Materials and Methods
Mice and fetal liver chimerizations
Foxj1 -/- (1), BXSB/MpJ, MRL/MpJ-CD95lpr/lpr (MRL/lpr), C57BL/6 (Jackson
Laboratory, Bar Harbor, ME), and Rag-2 -/- (Taconic, Germantown, NY) 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 (2). Briefly, fetal livers of embryos of ages E12.5-14.5 (from Foxj1 +/- x
+/- matings) were genotyped by PCR (1) and +/+ and/or -/- livers were used to
reconstitute 6 Gy-irradiated Rag-2 -/- animals. Recipients were then maintained on
trimethoprim/sulfamethoxazole-supplemented water for approximately 28 days before
analyses approximately 4-6 weeks post-chimerization. Foxj1 -/- fetal liver cells
reconstituted irradiated recipients with a similar efficiency as their Foxj1 +/+
counterparts, as judged by the presence of IgM+ peripheral blood lymphocytes (96%
(48/50) versus 92% (44/48), respectively, 3). As Foxj1 -/- chimeras became moribund,
they were euthanized for humane reasons. 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, San Diego, California) using splenocytes cleared of red blood cells by
osmotic lysis or lymph node cells. Antibodies used included: FITC-16A (anti-
CD45RB), APC-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, San Diego, CA). Where
indicated, cell sorting was performed by the High Speed Cell Sorter Core Facility of the
Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO.
Lymphocyte cultures and in vitro differentiation
Naive-enriched B cells were purified from C57BL/6 spleens by negative
selection against CD43 (Miltenyi Biotec, Auburn, CA, 4). For bulk CD4+ Th cell
analyses, splenocytes were cleared of erythrocytes by osmotic lysis, and CD4+ cells
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 medium
supplemented with 10% fetal calf serum (BioWhittaker, Walkersville, MD), 10 mM
HEPES, 1 mM sodium pyruvate, 2 mM glutamine, 50 µM β-mercaptoethanol, and 100
U penicillin/streptomycin (Sigma-Aldrich Chemical Co., St. Louis, MO) in 96-well U-
bottom plates pre-coated with anti-CD3 (145-2C11, Pharmingen) at the concentrations
indicated at 5 x 104 cells/well. Where indicated, 1 µg/mL soluble anti-CD28 (37.51,
Pharmingen) and/or 100 U/mL recombinant human IL-2 (PeproTech, Inc, Rocky Hill,
NJ) were added. For Th-neutral primary stimulation, 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
recombinant murine IL-12 10 ng/mL (PeproTech) and 10 µg/mL anti-IL-4 (11B11,
Pharmingen); for Th2 conditions, cultures were supplemented with 10 ng/mL
recombinant murine IL-4 (PeproTech), 10 µg/mL anti-IFN-γ (XMG1.2) and 10 µg/mL
anti-IL-12 (C17.8, 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. Where indicated, anti-IL-2 (SB46, Pharmingen) was added at 10
µg/mL; and phosphorothiate antisense or missense oligonucleotides against RELA,
which result in the specific knock-down of this NF-κB subunit, were added at 10 µM
(3, 5). Culture supernatants were assayed for IL-2, IL-4, IL-5, IL-6, IL-10, and/or IFN-γ
by ELISA (Pharmingen) after 72 hours for primary stimulations, or 20 hours for
secondary stimulations.
T cell proliferation assays
Proliferation was assessed by 5-bromo-2’-deoxy-uridine incorporation (BrdU
Labeling and Detection Kit III, Roche Molecular Biochemicals, Mannheim, Germany)
on day 3 of T cell stimulation after 4-5 hours of cell labeling. For autologous mixed
lymphocyte reactions, CD4 cells were stimulated under Th-neutral conditions, followed
by expansion in IL-2, as above. Antigen-presenting cells were prepared from Rag-2 -/-
chimera splenocytes, irradiated by 30 Gy, and combined with day 6 primarily-
stimulated T cells at a 1:1 ratio in complete medium, 5 x 104 cells per well in a 96-well
flat-bottom plate. Where indicated, concanavalin A (Calbiochem) was supplemented at
5 µg/mL. Proliferation was assessed by BrdU incorporation as above on day 3.
