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Nuclear Factor B Is Required for Peptide Antigen-Induced Differentiation of a CD4⁺CD8⁺ Thymocyte Line¹

Jorge Ochoa-Garay^*, Jonathan Kaye and John E. Coligan^2,*

^* Laboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852; and Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037

	Abstract

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NF-B transcription factors are known to regulate the expression of a number of genes involved in T cell activation and function. Some evidence has suggested that they also play a role in T cell development. However, the role of NF-B in Ag-induced thymocyte differentiation has not been directly addressed to date. Here we critically examine this role by employing DPK, a CD4⁺CD8⁺ thymocyte line that undergoes differentiation upon TCR engagement in a process that closely mimics positive selection. Expression of a degradation-resistant form of IB in DPK cells results in constitutive inhibition of NF-B activity. We find that in the absence of NF-B activity, MHC-peptide-induced differentiation of DPK is blocked. Furthermore, differentiation induced by a nonphysiologic stimulus, anti-TCR Ab, is greatly reduced. Altogether, our data indicate a requirement for NF-B in the developmental changes associated with positive selection.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tcell development in the thymus proceeds through a series of stages characterized by the expression of distinct sets of cell surface molecules (1). The major adult thymocyte population consists of double positive (DP)³ CD4⁺CD8⁺ cells, which derive from double negative (DN) CD4^-CD8^- precursors and differentiate into mature single positive (SP) CD4⁺CD8^- or CD4^-CD8⁺ cells. The DP stage is of key importance since the processes of positive and negative selection that shape the peripheral TCR repertoire take place at this stage.

In recent years, much has been learned about the affinity and specificity requirements of the TCR as well as the intracellular signaling events that lead to positive and negative selection of thymocytes (reviewed in Refs. 2–4). However, little is known about the transcription factors mediating the changes in phenotype and function that occur during the selection processes.

The mammalian Rel/NF-B family of transcription factors consists of five members (NF-B1 (p50), NF-B2 (p52), RelA, RelB, and c-Rel) that can bind to DNA as homo- or heterodimers (5) and are collectively referred to as NF-B. Of all dimers, only those containing RelA, RelB, or c-Rel are potent activators of transcription (6). In most cell types and under resting conditions, NF-B dimers are sequestered in the cytoplasm by IB proteins, inhibitory molecules that prevent translocation to the nucleus by masking the nuclear localization sequence on NF-B (5, 7). The best-characterized member of the IB family is IB, which binds tightly to RelB-, to c-Rel-, and particularly to RelA-containing dimers (6). NF-B becomes activated by a host of stimuli, some of which are potent inducers of immune responses, such as mitogens, bacterial products, viral products, and cytokines. Activation of NF-B is a process that involves phosphorylation of IB at Ser³² and Ser³⁶ (8, 9), followed by ubiquitination at Lys²¹ and Lys²² (10, 11), which target the inhibitory molecule for degradation by the proteasome (7). In the absence of cytoplasmic retention, NF-B is free to enter the nucleus where it binds to and regulates expression of target genes. Genes regulated in this way include a number of immune factors, such as cytokines, cytokine receptors, and adhesion molecules (reviewed in 6 .

Several studies have suggested a role for NF-B in thymocyte development (12, 13, 14, 15). However, studies in mice with targeted disruption of single Rel/NF-B family members have not found major defects in the establishment of T cell populations (16, 17, 18, 19, 20). A possible explanation for these results is functional redundancy among the various Rel/NF-B family proteins (21). In this respect, overexpression of IB offers an advantage over targeted disruption of single NF-B genes, since it allows for the simultaneous inhibition of RelA-, RelB- and c-Rel-containing dimers (6).

Recent reports have addressed the effect of trans-dominant IB on T cell development (21, 22). The observed phenotypes, even though variable, include a reduction in the number of TCR^high thymocytes and peripheral T cells, suggesting a developmental arrest at the DP stage caused by constitutive NF-B repression. However, these alterations could be due instead to more general defects in thymocyte proliferation or survival. Thus, the role of NF-B in Ag-induced TCR-mediated thymocyte differentiation has not been studied directly. For this, a system is required in which differentiation can be induced and analyzed in a controlled way while, at the same time, allowing for complete and specific abrogation of NF-B activity.

The cell line DPK was created from a spontaneous thymic lymphoma that originated in AND TCR transgenic mice (23). The AND TCR is specific for a fragment of pigeon cytochrome c (PCC) in the context of I-E^k molecules (24). DPK cells display the phenotype of a typical DP thymocyte and retain the ability to differentiate into CD4⁺ SP cells in vitro and in vivo upon TCR engagement. Because of this ability, DPK has been used as an in vitro model of positive selection (23, 25, 26). To study the role of NF-B in positive selection in a direct way, we constitutively repressed its activity in DPK cells by means of a mutated, degradation-resistant form of IB. Under these conditions, we find that differentiation induced by recognition of MHC-peptide, a ligand of physiologic affinity, is blocked. In addition, the extent of differentiation induced by TCR cross-linking with anti-TCR Ab, a ligand of supraphysiologic affinity, is considerably reduced. These results indicate that NF-B is required for the TCR-mediated induction of DPK differentiation and suggest a key role for this transcription factor in the developmental changes associated with positive selection of thymocytes.

