and Foxj1 expression raises the intriguing possibility that a network of Fox genes coordinately regulates the activity of activating transcription factors, like NF-{kappa}B and NF-AT. Supporting this concept, Foxo3a deficiency, which also results in a Th cell-related autoimmune phenotype, also leads to a Foxj1 deficiency in Th cells (6): this most likely accounts for the I{kappa}Bbeta deficits of Foxo3a-deficient Th cells, but unlike Foxd1, Foxo3a is unable to trans-activate a Foxj1 promoter construct (6) (our unpublished data). Thus, the forkheads appear to comprise an integrated, yet multilayered complex network in Th cells that modulate inflammatory responses.

Regardless of the specific pathways, the present findings are consistent with the long-established clinical efficacy of the NF-AT inhibitors cyclosporin A and FK506 in human and murine systemic autoimmunity (24, 25, 26, 27, 28) as well as the immunosuppressive utility of specific NF-AT inhibitors (29). They further are consistent with prior demonstrations of dysregulated NF-{kappa}B activity in both human and murine lupus (30, 31, 32), the inflammatory phenotypes of animals deficient in I{kappa}B activity (33), and the ability of ectopic expression of inhibitory regulators of NF-{kappa}B to suppress autoimmunity in vivo (31). Although relatively few studies have directly addressed the roles of the various NF-AT isoforms and their regulatory proteins in autoimmune diseases (3), the present findings suggest that abnormalities in NF-ATs and/or their signaling regulators are likely to play critical roles in tolerance loss and disease pathogenesis. In this sense, it is intriguing to note that Foxd1 is located on murine chromosome 13 at 52 cM, near a proposed Nba lupus susceptibility locus (34), and several of the genes that encode components of human CK1, CK1{delta} (17q25), CK1{gamma}1 (15q22), and CK1{gamma}2 (19p13), are located at proposed human lupus susceptibility loci (35). Therefore, continued investigation into the regulation and interaction of the Fox genes with each other as well as with other transcription factor families will hopefully continue to provide further insight into the regulation of T cell homeostasis, as well as the mechanisms of and potential therapeutic approaches to immunological tolerance.


   Acknowledgments
 
We are grateful to Peter Carlsson for the Foxd1 cDNA, Steven Brody for Foxj1 promoter constructs, Rebecca Lieberson and Laurie Glimcher for the NF-ATc2-GFP construct, as well as Andrey Shaw and Emil Unanue for critical commentary on the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI057471 and AI061478. Back

2 Address correspondence and reprint requests to Dr. Stanford L. Peng at current address, Inflammation, Autoimmunity, and Transplantation Research, Roche Palo Alto, 3431 Hillview Avenue, M/S R7-101, Palo Alto, CA 94304. E-mail address: stanford.peng{at}roche.com Back

3 Abbreviations used in this paper: CK1, casein kinase 1; AMLR, autologous MLR; FLC, fetal liver chimera; GSK3, glycogen synthase kinase-3; KO, knockout; WT, wild type. Back

