c-Rel is crucial for lymphocyte proliferation but dispensable for T cell effector function
Hsiou-Chi Liou,
Zhuang Jin,
Joseph Tumang,
Sofija Andjelic,
Kendall A. Smith and
Mei-Ling Liou
Cornell University Medical College, Department of Medicine, Division of Immunology, LC-929, 1300 York Avenue, New York, NY 10021, USA
Correspondence to:
H.-C. Liou
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Abstract
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The TCR signals are essential for T cell activation and proliferation, primarily through the induction of cytokine and cytokine receptors. Several transcription factor families, including NF-
B/Rel, have been implicated in the regulation of cytokine gene expression in T cells in response to antigen, cytokine and mitogenic stimulation. In this study, we show that the mice with a null mutation in the lymphoid-specific c-Rel gene have normal development of lymphoid tissues and T cell compartment. However, T cells derived from the c-Rel knockout mice have several functional abnormalities. The c-Rel-deficient T lymphocytes fail to respond to activation and proliferation signals mediated by the TCR and mitogens in vitro. This is attributed to an impaired production of cytokines IL-2, IL-3 and granulocyte macrophage colony stimulating factor. In addition, the induction of IL-2R
chain is impaired in the c-Rel/ T cells. The poor expression of cytokines and IL-2R
chain correlates with a reduced nuclear translocation of NF-
B components in c-Rel/ T cells. Since activation is prerequisite for differentiation into effector cells, c-Rel/ T cells failed to differentiate into cytotoxic T cells or Th cells without rescuing cytokines. However, upon supplement with exogenous IL-2, the c-Rel/ cytotoxic T lymphocytes are able to execute cytotoxicity and the c-Rel/ Th cells are capable of providing help to normal B cells. These data suggest that c-Rel is important for inducible cytokine and cytokine receptor expression, and a key regulator of early activation and proliferation in T cells.
Keywords: c-Rel, lymphocyte, proliferation, T cell
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Introduction
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The NF-
B/Rel transcription factor family is composed of five members of interacting proteins, c-Rel, p50, p65, p52 and RelB. They share a highly conserved Rel-homology domain which confers the ability to bind DNA, dimerize and interact with the specific cytoplasmic inhibitors, the I
Bs (18). Early in vitro studies in various cell types demonstrated that NF-
B/Rel transcription factors respond to a variety of stimuli. Those relevant to the immune system include antigen receptors on B and T cells, CD40, CD28, IL-1, tumor necrosis factor (TNF)-
, phytohemagglutinin, and lipopolysaccharide as well as other mitogens. Studies on NF-
B/Rel knockout mice confirmed general features of impaired immunity with distinct phenotypes (915) and were consistent with the observations that each member has distinct tissue distribution as well as target gene specificities (16).
The activation of T cells is a complex process that has been ascribed to three distinct steps. The first step involves signals transduced by the TCR, while the second step is supplied by accessory, co-stimulatory molecules, principally CD28. Together, signals emanating from these receptors synergize to activate the cell to move from a quiescent, resting G0 phase into the early, G1 phase of the cell cycle. The third step is triggered by IL-2, which promotes progression through G1 into the S phase (17,18).
In the dissection of the molecular events responsible for these steps, the regulation of the genes expressed as a consequence of the first two steps has been studied extensively and, thus far, perhaps the gene studied most thoroughly is the IL-2 gene. Exhaustive analysis of the enhancer region 5' upstream of the transcriptional start site has identified response elements for several well-characterized transcription factors. These include elements capable of binding AP-1 complexes, NF-AT, NF-
B/Rel and Oct-1 (1924). Of these various response elements and transcription factors, NF-AT has received the most attention as a key regulator of TCR-dependent activation of IL-2 gene expression, primarily because the immunosuppressive drug cyclosporin A has been shown to suppress TCR-promoted T cell proliferation by preventing NF-AT activation of IL-2 gene expression. By comparison with NF-AT, the importance of the NF-
B/Rel family of transcription factors in the regulation of IL-2 gene expression has remained obscure. However, the suppressive effect of glucocorticoids on T cell proliferation has recently been attributed to the induction of I
B expression (25,26). The increase in I
B results in the prevention of NF-
B/Rel activation and a marked inhibition of IL-2 gene expression upon TCR triggering. Supporting the role of NF-
B/Rel in cytokine gene expression, targeted deletion of the c-Rel gene results in a profound deficiency in TCR-triggered IL-2, IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF) cytokine production (13,27).
To investigate further the role of c-Rel in the regulation of T cell activation and effector function, we utilized gene targeting to disrupt the c-Rel gene. The absence of c-Rel expression results in a marked deficiency in the capacity of T cells to proliferate in response to T cell mitogens, thus confirming the results of Kontgen et al. (13,27). Although this deficiency is primarily attributable to the lack of IL-2 production, the expression of the IL-2R
chain gene was also found to be depressed compared to wild-type controls. As cytokines and receptors are key mediators of cellular and humoral immune responses, we also tested the effector cell function in c-Rel-deficient T lymphocytes.