	Materials and Methods

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mut-IB cloning and DPK transduction

A BamHI-BglII fragment containing a FLAG epitope-tagged S32A/S36A-mutated human IB cDNA (a gift from D.W. Ballard, Vanderbilt University, Nashville, TN) was cloned into the BamHI site of the retroviral vector pBabe Neo (a gift from S. Gutkind, National Institutes of Health, Bethesda, MD). The recombinant plasmid was transfected by calcium phosphate precipitation into the amphotropic packaging line PA317 (American Type Culture Collection, Rockville, MD). Stable transfectants were selected in medium containing 800 µg/ml G418 (Life Technologies, Grand Island, NY). For transduction, clone C4-C7-D12 of the CD4⁺CD8⁺ thymic lymphoma cell line DPK was cocultured overnight on a PA317/mut-IB transfectant monolayer in the presence of 8 µg/ml hexadimethrine bromide (Sigma, St. Louis, MO). After 24 h in fresh medium, DPK cells were transferred to 96-well plates and selected in G418 as above. Individual clones were expanded and analyzed by Western blot for the expression of mut-IBa. The DPK clone expressing the highest protein levels was selected for additional experiments (similar results were obtained when two other clones were analyzed). PA317 and DPK media consisted of DMEM or RPMI 1640, respectively, supplemented with 10% FCS (HyClone, Logan, UT), 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM glutamine (Biofluids, Rockville, MD).

DPK differentiation cultures

DCEK-ICAM is a murine fibroblast cell line transfected with I-E^k and ICAM-1 (27) that has been used as APC in the DPK differentiation assay (23). Our differentiation protocol is similar to that originally reported, with some modifications: 5 to 6 x 10⁶ DCEK-ICAM cells were treated with mitomycin C (Sigma) for 30 min, extensively washed with HBSS + 5% FCS and plated on 9-cm dishes (Becton Dickinson, San Jose, CA). After 24 to 48 h, either 1 to 3 µM PCC peptide, residues 88–104 (National Institute of Allergy and Infectious Diseases (NIAID) Peptide Synthesis Laboratory, Rockville, MD), or 50 to 150 ng/ml SEA (Sigma) was added and cells were returned to the incubator. DPK cells (3 x 10⁶) were added 1 to 2 h later and the mixed culture incubated for 3 days. For anti-TCR Ab-induced differentiation, 10 µg of anti-TCR Ab (H57–597) or control anti-TNP hamster IgG (PharMingen, San Diego, CA) in 500 µl of PBS were pipetted into each well of a 24-well plate (Costar, Cambridge, MA) and incubated overnight at 4°C. Wells were washed 2 to 3 times with PBS and blocked with culture medium for 1 to 2 h. DPK cells (2 x 10⁵) were then added to each well and incubated for 3 days.

Ab-mediated cell sorting

DPK cells from either DCEK-ICAM + PCC or anti-TCR Ab cultures were harvested by pipetting and resuspended in PBS + 1% BSA. To remove dead cells and DCEK-ICAM APCs, DPK cells were positively selected based on their expression of CD4. For CD4⁺ cell sorting, cells were incubated for 15 min at 10°C with anti-CD4-conjugated microbeads (Miltenyi Biotec, Auburn, CA), and run through a RS⁺ or VS⁺ separation column as specified by the manufacturer. For H-2D^d or CD69 sorting, cells were incubated with biotinylated anti-H-2D^d or anti-CD69 Abs, washed, incubated with streptavidin-conjugated microbeads, and run through a separation column as above. Positively selected cells were recovered into PBS + 1% BSA medium for further use.

Cytoplasmic and nuclear extracts

DPK cells were harvested and washed in PBS. Cell pellets were resuspended in ice cold lysis buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT) supplemented with a cocktail of protease inhibitors and incubated at 4°C for 15 min. Lysates were pelleted by centrifugation and the supernatant collected as cytoplasmic fraction. Nuclei were washed with lysis buffer, resuspended in high-salt buffer (20 mM HEPES (pH 7.8), 1.5 mM MgCl₂, 0.5 mM DTT, 25% glycerol, 0.2 mM EDTA, 420 mM NaCl) supplemented with protease inhibitors, and incubated with shaking at 4°C for 30 min. Membranes were pelleted and the supernatant collected as nuclear fraction. Nuclear extracts were dialyzed against dialysis buffer (20 mM HEPES (pH 7.8), 100 mM KCl, 0.5 mM DTT, 20% glycerol, 0.2 mM EDTA) for 2 to 4 h and stored at -70°C.

Electrophoretic mobility shift assay

EMSA reactions were conducted in a total volume of 20 µl. Typically, 5 µl of nuclear extracts were added to a reaction buffer containing 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA, 10% glycerol, 1 mg/ml BSA, 1 mM DTT, 0.1 µg/µl poly(dI-dC) (Sigma), 0.1 µg/µl herring sperm DNA (Promega, Madison, WI), and 10⁵ cpm of [-³²P]ATP-labeled ds-oligonucleotide probe (Amersham, Arlington Heights, IL). The oligonucleotide sequences used were: B, 5'-TGGGGAAGCCCAGGGCTGGGGATTCCCCAT-3' (H-2K^b promoter); and Sp-1, 5'-GATCGATCGGGGCGGGGCGATC-3'. To supershift reactions, 2 to 3 µl of rabbit polyclonal serum (Santa Cruz Biotechnology, Santa Cruz, CA) were added simultaneously with nuclear extracts. Reactions were performed at room temperature for 20 min. Samples were loaded onto a 5% polyacrylamide gel in 0.5x TBE buffer and run at 150 V, constant current, for 3 to 6 h. The gel was vacuum dried for 1 h at 80°C and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA).