RNA transcript analysis
For RNA analyses, RNA was prepared from cells at the times indicated in the
text with the RNeasy® Mini Kit (Qiagen, Inc., Valencia, CA), and first-strand cDNA
synthesized using oligo(dT) primers and SuperScript™ II reverse transcriptase
(Invitrogen Corp., Carlsbad, CA). Samples were then subjected to real-time PCR
analysis on an ABI PRISM® 7000 Sequence Detection System (Applied Biosystems,
Foster City, CA) under standard conditions with specificity reinforced via the
dissociation protocol. Foxj1-specific primers included 5’-
CACGGACAACTTCTGCTACTTCC and 5’-AGGACAGGTTGTGGCGGAT. T-bet,
GATA-3 (6), cyclinD1 (7), GADD45β (8) and cytokine-specific (9) primers have been
previously described. Relative mRNA abundance of each transcript was normalized
against tubulin (10), calculated as 2(Ct[tubulin] – Ct[gene]), where Ct represents the threshold
cycle for each transcript.
Luciferase assays and constructs
Reporter assays utilized pNFAT-luc (a 3X NF-AT reporter; Stratagene, La Jolla,
CA), 2X NF-κB-luc (11), T-box-luc (a T-box transcription factor reporter, 12), and
pRL-CMV (Renilla luciferase control reporter, Promega, Madison, WI). IκBβ-luc was
constructed by PCR from C57BL/6 genomic DNA, using primers 5’-
GGGGTACCAGAACTTGACATCGGACCCTTACATTTC and 5’-
GAAGATCTGCTCCAGTGCTTCCGCCCTATCG, which produced a ~598 bp
fragment corresponding to the IκBβ promoter (13), flanked by KpnI and BglII
restriction sites. The amplicon was cloned into the KpnI-BglII sites of TK-luc (12) and
then confirmed by routine sequencing (3). Of note, this fragment contains a single
sequence TGTGGTGC at b.p. 503-509 that resembles the in vitro-defined Foxj1
consensus binding site TGTNNTGT (14). For studies in the M12 murine B cell
lymphoma (4), 107 cells in 400 µL complete RPMI medium were electroporated in a 0.4
cm cuvette at 280 mV, 975 µF in the presence of 10 µg luciferase reporter, 40 ng pRL-
CMV, and 10 µg pCDNA3 (Invitrogen Corp., Carlsbad, CA) or pCDNA3-Foxj1
expression plasmid (15), and then returned to cell culture medium. After four hours,
reporter activity was determined by the Dual-Luciferase® Reporter Assay System
(Promega), and relative activity determined after normalization for Renilla luciferase.
For the 293-T transformed human embryonic kidney line (11), 0.1-0.2 x 106 adherent
cells in DMEM medium with 10% fetal calf serum were transfected with 10 ng pRL-
CMV, 2 µg pcDNA3 or pcDNA3-Foxj1, and the indicated amounts of NF-κB-luc using
FuGENE 6 (Roche). Twenty-four hours later, cells were stimulated with 20 ng/mL
recombinant human TNF-α (PeproTech), and 4 hours thereafter cells were analyzed via
the Dual-Luciferase assay, with relative activity determined after normalization for
Renilla. For primary T cells, assays were performed as described (16), except that we
used 2 x 107 purified CD4+ cells, 20 µg NF-κB- or NF-AT-luc and 0.4 µg of pRL-
CMV, with relative activity determined after normalization for Renilla.
Histopathology
Tissue histology was performed on buffered formalin-fixed, paraffin-imbedded
specimens with routine hematoxylin and eosin staining.
Immunoglobulin studies
Chimeric animals were assayed for serum immunoglobulin titers and/or
autoantibodies 4-6 weeks after chimerization by standard ELISA (Southern
Biotechnology Associates, Birmingham, AL) and/or immunofluorescence protocols, as
described (2, 4).