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Methods
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Gene targeting deletion of the c-Rel locus
Several overlapping phage clones which cover 20 kb of the c-Rel genomic locus were isolated from a 129/sv genomic library and were provided by Dr William Sha. The 3.0 kb BspHI restriction fragment (B3), containing exon 4, was subcloned into the blunted EcoRI site of the pNT-neo vector (28) to generate the pNTB3 construct. The EcoRV 7.6 kb fragment, containing exons 6 and 7, was then subcloned into the blunted XhoI site in the pNT-B3 to generate the final c-Rel targeting construct, pNTB3-RV7.6. This construct was transfected into the embryonic stem cell line, J1, kindly provided by Dr En Li (29), and selected with 180 µg/ml G418 and 2 µM ganciclovir. To screen for homologous recombination, genomic DNA from several hundreds of G418-resistant embryonic stem clones were digested with BamHI and analyzed by Southern blot analysis using a BamHIEcoRV 2.5 kb external probe which recognizes a region upstream of the B3 fragment (Fig. 1A
). This probe detects a 9 kb BamHI fragment of the wild-type allele and a 7 kb fragment of the disrupted allele. One of the embryonic stem cell clones, I-20, contained a copy of the disrupted c-Rel locus and was injected into pseudopregnant female C57/Bl6 mice in the transgenic/knockout mice facility at the Rockefeller University. Twenty-four chimeric mice were obtained displaying 8595% chimerism. Of these, 14 of the 19 male chimeras had germline transmission of the c-Rel gene-targeted allele. Heterozygotic F1 mice were obtained through the breeding of male chimeras with C57BL/6 females. Southern blot analysis and PCR were used for subsequent genotyping.




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Fig. 1. Generation of the c-Rel knockout mice. (A). The c-Rel locus and the targeting-deletion c-Rel construct. The structure of the c-Rel wild-type genomic DNA, the knockout targeting construct and the targeted locus is shown. Exons are represented by open boxes and numbered. Exons 4 and 5 encode amino acids 51101 and 102130 of the c-Rel protein respectively, which are located within the Rel-homologous domain. Homologous recombination between genomic and the deletional construct is expected to result in a deletion of exon 5 of the c-Rel gene and a replacement with the neo gene. The length of the diagnostic BamHI restriction fragments using the external probe (BamHIEcoRV 2.5 kb fragment) in Southern blot analysis is 9.0 kb for the wild-type locus and 7.0 kb for the gene-targeted c-Rel locus (B). Southern blot analysis of genomic DNA of the embryonic stem cell clones. Embryonic stem cell line, J1, was transfected with the c-Rel knockout targeting vector described in (A) by electroporation. Several hundreds of G418- and ganciclovir-resistant clones were analyzed by Southern blot analysis, following BamHI digestion, for homologous recombination at the c-Rel locus using the BamHIEcoRV 2.5 kb probe. A representative clone with the targeted deletion at one c-Rel allele as indicated by the presence of the 7.0 kb BamHI fragment is shown in lane 2. The wild-type allele generated the 9.0 kb BamHI fragment. (C) Southern blot analysis of genomic DNA of the F2 mice derived from inter-breeding of the heterozygous c-Rel-deficient F1 mice (c-Rel+/). Genomic DNAs were digested with BamHI and hybridized with the BamHIEcoRV 2.5 kb probe as described in (A). Results from five representative F2 mice are shown. c-Rel+/, heterozygote; c-Rel /, homozygote; WT, wild-type. As depicted, the mouse represented in lane 1 (c-Rel+/) is a heterozygote as it contains both the wild-type 9 kb and the deleted 7 kb bands. The mice represented in lanes 2 and 4 (c-Rel/) are homozygous c-Rel knockout mice as they contain only the 7 kb restricted fragment. Lastly, DNA from wild-type mice in lanes 3 and 5 (WT) display only the 9 kb band. (D) Western blot analysis of splenic lysates derived from the c-Rel knockout /, heterozygous (+/) and wild-type (+/+) F2 mice. Total spleen lysates obtained from the same F2 mice analyzed in (C) were separated by SDSPAGE, transferred onto nitrocellulose membrane and blotted with the anti-c-Rel-specific antibody (upper panel). The bottom panel shows the same filter that was subsequently re-probed with the anti-I B antibody to serve as protein loading control. Note that no c-Rel protein was detected in the c-Rel/ lysates, indicating a gene-targeted disruption of the c-Rel locus as expected.