Western blot analysis

Cytoplasmic extracts (5–50 µg of total protein) from DPK cells were electrophoresed on a 10% SDS-polyacrylamide gel at 200 V, constant current, for 4 h. Transfer to an Immobilon-P membrane (Millipore, Bedford, MA) was done in buffer containing 25 mM Tris, 0.2 M glycine, and 20% methanol at 20 V, constant current, for 2 to 3 h. The membrane was blocked for 1 h with TBS buffer containing 3% powdered milk (BLOTTO) and 0.1% Tween-20, incubated with polyclonal rabbit anti-IB serum (Santa Cruz Biotechnology) in the same buffer for 1 h, washed extensively, incubated for 1 h with donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham), washed extensively, incubated with chemiluminescence substrate as specified by the manufacturer (Amersham), and exposed to film.

Cell surface staining and flow cytometry analysis

DPK cells were harvested and washed in PBS supplemented with 1% BSA and 0.1% NaN₃. Incubation with biotinylated primary Abs was performed in the same medium at 4°C for 30 min. Abs used were: biotinylated anti-H-2K^d (SF1–1.1), anti-H-2D^d (34–2–12), anti-CD3, anti-CD8a, anti-CD24 (HSA), and anti-CD69 (all from PharMingen). After washing, cells were incubated with FITC-conjugated streptavidin in the same medium as above, and analyzed on a FACSort (Becton Dickinson). Dead cells were excluded by propidium iodide staining at 1 µg/ml.

DPK treatment with NF-B-, apoptosis-, or H-2D^d-inducing agents

For NF-B induction, 4 x 10⁷ DPK/neo or DPK/mut-IB cells were harvested and resuspended in PBS. PMA (100 ng/ml) (Sigma) and 1 µg/ml ionomycin (Calbiochem, La Jolla, CA) or 500 U/ml mouse TNF- (Genzyme, Cambridge, MA) were added and the cells incubated at 37°C for 30 min. After washing, protein extracts were prepared as indicated above. For apoptosis induction, 1 to 2 x 10⁵ DPK/neo or DPK/mut-IB cells in 1 ml of culture medium were incubated overnight (16 h) in 48-well plates in the presence of varying concentrations of mouse TNF-, dexamethasone, dibutyryl-cAMP (Sigma), or ionomycin. Cells were resuspended by pipetting, harvested, and analyzed on a FACSort (Becton Dickinson). Dead cells were excluded by propidium iodide staining at 1 µg/ml. For H-2D^d induction, 1 to 2 x 10⁵ DPK/neo or DPK/mut-IB cells in 1 ml of culture medium were incubated for 48 h in 48-well plates in the presence of varying concentrations of mouse IFN- (Amgen, Thousand Oaks, CA). Cells were resuspended by pipetting, harvested, stained for H-2D^d, and analyzed on a FACSort as above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased B binding activity in nuclei of DPK cells upon differentiation

Nuclear extracts from immature T cell lines contain lower levels of B binding activity than extracts from mature T cell lines (28). An analogous finding has been reported for extracts from immature CD69^- vs activated CD69⁺ thymocytes (14). To determine whether an increase in B binding activity takes place during the DP (CD4⁺CD8⁺) to SP (CD4⁺CD8^-) transition and as a consequence of TCR signaling (29), we have made use of DPK, a CD4⁺CD8⁺ thymocyte line that differentiates into a CD4⁺CD8^- mature phenotype upon recognition of MHC + peptide. DPK differentiation was induced in vitro by coculture with DCEK-ICAM APCs and PCC peptide. Similarly to what happens during thymocyte differentiation, DPK differentiation is accompanied by an up-regulation of MHC class I molecules (23). Thus, differentiated cells were purified away from undifferentiated cells based on their up-regulated expression of H-2D^d (see Materials and Methods). Nuclear extracts from undifferentiated and differentiated cells were incubated with a radiolabeled ds-oligonucleotide probe containing the palindromic B site of the murine MHC class I promoter (30), and analyzed by EMSA. As shown in Figure 1A, extracts from undifferentiated cells cause a low-intensity shift with the B probe (lane 1, lower arrow), whereas two intense bands (lane 2, lower and middle arrows) and a more diffuse band (lane 2, upper arrow; more distinctive in lane 5 where the middle band is absent) can be observed with extracts from differentiated cells. Incubation of differentiated cell extracts with polyclonal sera specific for the various NF-B family members allowed us to identify the factors contained in each band. Incubation with anti-p50 serum resulted in selective depletion of the faster mobility complex (lower arrow) and appearance of a supershifted band, indicating that it contains p50 (lane 3). Similarly, selective depletion of the intermediate complex (middle arrow) with anti-RelA Ab indicates that it contains RelA (lane 5). Incubation with anti-c-Rel serum (lane 7) depleted the third, slower-mobility complex (upper arrow), better discernible as a supershifted band, indicating the presence of c-Rel in this complex. Abs to p52 and RelB (lanes 4 and 6, respectively) did not cause observable changes. Given the almost complete abrogation of the lower, middle, and upper complexes by Abs to p50, RelA, and c-Rel, respectively, and the absence of an effect by Abs to p52 and RelB, it is likely that the complexes detected are predominantly homodimeric. Analysis of undifferentiated and differentiated cell extracts using an Sp-1 probe shows equal loading of the lanes (Fig. 1B).