Received for publication November 1, 2005. Accepted for publication January 31, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Li, Q., I. M. Verma. 2002. NF-{kappa}B regulation in the immune system. Nat. Rev. Immunol. 2: 725-734. [Medline]
  2. Rao, A., C. Luo, P. G. Hogan. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15: 707-747. [Medline]
  3. Peng, S. L.. 2004. Transcription factors in the pathogenesis of autoimmunity. Clin. Immunol. 110: 112-123. [Medline]
  4. Coffer, P. J., B. M. T. Burgering. 2004. Forkhead-box transcription factors and their role in the immune system. Nat. Rev. Immunol. 4: 889-899. [Medline]
  5. Jonsson, H., S. L. Peng. 2005. Forkhead transcription factors in immunology. Cell. Mol. Life Sci. 62: 397-409. [Medline]
  6. Lin, L., J. D. Hron, S. L. Peng. 2004. Regulation of NF-{kappa}B, Th activation, and autoinflammation by the forkhead transcription factor Foxo3a. Immunity 21: 203-213. [Medline]
  7. Lin, L., M. Spoor, A. J. Gerth, S. L. Brody, S. L. Peng. 2004. Modulation of Th1 activation and inflammation by the NF-{kappa}B repressor Foxj1. Science 303: 1017-1020. [Abstract/Free Full Text]
  8. Lin, L., S. L. Brody, S. L. Peng. 2005. Restraint of B cell activation by Foxj1-mediated antagonism of NF-{kappa}B and interleukin-6. J. Immunol. 175: 951-958. [Abstract/Free Full Text]
  9. Hatini, V., S. O. Huh, D. Herzlinger, V. C. Soares, E. Lai. 1996. Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev. 10: 1467-1478. [Abstract/Free Full Text]
  10. Brody, S. L., X. H. Yan, M. K. Wuerffel, S. K. Song, S. D. Shapiro. 2000. Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am. J. Respir. Cell Mol. Biol. 23: 45-51. [Abstract/Free Full Text]
  11. Khaled, A. R., E. J. Butfiloski, E. S. Sobel, J. Schiffenbauer. 1998. Use of phosphorothioate-modified oligodeoxynucleotides to inhibit NF-{kappa}B expression and lymphocyte function. Clin. Immunol. Immunopathol. 86: 170-179. [Medline]
  12. Overbergh, L., D. Valckx, M. Waer, C. Mathieu. 1999. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11: 305-312. [Medline]
  13. Wang, X., B. Seed. 2003. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 31: e154[Abstract/Free Full Text]
  14. Gerth, A. J., L. Lin, S. L. Peng. 2003. T-bet regulates T-independent IgG2a class switching. Int. Immunol. 15: 937-944. [Abstract/Free Full Text]
  15. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, L. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 100: 655-669. [Medline]
  16. Dahle, M. K., L. M. Gronning, A. Cederberg, H. K. Blomhoff, N. Miura, S. Enerback, K. A. Tasken, K. Tasken. 2002. Mechanisms of FOXC2- and FOXD1-mediated regulation of the RI{alpha} subunit of cAMP-dependent protein kinase include release of transcriptional repression and activation by protein kinase B{alpha} and cAMP. J. Biol. Chem. 277: 22902-22908. [Abstract/Free Full Text]
  17. Brody, S. L., B. P. Hackett, R. A. White. 1997. Structural characterization of the mouse Hfh4 gene, a developmentally regulated forkhead family member. Genomics 45: 509-518. [Medline]
  18. Furuyama, T., T. Nakazawa, I. Nakano, N. Mori. 2000. Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. Biochem. J. 349: 629-634. [Medline]
  19. Rengarajan, J., P. R. Mittelstadt, H. W. Mages, A. J. Gerth, R. A. Kroczek, J. D. Ashwell, L. H. Glimcher. 2000. Sequential involvement of NFAT and Egr transcription factors in FasL regulation. Immunity 12: 293-300. [Medline]
  20. Jonsson, H., P. Allen, S. L. Peng. 2005. Inflammatory arthritis requires Foxo3a to prevent Fas ligand-induced neutrophil apoptosis. Nat. Med. 11: 666-671. [Medline]
  21. Pohl, B. S., W. Knochel. 2001. Overexpression of the transcriptional repressor FoxD3 prevents neural crest formation in Xenopus embryos. Mech. Dev. 103: 93-106. [Medline]
  22. Graef, I. A., P. G. Mermelstein, K. Stankunas, J. R. Neilson, K. Deisseroth, R. W. Tsien, G. R. Crabtree. 1999. L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons. Nature 401: 703-708. [Medline]
  23. Zhu, J., F. Shibasaki, R. Price, J. C. Guillemot, T. Yano, V. Dotsch, G. Wagner, P. Ferrara, F. McKeon. 1998. Intramolecular masking of nuclear import signal on NF-AT4 by casein kinase I and MEKK1. Cell 93: 851-861. [Medline]
  24. Cranney, A., P. Tugwell. 1998. The use of Neoral in rheumatoid arthritis. Rheum. Dis. Clin. North Am. 24: 479-488. [Medline]
  25. Griffiths, B., P. Emery. 2001. The treatment of lupus with cyclosporin A. Lupus 10: 165-170. [Abstract/Free Full Text]
  26. Blank, M., M. Ben-Bassat, Y. Shoenfeld. 1992. The effect of cyclosporin A on early and late stages of experimental lupus. Arthritis Rheum. 35: 1350-1355. [Medline]
  27. Morland, C., J. Michael, D. Adu, T. Kizaki, A. J. Howie, A. Morgan, N. A. Staines. 1991. Anti-idiotype and immunosuppressant treatment of murine lupus. Clin. Exp. Immunol. 83: 126-132. [Medline]
  28. Mountz, J. D., H. R. Smith, R. L. Wilder, J. P. Reeves, A. D. Steinberg. 1987. CS-A therapy in MRL-lpr/lpr mice: amelioration of immunopathology despite autoantibody production. J. Immunol. 138: 157-163. [Abstract]
  29. Venkatesh, N., Y. Feng, B. DeDecker, P. Yacono, D. Golan, T. Mitchison, F. McKeon. 2004. Chemical genetics to identify NFAT inhibitors: potential of targeting calcium mobilization in immunosuppression. Proc. Natl. Acad. Sci. USA 101: 8969-8974. [Abstract/Free Full Text]
  30. Burgos, P., C. Metz, P. Bull, R. Pincheira, L. Massardo, C. Errazuriz, M. R. Bono, S. Jacobelli, A. Gonzalez. 2000. Increased expression of c-rel, from the NF-{kappa}B/Rel family, in T cells from patients with systemic lupus erythematosus. J. Rheumatol. 27: 116-127. [Medline]
  31. Vallabhapurapu, S., R. P. Ryseck, M. Malewicz, D. S. Weih, F. Weih. 2001. Inhibition of NF-{kappa}B in T cells blocks lymphoproliferation and partially rescues autoimmune disease in gld/gld mice. Eur. J. Immunol. 31: 2612-2622. [Medline]
  32. Liu, J., D. Beller. 2002. Aberrant production of IL-12 by macrophages from several autoimmune-prone mouse strains is characterized by intrinsic and unique patterns of NF-{kappa}B expression and binding to the IL-12 p40 promoter. J. Immunol. 169: 581-586. [Abstract/Free Full Text]
  33. Cheng, J. D., R. P. Ryseck, R. M. Attar, D. Dambach, R. Bravo. 1998. Functional redundancy of the nuclear factor {kappa}B inhibitors I{kappa}B{alpha} and I{kappa}Bbeta. J. Exp. Med. 188: 1055-1062. [Abstract/Free Full Text]
  34. Wakeland, E. K., K. Liu, R. R. Graham, T. W. Behrens. 2001. Delineating the genetic basis of systemic lupus erythematosus. Immunity 15: 397-408. [Medline]
  35. Kelly, J. A., K. L. Moser, J. B. Harley. 2002. The genetics of systemic lupus erythematosus: putting the pieces together. Genes Immun. 3: S71-S85.



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