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Western blot analysis
Splenic lysates were prepared with 1xRIPA buffer (16), resolved on SDSPAGE, transferred to nitrocellulose filter and subjected to Western blot analysis (16). Equal amounts of protein (40 µg) were loaded in each lane as was confirmed by Ponceau S staining of the membrane. The filter was first probed with the anti-c-Rel antibody at 1:1000 dilution (or 100 ng/ml). Following the incubation with the secondary antibody (horseradish peroxidase conjugated anti-rabbit antibody), specific bands were visualized via the ECL kit according to the manufacturer's instruction (Amersham). The same filter was subsequently re-probed with anti-I
B
antibody, anti-p50 and anti-p65 antibodies as internal protein loading controls.
Flow cytometric analysis
Splenic single-cell suspensions (1x106 cells) were generated and stained with directly FITC- or phycoerythrin (PE)-conjugated antibodies for 30 min at 4°C in 1xHBSS (with 2% FCS, 0.1% sodium azide and 10 mM HEPES, pH 7.2), washed extensively and analyzed using a Coulter XL flow cytometry analyzer. Antibodies utilized for phenotypic characterization include: FITCanti-CD3, FITCanti-CD4, FITCanti-CD8, FITCanti-IL-2R
and PEanti-B220 (PharMingen, San Diego, CA). An Epics XL flow cytometer and XL software was used to collect data while WinMDI software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA) was used for analysis.
Analysis of cell proliferation
Partial purified T cells (8085%) were obtained from crude spleen populations by passing through a nylon wool column to enrich for T cells. The T cells were further enriched (>95%) through antibody-complement-mediated lysis using HB38 (anti-MHC class II) and M3/84 (anti-Mac-3) hybridoma supernatants and Low-Tox M Rabbit complement (Cedarlane, Ontario Canada). Proliferation assays were performed by plating out 1x105 purified B or T cells in triplicate for each condition in 96-well plates. The concentrations of each stimulus for the proliferation and functional studies were: anti-CD3 (2C11), 1.0 µg/ml; phorbol myristate acetate (PMA), 10 µg/ml; ionomycin, 100 µg/ml; IL-2 (Endogen, Cambridge, MA), 125 pM. Prior to indicated time points, cultured cells were incubated with 1 µCi [3H]thymidine for 6 h and incorporation into DNA quantified by scintillation.
ELISA
Splenic T cells were stimulated with 1.0 µg/ml of anti-CD3 (2C11) for indicated time points and culture supernatants were utilized for ELISA following the manufacturer's protocol (Endogen). For the expression of various cytokines (IL-2, IL-3 and GM-CSF), plates were coated with 100 µl of diluted coating antibodies directed against the specific cytokine overnight at room temperature followed by blocking with 200 µl of 10% FCS in PBS at room temperature for 3 h. After extensive washing with 50 mM Tris (pH 7.0) plus 0.2% Tween 20, lymphocyte supernatant was added to each well in serial dilutions in triplicate and incubated overnight at 4°C. The plates were then incubated with the biotinylated detecting antibody (100 µl/well) and incubated for 1 h at room temperature. After extensive washing, visualization of the reaction results was achieved by incubation with horseradish peroxidase-conjugated streptavidin and development of the colorimetric reaction in the presence of appropriate substrate, tetramethylbenzidine. For all cytokines tested, standard curve with ranges of 1.065 pM (IL-2), 0.413 pM (IL-3) and 1.585 pM (GM-CSF) were determined using the recombinant cytokines provided in the ELISA Minikit.
Electrophoretic mobility shift assay (EMSA)
The nuclear and cytosolic extracts were prepared as the following (16). Purified T lymphocytes (10x106 cells per treatment) were harvested, washed once with PBS, resuspended into 100 µl of Buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl and 0.5 mM DTT] plus 0.1% NP-40. Cells were lysed on ice for 2 min and checked for complete lysis under the microscope. Nuclei were spun down and the supernatant was saved as cytosolic fraction. The nuclear pellet was resuspended into 100 µl of Buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT], sonicated for 5 s to decrease viscosity and rotated in the cold room for 30 min. Ultracentrifugation was performed to remove insoluble debris. The supernatant represented nuclear extract.
The EMSA was performed as described previously (16). For antibody supershift and inhibition experiments, the p50 and p65 and control antibodies was purified by ammonium sulfate precipitation of crude immune rabbit serum. C-Rel, p52 and RelB antibodies were commercially available (Santa Cruz Biotechnology, Santa Cruz, CA). The optimal amount of antibodies used in EMSA inhibition was pre-determined using purified proteins. For a 20 µl DNA binding reaction, 0.1 µg of purified antibody was incubated with 10 µg of nuclear extract and 1 µg of poly(dIdC) in 1xDNA binding buffer on ice for 30 min before the addition of 32P-labeled Ig
B probe. The reaction was incubated at room temperature for 15 min and loaded onto a 6% native polyacrylamide gel. For high-resolution electrophoresis, gels were pre-run at 160 V for 2.5 h and continued for 33.5 h after the samples were loaded.