View larger version (83K): [in this window] [in a new window]   FIGURE 1. A, NF-B activity is up-regulated upon DPK differentiation. EMSA was performed with nuclear extracts from equal numbers of undifferentiated (lane 1) or differentiated DPK cells (lanes 2–7) and the MHC class I B probe. Differentiated cells were purified by magnetic separation after labeling with biotinylated anti-H-2Dd Ab and streptavidin-conjugated beads. Identity of the complexes was determined by incubation of extracts with specific sera, which include anti-p50 (lane 3), anti-p52 (lane 4), anti-RelA (lane 5), anti-RelB (lane 6), anti-c-Rel (lane 7). Arrows indicate the different complexes described in the text. B, EMSA using the Sp-1 probe with the same nuclear extracts as in A (lanes 1–2) to show the amount of sample loaded.

Phenotypic analysis of DPK cells transduced with mut-IB

To address the role of NF-B in thymocyte differentiation, DPK cells were transduced with pBabe Neo, a Moloney murine leukemia virus LTR-driven retroviral vector (31) into which a mutated form of human IB (mut-IB) (8) had been cloned (see Materials and Methods). The introduced mutations, two Ser-to-Ala substitutions at residues 32 and 36, prevent phosphorylation and subsequent degradation of this molecule. As a consequence, mut-IB constitutively represses NF-B activity and inhibits its induction by a number of stimuli (9, 32, 33). To test for mut-IB protein expression, cytoplasmic extracts were obtained from mut-IB-transduced (DPK/mut-IB) or control-transduced cells (DPK/neo) and analyzed by Western blotting. These experiments revealed significant mut-IB expression, which could easily be distinguished from endogenous IB due to its slower migration (data not shown, but see Fig. 3A). DPK/neo and DPK/mut-IB cells were analyzed by flow cytometry for T cell surface molecules H-2D^d, CD2, CD3, CD4, CD8a, CD11a, CD24, CD25, CD28, CD44, CD54, CD62L, and CD69 to verify that there were no significant phenotypic differences. Most of these molecules were expressed at similar levels on both cell types (not shown), although small variations could be observed in some of them (see Discussion). Figure 2 shows the expression pattern of a set of these molecules, including those used as differentiation markers in the ensuing experiments (H-2D^d, CD8, CD24, and CD69).

View larger version (72K): [in this window] [in a new window]   FIGURE 3. A, Resistance of mut-IB to induced degradation. Western blot with IB-specific polyclonal serum was conducted on cytoplasmic extracts from DPK/neo (lanes 1–3) or DPK/mut-IB cells (lanes 4–6) untreated (lanes 1 and 4), treated with 100 ng/ml PMA and 1 µg/ml ionomycin (lanes 2and 5), or treated with 500 U/ml of mouse TNF- (lanes 3 and 6) for 30 min at 37°C. Extracts from equal numbers of cells were loaded into each lane. The identity of the bands is indicated. The film was overexposed to show IB in DPK/mut-IB extracts. B, Lack of RelA activation in stimulated DPK/mut-IB cells. EMSA was performed on the MHC class I B probe with nuclear extracts from equal numbers of DPK/neo (lanes 1–3) or DPK/mut-IB cells (lanes 4–6) untreated (lanes 1 and 4), treated with 100 ng/ml PMA and 1 µg/ml ionomycin (lanes 2 and 5), or treated with 500 U/ml of mouse TNF- (lanes 3 and 6) for 30 min at 37°C. To determine the identity of the complexes, DPK/neo extracts treated with PMA and ionomycin (lanes 7–8) or TNF- (lanes 9–10) were incubated with anti-p50 (lanes 7 and 9) or anti-RelA serum (lanes 8 and 10). Arrows indicate the different complexes observed (see text for details). C, EMSA using the Sp-1 probe with the same nuclear extracts as in B (lanes 1–6) to show the amount of sample loaded.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Surface phenotype similarity of DPK/neo and DPK/mut-IB cells. Flow cytometry of DPK/neo (left panels) and DPK/mut-IB cells (right panels) was performed for a set of T cell surface markers. The dotted line represents cells stained with biotinylated control Ab and FITC-conjugated streptavidin, and the solid line cells stained with biotinylated Abs of the indicated specificity and FITC-conjugated streptavidin. Fluorescence intensity is represented in arbitrary units.

mut-IB is resistant to induced degradation and functions to suppress NF-B activation

To test for resistance of mut-IB to induced degradation, DPK/neo and DPK/mut-IB cells were incubated with PMA and ionomycin or mouse TNF-. Equivalent amounts of cytoplasmic extracts were resolved by SDS-PAGE and subjected to Western blotting with an anti-IB serum. As shown in Figure 3A, both stimuli caused a decrease in the amount of endogenous IB in DPK/neo (lane 1 vs lanes 2 and 3) as well as in DPK/mut-IB cells (lane 4 vs lanes 5 and 6), but did not affect the levels of mut-IB (lanes 5 and 6). This result confirms that, in our system, stability is conferred to mut-IB by the S32A/S36A mutations under conditions that cause degradation of wild-type protein. The reduced level of endogenous IB in unstimulated DPK/mut-IB extracts (compare lanes 1 and 4) parallels a similar decrease observed in thymocytes from IB transgenic mice (21), and may be due to indirect inhibition by mut-IB, since IB expression is regulated by RelA (34, 35, 36).