Immunohistochemistry
The retroviral pMX-Foxj1-IRES-GFP expression vector was generated by
subcloning a ~1.7 kB KpnI-XbaI fragment containing the Foxj1 coding sequence from
pCDNA3-Foxj1 into the XhoI site of pMX-IRES-GFP (17). For infection, 293-T cells
were plated at 0.1 x 106 cells per well of a 6-well tissue culture plate, each well
containing a cover slip. After growth for 16-20 hours, the cells were infected with fresh
retroviral supernatants of pMX-IRES-GFP versus pMX-Foxj1-IRES-GFP viruses
generated in the PlatE packaging line (18) by centrifugation at 1000g, 45 minutes in the
presence of 4 µg/mL polybrene (Sigma). Twenty-four hours after infection, cells were
treated with or without 20ng/mL TNF-α for 20 minutes prior to fixation in 100%
methanol (Sigma) for 5 minutes, -20°C. The cover slips were then washed thrice with
PBS, blocked with 10% normal goat serum in PBS, incubated with primary antibody at
1:100 dilution in PBS for 60 minutes in a humidified chamber, washed thrice with PBS,
incubated with PE goat anti-rabbit IgG (Southern Biotechnology) at 1:100 dilution in
PBS for 45 minutes, mounted on glass slides and visualized by fluorescence
microscopy. Nuclear versus cytoplasmic staining of NF-κB proteins was determined in
GFP-positive cells. Primary antibodies included C-20 (rabbit anti-RELA), NLS (rabbit
anti-p50), and C (rabbit anti-c-REL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Western blotting
293-T cells were transfected with pCDNA versus pCDNA-Foxj1 and treated
with TNF-α as described above. After two hours, total cell lysates were resolved by
7.5% SDS-PAGE electrophoresis and blotted to nitrocellulose. Membranes were
blocked with 5% nonfat dried milk (Sigma), incubated with primary antibody at 1:200
dilution for 1 hour, washed thrice with PBS containing 0.05% Tween-20 (Sigma),
incubated with HRP-conjugated mouse anti-goat or donkey anti-rabbit IgG (Pierce,
Rockford, IL) at 1:5000 dilution for 1 hour, washed thrice with PBS-Tween, and then
developed using ECL Western Blotting Detection Reagents (Amersham Biosciences,
Piscataway, NJ) and BioMax MR film (Eastman Kodak Co., Chicago, IL). Primary
antibodies included I-19 (goat anti-actin) FL (rabbit anti-IκBα), C-20 (rabbit anti-IκBβ)
and M-364 (rabbit anti-IκBε; Santa Cruz).
EMSA
For detection of NF-κB activity, the NF-κB target sequence oligo 5’-
AGTTGAGGGGACTTTCCCAGGC was incubated with its complementary sequence
for 5 minutes at 100°C in 50 mM NaCL, cooled overnight to room temperature, and
then end-labeled with 32P using polynucleotide kinase (New England Biolabs, Beverly,
MA). The labeled oligo was then incubated for 5 minutes, room temperature with
nuclear extracts derived from 293-T or BEAS-2B cells previously transfected with
pCDNA versus pCDNA-Foxj1 (19), and treated with or without TNF-α for 30-45
minutes (20). The oligo-extract mixture was then resolved on a non-denaturing 5%
acrylamide gel, which was then dried and developed by routine autoradiography.
Supershift experiments involved the pre-incubation of appropriate antibody prior to
electrophoresis: C-20, NLS, C, or a rabbit polyclonal antibody against Foxj1 (19).
Supporting Figure Legends
Figure S1. Foxj1 is downregulated by T cell stimulation. Naive CD4+ T cells were
isolated from wild-type C57BL/6 mice (left graph) and incubated in the presence (black
bars) or absence (open bars) of 1 µg/mL plate-bound anti-CD3, as well as, where
indicated, soluble anti-CD28 and/or recombinant IL-2. After 24 hours, Foxj1 expression
was determined by real-time PCR. For comparison, expression of Foxj1 is shown for
naive CD4 cells stimulated for 3 days under Th1 or Th2 conditions, as well as naive B
cells (middle graph). Also shown are expression levels for Foxj1 in naive CD4 T cells
derived from lupus-prone BXSB or MRL/lpr mice, incubated for 24 hours in the
presence (black bars) or absence (open bars) of IL-2. Shown are data based upon three
separately tested mice, with standard deviations shown, representative of two
experiments.
Figure S2. Relationship of Foxj1 expression to IκBβ. A, Foxj1 is downregulated during
T cell activation. Naive wild-type (WT) Th cells, as well as WT cells stimulated
overnight with plate-bound anti-CD3 or IL-2, were assessed for Foxj1 expression by
Western blotting. Foxj1 runs as a doublet (arrows). B, Expression of IκBβ correlates
with that of Foxj1. Naive wild-type Th cells were stimulated as in Fig. S1 and then
analyzed for IκBβ expression by real-time PCR. This expression pattern is highly
reminiscent of Foxj1 (Fig. S1). C, Foxj1 induces IκBβ gene transcription. Wild-type Th
cells were retrovirally transduced with pMX-IRES-GFP (control) or pMX-Foxj1-IRES-
GFP (Foxj1) retroviruses, sorted for GFP-positive cells, and then assessed for IκBβ
expression by real-time PCR. D, Foxj1 transactivates the IκBβ promoter. The
responsiveness of an IκBβ promoter-luciferase construct was assessed in wild-type Th
cells co-transfected with pCDNA (control) or pCDNA-Foxj1 (Foxj1) vectors.
Figure S3. Developmental lymphoid phenotype in the absence of Foxj1. Splenic
lymphoid populations were examined by flow cytometry for the cell populations
indicated, gated on the cell marker indicated (right). Numbers indicate percentage of
gated cells, representative of 5 animals tested for each genotype. Findings were similar
in lymph nodes (3).