Generation of cytotoxic T lymphocytes (CTL) and Th clones
To generate the CTL cell lines, we took 5x106 whole splenocytes from either wild-type, heterozygous or c-Rel null mice and co-cultured them in final media (RPMI 1640, 10% FCS, 50 µM ß2-mercaptoethanol) with equivalent numbers of
-irradiated whole BALB/c splenocytes in the presence of recombinant IL-2 (125 pM) for 10 days. Cells were then harvested, and residual CD4+ cells depleted using an anti-L3T4 mAb (GK1.5) and anti-mouse IgG magnetic beads (Perceptive Diagnostics, Cambridge MA).
To generate the Th cell line, we took 5x106 splenocytes from either wild-type or c-Rel null mice and cultured in final media in the presence of 1 µg/ml staphyloccocal enterotoxin B (SEB) (Toxin Technologies, Madison, WI) and 125 pM IL-2. After 10 days, residual CD8+ cells were depleted by treatment with an anti-LY2 mAb (TIB 105) and anti-mouse IgG magnetic beads. Cells were subsequently re-stimulated and expanded in the presence of IL-2.
Measurement of cytotoxicity and helper functions of T cell clones
Cytotoxicity assays were performed as follows. Target cells bearing either the allogeneic haplotype (P815; MHC d haplotype) or autologous haplotype (EL-4; MHC b haplotype) were labeled for 1 h in 100 µl of 1 µCi/ml Na51Cr (Amersham). Cells were then extensively washed and 5x103 cells cultured per well in triplicate in the presence of increasing numbers of the CTL effector clones. After 4 h of incubation at 37°C, cells were pelleted and 100 µl of supernatant recovered for scintillation counting. Percent specific lysis was calculated as the percent c.p.m. of maximum (1% Triton-X control) obtained per sample after subtracting away the minimum control (media only). Percent lysis = 100x(sample release spontaneous release)/(maximum release spontaneous release).
Th cell assays were performed as follows. Splenic B cells were purified from wild-type mice by mAb and complement-mediated lysis of T cells with a concurrent adherence step to deplete macrophages. Briefly, splenocytes were treated with mAb directed against Thy1 (J1J), L3T4 (GK1.5) and Ly2 (TIB 105), and subjected to two rounds of Low Tox M complement (Cedarlane). Cells were verified by flow cytometry to be >98% B220+. Responder B cells (1x105) were then co-cultured in triplicate, alone or with 1x104
-irradiated Th cells, in the presence or absence of 1 µg/ml SEB. At the times indicated, cultures were pulsed with 1 µCi [3H]thymidine for 6 h before termination of culture. Cultures were harvested onto glass fiber filters and incorporation measured by liquid scintillation.
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Results
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Phenotypic characterization of the c-Rel knockout mice
To generate mice deficient for c-Rel, a genomic DNA fragment containing exon 5, which encodes amino acids 102130 of the highly conserved Rel homologous domain, was targeted for deletion and replaced by an insertion of the neomycin resistance gene (Fig. 1A
). The c-Rel deletional gene-targeting construct (Fig. 1A
) was transfected into the embryonic stem cell line, J1 (29). One of the c-Rel-knockout embryonic stem cell clones, I-20, was found to contain a copy of the disrupted c-Rel locus (Fig. 1B
) and was subsequently injected into pseudopregnant female C57BL/6 mice. Through breeding of these chimeras with C57BL/6 females, 10 out of the 14 male chimeras generated had germline transmission of the disrupted c-Rel gene as assessed by the transmission of the targeted allele to the F1 offspring. Further breeding of the heterozygous F1 mice generated c-Rel homozygous knockout mice in F2 (Fig. 1C
). All genotypic analyses were performed by Southern blot analysis of the tail-tip DNAs from 4- to 5-week-old mice using a diagnostic external probe, the 2.5 kb BamHIEcoRV fragment, located N-terminal to the targeted region (Fig. 1A
).
The spleen cell lysates extracted from five representative mice were subjected to Western blot analysis using a polyclonal anti-c-Rel antibody. While the splenic lysates from two representative wild-type mice express high levels of the c-Rel protein, the lysates obtained from c-Rel homozygous knockouts have no expression of the c-Rel protein (Fig. 1D
), confirming a successful deletion in the c-Rel gene locus. Interestingly, a gene-dosage effect was observed, in that the c-Rel heterozygote lysates contain about half the amount of the c-Rel protein as do wild-type lysates. As a protein loading control, the same filter was subsequently reprobed with antibodies recognizing other proteins, including I
B
, p50 and p65. The expression levels of I
B
, p50 and p65 are comparable in all lysates (Fig. 1D
, bottom panel; data not shown), suggesting that: (i) the loss of c-Rel had no demonstrable effect on these family members and (ii) the proteins were equally loaded in all samples analyzed. Taken together, these experiments indicate that we have successfully created c-Rel knockout mice which do not express the c-Rel protein.