To verify the ability of mut-IB to inhibit NF-B binding to DNA, nuclear extracts from DPK/neo and DPK/mut-IB cells either untreated or treated with NF-B-activating stimuli were analyzed by EMSA using the MHC class I B probe (Fig. 3, B and C). Extracts from untreated DPK/neo cells show undetectable B-binding activity (lane 1), but, upon stimulation with PMA and ionomycin or TNF-, a prominent band appears (lanes 2 and 3, respectively, upper arrow). A weaker intensity band (lower arrow) can also be observed in these lanes. Reportedly, the B binding activity induced by these stimuli is due to their ability to induce both degradation of IB and processing of p105, the p50 precursor (37). In fact, incubation of activated DPK/neo extracts with anti-p50 serum (lanes 7 and 9) supershifts the fast-mobility band, revealing the presence of p50 in this complex. Incubation with anti-RelA serum (lanes 8 and 10) depletes the slow-mobility band and yields a weak supershifted band, indicating that it primarily contains RelA. When extracts from DPK/mut-IB cells are analyzed, however, PMA and ionomycin or TNF- stimulation do not result in the formation of a RelA-containing complex (lane 4 vs lanes 5 and 6). This indicates efficient repression of RelA by mut-IB. In contrast, the fast-mobility complex (lower arrow) increases in intensity upon stimulation (lanes 5 and 6), as occurs in DPK/neo cells. A similar effect observed in pre-B lymphocyte lines transfected with a mutated form of IB (38) was attributed to the low affinity of IB for p50 molecules (39, 40). The fast-mobility complex is more abundant in DPK/mut-IB than DPK/neo extracts (compare lanes 2 and 5, lower arrow). This could also result from the low affinity of mut-IB for p50, since preferential cytoplasmic retention of RelA-containing dimers would increase the nuclear p50:RelA ratio, and consequently the relative concentration of p50 homodimers. As a loading control, the extracts used in lanes 1–6 were tested on the Sp-1 probe (Fig. 3C).

Increased susceptibility of DPK/mut-IB cells to apoptosis

NF-B has been shown to prevent the induction of apoptosis by TNF-, ionizing radiation, or the anti-tumor agent daunorubicin in primary cultures and cell lines (41, 42, 43, 44). To examine the ability of mut-IB to inhibit NF-B activity in vivo, we compared the susceptibility of DPK/neo and DPK/mut-IB cells to TNF--induced apoptosis. Figure 4 shows that mut-IB expression increases the susceptibility of DPK cells to TNF--induced cell death in a dose-dependent manner, as determined by propidium iodide staining (upper left panel). The characteristic pattern of DNA fragmentation detected by agarose gel electrophoresis (not shown) indicated that cell death is indeed apoptotic.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Increased susceptibility of DPK/mut-IB cells to TNF--induced apoptosis. Viability of DPK/neo (triangles) and DPK/mut-IB cells (circles) was determined by propidium iodide exclusion and flow cytometry after overnight treatment at 37°C with 3 to 3,000 U/ml of mouse TNF- (upper left panel), 3 to 100 µM dibutyryl cAMP (upper right panel), 0.1 to 3 nM dexamethasone (lower left panel), or 30 to 1,000 nM ionomycin (lower right panel).

mut-IB inhibits PCC-induced DPK differentiation

Overexpression of IB in transgenic mice results in a reduction in the number of mature SP thymocytes and peripheral T cells (21, 22). This phenotype suggests a block in thymocyte maturation at the DP to SP transition as a consequence of NF-B inhibition. To determine whether NF-B inhibition affects the ability of DPK to differentiate, we incubated DPK/neo and DPK/mut-IB cells with DCEK-ICAM + PCC peptide. DPK cells harvested from these cultures, along with unstimulated DPK controls, were stained for a subset of surface markers that define the differentiated vs undifferentiated phenotypes (H-2D^d, CD8, CD24, CD69). We chose to analyze these markers in particular because they show the largest variation between the undifferentiated and differentiated states (our unpublished observations), and because they include molecules that are both up-regulated (H-2D^d, CD69) and down-regulated (CD8, CD24). Figure 5A shows flow cytometric analysis of unstimulated (dotted lines) and DCEK-ICAM + PCC-stimulated cells (solid lines). Most DPK/neo cells reach a differentiated phenotype: decreased CD8 and CD24 levels and increased H-2D^d and CD69 levels (left panels), while CD4 levels remain unchanged (not shown). In contrast, in DPK/mut-IB cells, CD8 and CD24 levels remain high, and H-2D^d and CD69 levels stay low, indicating that expression of mut-IB blocks the differentiation process (right panels). This effect was consistently observed when three independently generated DPK/mut-IB clones were analyzed (not shown). That the differentiation block was not due to a gross, nonspecific defect caused by mut-IB overexpression is indicated by the similar kinetics of H-2D^d induction on DPK/neo and DPK/mut-IB cells after treatment with IFN- (Fig. 5B, left and right panels, respectively).