Figure S4. Exaggerated Th2 cytokine production by Foxj1 -/- Th cells. Th2-
differentiated Th cells from Foxj1 -/- (circles) and –sufficient (squares) chimeric
animals were stimulated with the indicated amounts of plate-bound anti-CD3. After 20
hours, culture supernatants were assessed for specific cytokines by ELISA. Foxj1 -/-
Th2 cells were significantly different than their –sufficient counterparts for both
cytokines at all anti-CD3 doses above 0 µg/mL (p<0.001). Error bars indicate standard
deviations.
Figure S5. Relevance of Foxj1 to NF-κB activities. A, Foxj1 inhibits NF-κB DNA
binding activity. Nuclear extracts of BEAS-2B cells stably transfected with a control or
Foxj1-expressing vector were prepared after 1 hour of TNF-α stimulation, and 10 µg of
each sample was used for gel-shift. Where indicated anti-RELA or anti-Foxj1 antisera
were added for supershift analysis. B, NF-κB target gene expression in Foxj1 -/- T cells.
Naive Th cells from Foxj1 -/- or +/+ chimeras were stimulated with plate-bound anti-
CD3 only for 24 hours, and then analyzed for the expression of IL-2, IFN-γ, cyclin D1,
and GADD45β by real-time PCR analysis. C, NF-κB hyperactivation accounts for the
hyperproliferation of Foxj1 -/- T cells. Naive T cells from Foxj1 -/- or –sufficient
chimeras were stimulated in vitro with plate-bound anti-CD3 (1 µg/mL) in the presence
or absence of missense or antisense RELA (p65) oligonucleonucleotides. On day 3,
proliferation was evaluated by BrdU incorporation, and IFN-γ secretion was evaluated
by ELISA on culture supernatants.
Figure S6. Foxj1 is a distinct quiescence pathway in T cells. Unlike lung kruppel-like
factor (LKLF) and Tob/Smad, which have no known effect on NF-κB activity (21, 22),
Foxj1 is required to suppress spontaneous NF-κB activation, at least in part via IκB
genes, and prevent the subsequent development of activated T cells and tolerance loss.
Supporting References and Notes
1. S. L. Brody, X. H. Yan, M. K. Wuerffel, S. K. Song, S. D. Shapiro, Am. J. Respir.
Cell. Mol. Biol. 23, 45 (2000).
2. S. L. Peng, A. J. Gerth, A. M. Ranger, L. H. Glimcher, Immunity 14, 13 (2001).
3. L. L., A. J. G., S. L. P., Unpublished observations.
4. S. L. Peng, S. J. Szabo, L. H. Glimcher, Proc. Natl. Acad. Sci. USA 99, 5545 (2002).
5. A. R. Khaled, E. J. Butfiloski, E. S. Sobel, J. Schiffenbauer, Clin. Immunol.
Immunopathol. 86, 170 (1998).
6. J. L. Grogan et al., Immunity 14, 205 (2001).
7. G. Schneider et al., J. Biol. Chem. 277, 43599 (2002).
8. M. Iida et al., Carcinogenesis 24, 757 (2003).
9. L. Overbergh, D. Valckx, M. Waer, C. Mathieu, Cytokine 11, 305 (1999).
10. A. J. Gerth, L. Lin, S. L. Peng, Int. Immunol. 15, 937 (2003).
11. M. Oukka et al., Mol. Cell 9, 121 (2002).
12. S. J. Szabo et al., Cell 100, 655 (2000).
13. L. M. Budde, C. Wu, C. Tilman, I. Douglas, S. Ghosh, Mol. Biol. Cell 13, 4179
(2002).
14. L. Lim, H. Zhou, R. H. Costa, Proc. Natl. Acad. Sci. USA 94, 3094 (1997).
15. S. L. Brody, B. P. Hackett, R. A. White, Genomics 45, 509 (1997).
16. J. Rengarajan et al., Immunity 12, 293 (2000).
17. M. Onishi et al., Exp. Hematol. 24, 324 (1996).
18. S. Morita, T. Kojima, T. Kitamura, Gene Ther. 7, 1063 (2000).
19. Y. You et al., Am. J. Physiol. 20, in press and available online at
ajplung.physiology.org/cgi/reprint/00170.2003v1 (2003).
20. E. Schreiber, P. Matthias, M. M. Muller, W. Schaffner, Nuc. Acids Res. 17, 6419
(1989).
21. C. T. Kuo, M. L. Veselits, J. M. Leiden, Science 277, 1986 (1997).
22. D. Tzachanis et al., Nature Immunol. 2, 1174 (2001).