The c-Rel/ mice are fertile and appeared overtly normal at a Mendelian ratio when housed in a pathogen-free facility. Phenotypic characterization of the lymphoid tissues demonstrated a normal development of T and B cell compartments. In particular, c-Rel knockout mice have normal sized thymi and spleens. Flow cytometric analysis of the c-Rel knockout thymus indicated a normal distribution of CD4 and CD8 single-positive T cells, as well as double-positive and double-negative T cell populations when compared to the wild-type mice (data not shown). In cells from spleens and lymph nodes, the number and ratio of B and T cells were also comparable to the normal mice (data not shown). These analyses indicated that B and T lymphocyte development is not apparently disturbed in the absence of c-Rel protein.
Activation of proliferation in c-Rel deficient T lymphocytes
Since members of the NF-
B/Rel family are activated by TCR signals, we investigated whether the disruption of the c-Rel gene affected TCR-mediated activation of proliferation. Consistent with previous observations (13), we found that T cells purified from the c-Rel/ mice had defective proliferative responses to both anti-CD3, as well as PMA/ionomycin stimulation, as compared to the wild-type cells (Fig. 2
). The response of c-Rel heterozygous T cells were intermediate between the wild-type and the homozygous c-Rel knockout T cells, thus demonstrating a clear gene dosage effect. By comparison, there was no defect in proliferative responses of c-Rel knockout T cells, provided IL-2 was supplied exogenously (Fig. 2B and 2D
).

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Fig. 2. c-Rel-deficient T cells fail to proliferate in response to various stimuli. Purified T cells were stimulated with 1 µg/ml of anti-CD3 (A and B) or PMA/ionomycin (10 and 100 µg/ml) respectively (C and D). Recombinant IL-2 (125 µM) was added in experimental groups in (B) and (D). Each day, one of the four replicate plates was assayed for [3H]thymidine incorporation as a measure of cell proliferation. Each data point represents triplicate samples. For the anti-CD3 experiment group, the T cells were co-cultured with irradiated syngeneic splenic cells as a source of co-stimulatory signals.
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Impaired cytokine production in c-Rel deficient T lymphocytes
T cell activation through the TCR and co-stimulatory molecules results in the production of cytokines. Therefore, the proliferative defects in c-Rel/ T cells could be explained simply by impaired cytokine production, as NF-
B/Rel binding sites are present in the promoter regions of several cytokines and cytokine receptors, including IL-2, IL-3, GM-CSF, TNF-
, IL-6, and IL-2R
chain (6,12,27,3033). To focus specifically on the contribution of c-Rel in cytokine production, we took culture supernatants from wild-type or knockout splenocytes cultured with or without anti-CD3 antibody in the presence of irradiated antigen-presenting cells and analyzed for cytokine production by ELISA (Fig. 3A
C). Wild-type splenocytes treated with anti-CD3 produced detectable levels of IL-2, IL-3 and GM-CSF, although with varied kinetics. In cultures of wild-type cells, IL-2 levels peaked at 24 h (~186 pM) and declined by 48 h (~33 pM); whereas IL-3 and GM-CSF levels became detectable only after 48 h of stimulation (~8 pM for IL-3; ~54 pM for GM-CSF). By comparison to wild-type, splenocytes from c-Rel knockout mice produced <25% normal amounts of IL-2 (~30 pM), IL-3 (~2 pM) and GM-CSF (~8 pM).
Impaired IL-2R
expression in c-Rel-deficient T lymphocytes
Since the IL-2R
promoter contains an NF-
B/Rel regulatory element (31,32,34), we chose to follow the cell surface expression of the IL-2R
chain on wild-type and c-Rel/ T cells. As expected, anti-CD3 ligation of wild-type T cells induced 26.6% of the cells to become IL-2R
+ by 24 h (Fig. 4
, top two panels), and by 36 h, this expression level had decreased to 15.0%. In contrast to wild-type cells, only 5.5% of c-Rel/ T cells became IL-2R
+ by 24 h, with little change was observed at 36 h (Fig. 4
, bottom two panels).
IL-2 up-regulates IL-2R
by activating STAT5-responsive elements in the IL-2R
promoter (32). Therefore, we examined the effect of adding exogenous IL-2 to our culture system, particularly as c-Rel/ cells were deficient in IL-2 production. The addition of 125 pM IL-2 resulted in a slight up-regulation of IL-2R
expression by wild-type cells (30% at 24 h and 36% at 36 h), but enhanced expression by the c-Rel/ T cells was even more noticeable (20% at 24 h and 26% at 36 h) when stimulated with anti-CD3 antibody (Fig. 4
). Consistent with the lack of functional IL-2R
expression by resting T cells, IL-2 alone had no effect on IL-2R
expression. Therefore, these data indicated that c-Rel/ T cells have an impaired proliferative response to TCR signals as a consequence of the decreased production of IL-2 and IL-2R
, but supplement with exogenous IL-2 circumvents such deficiency.