View larger version (24K): [in this window] [in a new window]   FIGURE 5. A, Expression of mut-IB blocks DCEK-ICAM + PCC-induced DPK differentiation. Flow cytometry was performed on cloned DPK/neo (left panels) and DPK/mut-IB cells (right panels) after a 3-day incubation in the absence (dotted lines) or presence (solid lines) of DCEK-ICAM APCs + PCC peptide, and staining with biotinylated Abs of the indicated specificity and FITC-conjugated streptavidin. Fluorescence intensity is represented in arbitrary units. B, Absence of a general defect in H-2Dd up-regulation in DPK/mut-IB cells. Flow cytometry of DPK/neo (left panel) and DPK/mut-IB cells (right panel) incubated overnight with increasing doses of IFN-, and stained with biotinylated H-2Dd-specific (black columns) or control H-2Kd-specific Abs (white columns) and FITC-conjugated streptavidin. Fluorescence intensity is represented in arbitrary units.

mut-IB partially inhibits DPK differentiation induced by anti-TCR Ab

In addition to PCC peptide, DPK cells can be induced to differentiate in vitro by SEA superantigen (23) or by plate-bound anti-TCR Ab (our unpublished observations). Given its particularly high affinity for the TCR (see Discussion), we examined the ability of anti-TCR Ab to induce DPK/mut-IB differentiation. As shown in Figure 6, stimulation of control DPK/neo cells with plate-bound anti-TCR Ab also induces a differentiated CD8^neg CD69^high phenotype, but this phenotype is distinct from that induced by DCEK-ICAM + PCC, since H-2D^d levels stay low, and CD24 levels only decrease moderately (left panels). In the case of DPK/mut-IB cells, anti-TCR Ab-induced differentiation is not blocked, although the efficiency of the process is reduced. This reduction is manifested both as a bimodal distribution of cells stained with anti-CD8 or anti-CD69 Abs, and as practically unchanged H-2D^d and CD24 levels (right panels). This indicates that a mechanism of differentiation is still operative in DPK/mut-IB cells and suggests that NF-B is required under physiologic stimulatory conditions (TCR recognition of I-E^k + PCC complexes) but is not so critical after nonphysiologic stimulation (TCR cross-linking by anti-TCR Ab).

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Expression of mut-IB partially blocks anti-TCR Ab-induced DPK differentiation. Flow cytometry was performed on DPK/neo (left panels) and DPK/mut-IB cells (right panels) after incubation on plate-bound anti-TCR Ab (solid lines) or control hamster IgG (dotted lines), and staining with biotinylated Abs of the indicated specificity and FITC-conjugated streptavidin. Fluorescence intensity is represented in arbitrary units.

Anti-TCR Ab-induced DPK/mut-IB differentiation is NF-B independent

One interpretation of the results with DPK/mut-IB cells above is that TCR stimulation by ligands of different affinity (see Discussion) can result in distinct signaling pathways. Signaling induced by DCEK-ICAM + PCC peptide is clearly NF-B dependent, since no differentiation is induced by this stimulus, whereas signaling induced by anti-TCR Ab appears to be NF-B independent. Alternatively, DPK/mut-IB differentiation induced by anti-TCR Ab could still be NF-B dependent if IB inactivation occurred by a Ser³²/Ser³⁶ phosphorylation-independent mechanism. Alternative pathways of IB inactivation have been previously reported, such as amino-terminal truncation (45) or Tyr-42 phosphorylation (46).

To distinguish between these possibilities, DPK/neo and DPK/mut-IB cells were induced to differentiate with anti-TCR Ab. Differentiated cells were isolated based on their expression of CD69, which is up-regulated in both cell types (see Fig. 6). The purity of the preparations was higher than 95% as determined by FACS analysis (not shown). Nuclear extracts from these cells and a fourfold excess of extracts from untreated cells as controls were analyzed by EMSA on the MHC class I B probe as before. As shown in Figure 7A, DPK/neo differentiation induced by anti-TCR Ab stimulation is accompanied by NF-B activation (compare lanes 1 and 2). Treatment with anti-p50 (lane 3) or anti-RelA sera (lane 4) indicates that the fast-mobility complex (lower arrow) contains p50, and the slow-mobility complex (upper arrow) primarily RelA. In contrast, no upper complex can be observed when DPK/mut-IB extracts are analyzed (compare lanes 6 and 7), indicating efficient RelA inhibition by mut-IB. A fast-mobility complex is discernible in lanes containing differentiated DPK/mut-IB extracts (lower arrow). However, this complex is unlikely to be of relevance to the differentiation process, since its intensity is comparable in undifferentiated and differentiated cells (lanes 6 and 7), and since it primarily contains p50 (lane 8), a subunit with very low, if any, trans-activating potential (47). These results indicate the existence of an NF-B-independent mechanism of differentiation in DPK/mut-IB cells. On the other hand, since the extent of DPK/mut-IB differentiation is limited (see Fig. 6), the data are consistent with a partial involvement of NF-B in anti-TCR Ab-induced differentiation of DPK/neo cells. Interestingly, the RelA-containing complex is relatively abundant, as compared with DCEK-ICAM + PCC-stimulated cells where p50 predominates (compare Fig. 7A, lane 2 and Fig. 1, lane 2). Furthermore, anti-TCR Ab stimulation of DPK/neo cells, in contrast to DCEK-ICAM + PCC-stimulation (Fig. 1), does not induce c-Rel activation, as revealed by the absence of an effect (depletion or supershift) by anti-c-Rel serum (Fig. 7 lane 5). EMSA on the control Sp-1 probe shows the relative amounts of nuclear extracts loaded (Fig. 7B).

View larger version (107K): [in this window] [in a new window]   FIGURE 7. A, Induction of NF-B activity in anti-TCR Ab-stimulated DPK/neo but not DPK/mut-IB cells. EMSA was conducted on the MHC class I B probe with nuclear extracts from untreated DPK/neo (lanes 1–5) or DPK/mut-IB cells (lanes 6–10) after incubation in the absence (lanes 1 and 6) or presence (lanes 2–5 and 7–10) of plate-bound anti-TCR Ab for 3 days at 37°C. A fourfold excess of extracts from untreated over anti-TCR Ab-treated cells was loaded to improve visualization. Identity of the complexes was determined by incubation with specific sera, which include anti-p50 (lanes 3 and 8), anti-RelA (lanes 4 and 9), and anti-c-Rel (lanes 5 and 10). Arrows indicate the different complexes described in the text. B, EMSA using the Sp-1 probe with the same nuclear extracts as in A (lanes 1–2and 6–7) to show the amount of sample loaded.

	Discussion

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Our data are in agreement with recent phenotypic analysis of thymocyte and mature T cell populations in IB and mutant-IB transgenic mice (21, 22). In particular, the decrease in SP mature thymocytes observed in these mice suggested a block in the DP to SP transition, a notion supported by the dramatic decrease in peripheral T cell numbers (22). Suggestive evidence for the involvement of NF-B in thymocyte development had also come from in vitro as well as in vivo studies where its activation was inhibited by antioxidants (12) or cyclosporin A (15), conditions under which a block in thymocyte development was observed (12, 48).

The three DPK/mut-IB clones analyzed in our studies were essentially normal in their surface phenotype (Fig. 2 and data not shown), with minor variations. Among them, reduced CD3/TCR, increased CD8, and increased CD28 expression were consistently observed. These variations are not unique to transduced cells, since DPK subclones also tend to display variable phenotypes (our unpublished observations). Nonetheless, the observed variations are unlikely to affect DPK/mut-IB differentiation because of the following: 1) Relatively high levels of CD3/TCR are still expressed by DPK/mut-IB cells (Fig. 2). Also, DPK clones with low TCR levels differentiate normally under standard conditions (our unpublished observations). This is probably so because of the high ligand density (I-E^k, ICAM-1) expressed by DCEK-ICAM APCs and the saturating amounts of PCC peptide used. 2) Increased CD8 levels should not interfere with DPK differentiation, since PCC peptide recognition is MHC class II restricted. 3) Likewise for increased CD28 levels, since B7, the CD28 ligand, is not expressed by DCEK-ICAM cells.

In contrast to the substantial decrease in TCR^high thymocytes observed in wild-type IB transgenics (22), mice expressing a trans-dominant, N-terminally truncated, degradation-resistant form of IB show no decrease in total TCR^high thymocytes and only moderately reduced numbers of SP TCR^high thymocytes (21). This variability may reflect differences in the levels of transgene expression, which in turn results in differences in the extent of NF-B repression. In our in vitro system, mut-IB expression from retroviral Moloney murine leukemia virus LTR resulted in high protein levels as determined by Western blotting (Fig. 3A), and a complete inhibition of NF-B activation by inducing agents such as PMA and ionomycin or TNF- as determined by EMSA (Fig. 3B). Thus, high level expression of fully functional mut-IB may explain the practically complete block of DPK differentiation that we observe. In addition to a direct mechanism of cytoplasmic retention of NF-B, inhibition of RelA activity by mut-IB indirectly results in inhibition of RelA-dependent transcription of other Rel/NF-B family genes (49, 50). Also, the reduced ability of mut-IB to retain p50 in the cytoplasm increases the nuclear p50:RelA ratio, which may contribute to transcriptional repression (22).

Phenotypic alterations in mice transgenic for the N-terminally truncated form of IB include increased susceptibility to apoptosis of T cells, as well as a proliferative defect in thymocytes and T cells in response to mitogenic stimuli (21). DPK/mut-IB cells also show an increased susceptibility to TNF--induced apoptosis (Fig. 4), as do a number of NF-B-defective primary cultures and cells lines (41, 42, 43, 44). Proliferation rates of DPK/mut-IB cells are comparable to those of DPK/neo cells, as measured in a [³H]thymidine incorporation assay (not shown). However, the growth rate of DPK/mut-IB cells during the cloning procedure was considerably slower (data not shown), suggesting an initial defect in proliferation that is overcome by continuous in vitro passage. It is noteworthy also that DPK/mut-IB clones expressing the highest levels of mut-IB display a reduced survival compared with DPK/neo cells after the 3-day in vitro culture with DCEK-ICAM APCs and PCC peptide (data not shown). This reduction in survival, however, is not observed when clones expressing lower levels of mut-IB are analyzed, in spite of the fact that these clones are also unable to differentiate. Therefore, viable cell survival is virtually the same under conditions that promote the differentiation of DPK/neo but not of DPK/mut-IB cells.

NF-B plays a role in transcriptional regulation of several cytokine and cytokine receptor genes (6), and there is a requirement for certain cytokines in thymocyte development (51, 52, 53, 54, 55). Moreover, a role has recently been found for extracellular factors in the NF-B-dependent development of lymphocytes from early precursors (56). Therefore, the block in DPK differentiation caused by mut-IB could be due to an indirect effect, e.g., absence of a NF-B-regulated secretory factor, rather than a direct one, e.g., lack of expression of NF-B-regulated developmental genes. We tested this possibility in a mixed culture, where factors secreted by differentiating DPK/neo cells should be available to DPK/mut-IB cells as well, providing for the differentiation of the latter. Western blotting for IB of whole cell extracts from sorted CD4⁺ (total), H-2D^d+ (differentiated), or CD8⁺ (undifferentiated) populations from mixed cultures showed a perfect correlation between expression of mut-IB and expression of an undifferentiated phenotype (not shown). Although this approach does not address the possibility of a defect in cytokine receptors, the result makes it unlikely that the absence of a secretory factor is the only cause of the inability of DPK/mut-IB cells to differentiate.