Reduced nuclear translocation of NF-
B/Rel components
To determine whether the decreased cytokine and receptor production results from a diminution in the NF-
B/Rel nuclear complexes, we performed EMSA on nuclear extracts prepared from stimulated T cells. Ligation of the TCR receptor alone, or in conjunction with CD28 co-stimulation, caused a 3- and 8-fold induction of nuclear translocation of NF-
B/Rel binding complexes in wild-type T cells (Fig. 5A
). By comparison, the same stimulation of c-Rel/ T cells, caused only a modest induction of NF-
B/Rel binding complexes, with a 1.5- to 3-fold increase over the unstimulated control.
The composition of the NF-
B/Rel complexes was assessed by using antibodies in which family member specificity was verified and optimized by reactions with purified NF-
B/Rel proteins (16). The results demonstrated that the NF-
B/Rel complexes in the anti-CD3 or anti-CD3 + anti-CD28 stimulated wild-type nuclear extracts were supershifted or inhibited by anti-p50, anti-p65 and anti-c-Rel antibodies, but unaffected, or affected only slightly by anti-RelB or anti-p52 antibodies (Fig. 5B and C
). As expected, in c-Rel/ nuclear extracts the NF-
B/Rel complexes were supershifted only by the anti-p50 and anti-p65 antibodies, but not by anti-c-Rel antibody. The EMSA results implicate that, in the c-Rel deficient T cells, there was a decreased NF-
B nuclear translocation upon TCR or TCR/CD28 stimulation, which correlates with the impaired cytokine and receptor gene expression.
Cytotoxicity of c-Rel-deficient CD8+ T cells
T lymphocytes require TCR and co-stimulation signals in order to differentiate into CD8+ effector cytotoxic or CD4+ Th cells (3538). Since c-Rel/ T cells are deficient in cytokine production in the initial activation phase, they may be unable to differentiate into functional effector cells (38). However, since IL-2 can restore the proliferation defects, we tested whether c-Rel is required for effector function by using IL-2 supplement medium to bypass the activation requirement for T cell proliferation and differentiation.
To assess the role of c-Rel in CTL function, we generated allo-specific CD8+ T cells by co-culturing either wild-type or c-Rel null whole splenocytes (H-2b) with irradiated BALB/c (H-2d) splenocytes in the presence of 125 pM IL-2. After 10 days of culture and an enrichment step for CD8+ (to >95%), cells were assayed for their ability to lyse either MHC-matched or mismatched haplotypic targets, EL-4 (H-2b) or P815 (H-2d) respectively. As shown in Fig. 6
(A), the wild-type CD8+ T cells lysed the allotypic-target cells P815 effectively, but not the MHC-matched EL-4 cells, as expected. The c-Rel/ CD8+ cells had slightly reduced cytolytic capacity. However, the target cell specificity was comparable to that of wild-type cells in that they only lysed the target cells, P815, with allotypic MHC. Thus, in the presence of IL-2, c-Rel/ CD8 cells are capable of differentiating into fully functional cytotoxic T cells, suggesting that c-Rel plays little role in cytotoxicity effector function. Since cytotoxicity requires preactivation of lymphocytes, we could not detect cytotoxicity in freshly purified CD8+ cells of either wild-type or c-Rel/ origins without IL-2 pre-activation (Fig. 6B
).


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Fig. 6. c-Rel-deficient CTL have normal cytotoxicity. (A) Wild-type and c-Rel/ CTL lines were generated against irradiated BALB/c splenocytes (H2-Kd) as stimulators in the presence of exogenous IL-2. 51Cr-labeled H2-Kd-bearing (P815) or H2-Kb-bearing (EL-4) target cells were co-cultured with either wild-type or c-Rel/ CTL lines, and cytotoxicity was assayed at the indicated effector (CTL):target ratios. After 4 h of co-culture, supernatants were collected and specific cell lysis assessed by scintillation counting. (B) Cytotoxicity assay was performed using freshly purified naive wild-type and c-Rel/ CD8+ T cells.