The differentiated DPK/neo phenotype induced by DCEK-ICAM + PCC is different from the phenotype induced by anti-TCR Ab (compare Figs. 5A and 6). Similarly, a distinct mature phenotype has been identified for anti-TCR Ab-stimulated thymocytes (57). Interestingly, DPK differentiation induced by DCEK-ICAM + PCC is accompanied by an increase in activated RelA and c-Rel (Fig. 1), whereas differentiation induced by anti-TCR Ab occurs in the absence of up-regulated c-Rel activity (Fig. 7A). Although targeted disruption of c-Rel by itself does not result in a developmental defect in thymocytes (19), the results above are consistent with a requirement for c-Rel, together with other Rel/NF-B dimers inhibited by mut-IB, in the induction of a normal mature phenotype, a possibility that we are currently investigating. In support of this idea, c-Rel has been suggested to play a role in the signals involved in the DP to SP transition (14, 15).

Anti-TCR Ab induces partial differentiation of DPK/mut-IB cells relative to DPK/neo cells, as shown by CD8 down-regulation and CD69 up-regulation in 30 to 70% of the cells (Fig. 6). This suggests that NF-B participates, at least partially, in the anti-TCR Ab-induced pathway of differentiation. In support of this conclusion, anti-TCR Ab-induced differentiation of DPK/neo cells results in an increase in nuclear NF-B levels (Fig. 7A). As a corollary, the partial CD8 down-regulation and CD69 up-regulation in DPK/mut-IB cells follows a bimodal distribution, suggestive of the existence of a transcriptional threshold for the initiation of the differentiation program that is only exceeded in a limited number of cells (58). It is, perhaps, the contribution from NF-B-mediated signaling that allows differentiation of practically all DPK/neo cells to occur (Fig. 6, CD8 and CD69 panels).

The ability of anti-TCR Ab to induce DPK/mut-IB differentiation, as opposed to the inability of DCEK-ICAM + PCC, could have a qualitative and/or a quantitative basis. The affinity of anti-TCR Abs for the TCR (in the same range as for other Ab-Ag interactions) is 10^-8 to 10^-10 M (59, 60), whereas the interaction of specific TCRs with I-E^k/PCC complexes is 10^-4 to 5 x 10^-5 M (61), that is 3 to 6 orders of magnitude lower. This difference in affinity is consistent with the induction of a greater-intensity signal by anti-TCR Ab. However, anti-TCR Ab stimulation of DPK/neo cells only causes a reduced shift in CD24 and H-2D^d levels as compared with DCEK-ICAM + PCC (compare Figs. 5 and 6), suggesting a qualitative difference between the two stimuli. Interestingly, qualitative differences in TCR signaling induced by ligands of physiologic (MHC + peptide) vs supraphysiologic affinity (anti-TCR Ab) have recently been observed in an in vitro system that uses the same TCR/I-E^k combination as DPK/DCEK-ICAM (62), as well as in earlier reports (63). Thus, the ability of anti-TCR Ab to induce DPK/mut-IB differentiation appears to be NF-B independent (Fig. 7A), and suggests the existence of an alternative mechanism of differentiation.

Since IB inactivation can take place by alternative mechanisms, including amino-terminal truncation (45) and Tyr-42 phosphorylation (46), we stimulated DPK/mut-IB cells with anti-TCR Ab and analyzed cytoplasmic extracts by Western blotting for the presence of truncated/phosphorylated forms of mut-IB. We found that mut-IB from these cells is not reduced in amount or altered in its migration (data not shown), even though 1) endogenous IB from differentiated DPK/neo cells is largely degraded, and 2) treatment of DPK/mut-IB cells with the tyrosine phosphatase inhibitor pervanadate resulted in the formation of a slowly migrating form of mut-IB similar to the inactive Tyr-42 phosphorylated form of IB described in pervanadate-treated Jurkat cells (46). This result is consistent with the inability of anti-TCR Ab to induce mut-IB inactivation by an alternative mechanism, and therefore with its inability to induce NF-B activity in DPK/mut-IB cells.

In summary, our data have identified a major role for NF-B transcription factors in the process of positive selection induced by TCR recognition of MHC-peptide. A challenging question raised by our findings that we are currently pursuing concerns the identity of the genes regulated by NF-B that participate in the thymocyte differentiation process.

	Acknowledgments

 
We thank Drs. D.W. Ballard and S. Gutkind for providing reagents. We also thank Drs. A. Brooks, E. Fernández, W. Magner, K. Ozato, K. Parker, J. Shuman, and U. Siebenlist for critically reviewing the manuscript.

	Footnotes

 
¹ This work was supported in part by a postdoctoral fellowship from the Basque Government to J.O.-G.

² Address correspondence and reprint requests to Dr. John E. Coligan, Laboratory of Molecular Structure, National Institute of Allergy and Infectious Diseases, Twinbrook II, Room 103, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address:

³ Abbreviations used in this paper: DP, double positive; EMSA, electrophoretic mobility shift assay; PCC, pigeon cytochrome c; SEA, Staphylococcus enterotoxin A; SP, single positive.

Received for publication October 29, 1997. Accepted for publication December 22, 1997.

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