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Helper function of c-Rel deficient CD4+ T cells
It has been well-documented that TB collaboration provides an important mechanism for T cells to provide help to enhance B cell proliferation and antibody production. The key surface molecules involved in TB collaboration are TCR/CD28/CD40L on T cells and MHC/B7/CD40 on B cells (3942). Since TB collaboration involves multiple surface ligandreceptor interactions, it was of interest to determine whether c-Rel influenced, in any way, the differentiation of CD4+ T cells into fully functional Th lymphocytes. Because antigen-specific Th cells are found at a much lower frequency than allo-reactive T cells, we utilized the phenomenon of superantigen-mediated cognate TB interaction (3638) to assess whether SEB-reactive Th cells derived from either the wild-type or a c-Rel knockout mice were capable of inducing B cell proliferation. Since we knew that c-Rel/ T cell clone could not be generated in the absence of IL-2, we cultured splenocytes of both wild-type and c-Rel/ mice in the presence of SEB plus exogenous IL-2. After several days of culture and a subsequent enrichment for CD4+ (to >95%), cells were allowed to quiescence upon the withdrawal of IL-2 for 48 h before harvested for
-irradiation treatment. Normal splenic B cells were then purified and co-cultured with the irradiated Th cells in the presence or absence of SEB. In this setting, functional Th cells drive B cell proliferation only in the presence of the appropriate superantigen (3638). Our studies indicated that both wild-type and c-Rel knockout CD4+ lymphocytes are fully capable of inducing wild-type B cell proliferation in the presence of the superantigen (Fig. 7
). This activity was normal in the regard that the amplitude and the specificity of the response induced by the knockout cells was comparable to that of wild-type. In supporting this observation, the expression level of CD28 and CD40L on c-Rel/ T cells treated with anti-CD3 is comparable to that of wild-type T cells (data not shown). Taken together, our studies on the CTL and Th function indicated that c-Rel is crucial for the proliferation and differentiation into these effector cells. However, once the proliferation is overcome by the addition of exogenous IL-2, c-Rel/ T cells can fully execute cytotoxicity or helper function.

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Fig. 7. CD4+ Th cells derived from the c-Rel knockout mice provide help to B cells. Purified wild-type responder B cells (1x105) were co-cultured in triplicate alone or with 1x104 -irradiated Th cells from wild-type (+/+), c-Rel heterozygotes (+/) or c-Rel knockout homozygotes (/) mice in the presence or absence of 1 µg/ml SEB in 96-well plates. At the times indicated, cultures were pulsed with 1 µCi [3H]thymidine. After 6 h of incubation, thymidine incorporation was measured by liquid scintillation.
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Discussion
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The results of this investigation indicate that c-Rel is the predominant member of the NF-
B/Rel family of transcription factors that are involved in the promotion of IL-2, IL-3, and GM-CSF and IL-2R
expression. A deficiency of c-Rel is not compensated for by the other two members of the NF-
B/Rel family, p50 and p65, which are also activated by the TCR and accessory molecules. Our studies further demonstrated that once the activation block is corrected with the addition of IL-2, c-Rel-deficient T cells can differentiate into functional cytotoxic T cells and Th, suggesting its specific window of action at the immediate early phase of T cell activation.
c-Rel is essential for IL-2 and IL-2R
expression
The profound deficiency of expression of the IL-2 gene by cells from c-Rel/ mice was unanticipated, given the established relative importance of NF-AT in the activation of IL-2 gene expression. However, the importance of NF-
B/Rel in the activation of IL-2 gene expression could have been predicted, given the profound effect of glucocorticoids on IL-2 production. Recent reports have now shown quite convincingly that glucocorticoids inhibit cytokine gene expression by inducing the expression of I
B, which then prevents NF-
B/Rel activation by sequestering NF-
B/Rel factors in the cytoplasm (43,44). In addition, activated glucocorticoid receptors can prevent NF-
B/Rel transcriptional activity by interacting directly with NF-
B/Rel proteins, prior to DNA binding (45).
Equally as unanticipated was the lack of an effect on IL-2 gene expression with disruption of either the NF-ATp or NF-ATc transcription factor. In particular, NF-ATp deficient T cells have dysregulated IL-4 production (46,47), whereas NF-ATc deficiency led to a decrease in IL-4 and IL-6 production (48,49). In both cases, NF-AT-deficient cells seem to have normal production of IL-2. These studies indicated that either disruption of these NF-AT members alone is not sufficient for interfering with IL-2 production or that other transcription factors, such as NF-
B/Rel, can compensate for its expression.
The in vivo footprinting studies performed by Dr Rothenberg and colleagues clearly indicated that individual IL-2 regulating transcription factors, NF-AT, AP-1 and NF-
B/Rel, cannot stably bind their target sequences in vivo unless all the other factors are present at the neighboring sites (22,24). Recent X-ray crystal structure analysis demonstrated that the NF-AT, Fos and Jun, could form a stable tertiary complex which tightly associates with the composite NF-AT/AP-1 site in the IL-2 promoter (50). As the regulation of cytokine promoters is most likely coordinated by the combinatorial transcription factors, it should be acknowledged that NF-
B/Rel transcription factors may act synergistically with NF-AT and AP-1 in modulating the level of cytokine production in T cells (21,5153). Therefore, it will now be interesting to extend this kind of structural analysis to include the NF-
B/Rel binding site and the NF-
B/Rel family members, to visualize the conformation of the entire `enhansasome' bound to the IL-2 promoter.
It is intriguing to observe that the expression of the IL-2R
chain is also dependent on the presence of c-Rel protein. The regulation of the IL-2R
chain gene is distinct from the IL-2 gene itself, in that IL-2R
chain gene expression is controlled by both the TCR and IL-2 (54). Resting T cells express no detectable IL-2R
chain, but within 36 h of stimulation via the TCR, the
chain first becomes detectable. Subsequently, the secretion of IL-2 results in a further activation of the IL-2R
chain gene. The promoter region of IL-2R
gene contains bona fide NF-
B/Rel and GAS sites; the latter interacts with the STAT5 transcription factor (55). The fact that IL-2 supplement, which induces STAT5 transcription factor, can fully restore the expression of IL-2R
chain expression in the c-Rel/ T cells suggest that both NF-
B/Rel and STAT5 transcription factors contribute to its expression, although in response to distinct signals: NF-
B/Rel binding site is crucial for the initial induction phase by TCR signal, whereas the STAT5 site would be important only after IL-2 becomes available (55).
c-Rel and T cell effector function
By taking advantage of superantigen-mediated T cell help, we were able to assess whether the c-Rel/-derived CD4+ Th cells were capable of providing help to normal resting B cells. It needs to be emphasized that IL-2 was supplemented in culture medium in the generation of these Th cell clones, as we will not be able to generate T cell clones in the absence of the proliferation cytokine. Yet, our studies indicated that once the activation and proliferation requirements are fulfilled, The T cell differentiation program can proceed without c-Rel protein. Superantigen-mediated interaction between T and B cells is in its nature mimicking the cognate interaction between Th and B cells: both are engaged through interaction of TCR and MHC, as well as other co-stimulatory molecules, such as CD28/B7 and CD40 ligandCD40 interaction. Indeed, we found that the expression levels of CD28 and CD40 ligand was normal in the c-Rel/ T cells upon stimulation with anti-CD3 (data not shown), confirming the notion that these c-Rel-deficient T cells are capable of helping B cell proliferation
It was also surprising that disruption of the c-Rel locus seems to exert little effect on the ability of T lymphocytes to execute cytotoxicity function. It has been well-documented that the process that drives CTL precursors (CTLp) to the blastoid cytotoxic effector cells requires the signals from TCR and CD28 co-stimulation. In addition, the production of IL-2 and IL-2 receptors is crucial to the maturation and differentiation of CTL effectors, because the transformation of small non-granular CTL can be induced to large granular CTL by IL-2 (56). Similar to the approach described for the generation of Th clones, we generated the CTL clones in the presence of IL-2 to circumvent the intrinsic proliferation defects of the c-Rel/ T cells. Our studies indicated that once the activation phase was elicited, c-Rel/ CTL has the full capacity to kill the allogeneic cell targets, suggesting the integrity of cytotoxicity machinery. This collective information thus implies that c-Rel is only required for the TCR-mediated blastoid transformation of CTLp and is not required for the second phase of maturation of CTL effectors triggered by IL-2.
In conclusion, c-Rel is critical for controlling the early activation events mediated by the antigen receptor signals in T lymphocytes, but it is dispensable for the execution of the effector function. Its effects, in the case of T lymphocytes, are mostly grossly manifested in proliferative defects resulting directly from a defined impairment in cytokine production and receptor expression. Future work will be necessary to explore the physiological function of the c-Rel gene in the host defense network against pathogenic infection.
 |
Acknowledgments
|
---|
We would like to thank Drs David Baltimore, William Sha and En Li for providing us with the c-Rel genomic clones and J1 embryonic stem cell line respectively. We also thank Drs David Posnett, Patricia Finn and David Perkins for their valuable comments on this manuscript. This work was supported by grants from NIH (RO1-68155), Leukemia Research Foundation, Cancer Research Foundation of America. H.-C. L. is a recipient of the Shannon Award (NCI), Junior Investigator Award (American Cancer Society) and Basil O'Connor Scholar Award (March of Dimes Foundation).
 |
Abbreviations
|
---|
EMSA | electrophoretic mobility shift assay |
CTL | cytotoxic T lymphocyte |
CTLp | precursor cytotoxic T lymphocyte |
GM-CSF | granulocyte macrophage colony stimulating factor |
PE | phycoerythrin |
SEB | staphylococcal enterotoxin B |
TNF | tumor necrosis factor |
 |
Notes
|
---|
Transmitting editor: L. H. Glimcher
Received 16 October 1998,
accepted 9 November 1998.
 |
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