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BNS in T Cell Cytokine Regulation As Revealed by Targeted Gene Disruption1
* Laboratory of Immunobiology, Department of Medical Oncology, Dana Farber Cancer Institute, and
Department of Medicine, Harvard Medical School, Boston, MA 02115;
Baylor Institute for Immunology Research, Dallas, TX 75204; and
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BNS, a member of the IB family of NF-B inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of IBNS in TCR triggering, we created a targeted disruption of the IBNS gene. Surprisingly, mice lacking IBNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, IBNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-
than wild-type cells. Transfection analysis demonstrates that IBNS and c-Rel individually increase IL-2 promoter activity. The effect of IBNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-B rather than the CD28RE site; mutation of the NF-B site extinguishes the induction of transcription by IBNS in transfectants and prevents association of IBNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN--linked transcripts in IBNS knockout T cells. Collectively, our findings demonstrate that IBNS regulates production of IL-2 and other cytokines induced via "strong" TCR ligation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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pMHC engagement of the TCR induces multiple posttranslational biochemical modifications and new gene transcription (3) and the ubiquitous transcription factor, NF-B, has been implicated in both survival and death of thymocytes (4, 5, 6). Previously, our analysis of genes induced in DP thymocytes undergoing cognate peptide-induced apoptosis identified a novel member of the IB family of NF-B inhibitors, IBNS (7). Induction of IBNS expression in the thymus correlated with TCR signal strength and relatively avid pMHC ligands triggered expression of IBNS whereas weaker ligands did not. In fetal thymic organ cultures (FTOC) retrovirally infected to overexpress IBNS, development of double-negative (DN) to DP thymocytes was reduced and addition of anti-CD3
mAb had a much more dramatic apoptotic effect on IBNS-expressing than control DP thymocytes. Thus, expression of IBNS alone is not sufficient for DP thymocyte death, rather additional signaling via the TCR complex is required to regulate the response outcome.
The NF-B protein family plays a key role in the overall regulation of the immune system. Activation through the TCR is dependent on NF-B for proliferation and cytokine production (8, 9, 10). The pathway leading from the TCR to NF-B activation involves multiple components culminating in the ubiquitination and degradation of IB inhibitors, thereby releasing NF-B to enter the nucleus and direct gene transcription. This process is complicated by the presence of five NF-B family members, each capable of forming multiple hetero- and homodimers (11). The complexity is amplified by the fact that while specific NF-B-binding DNA sites are preferentially bound by particular dimers, multiple varieties of dimers can also bind with comparable affinities to the same DNA sequence (12, 13, 14). Functions unique to one NF-B dimer may result from the range of DNA sequences bound at high affinity by that protein dimer (15). Thus, gene regulation at a specific NF-B site is dependent on the identities and amounts of dimers expressed in the cell; this can also vary over time as the accumulation or destruction of some dimers results in NF-B dimer exchange on DNA (16).
Until recently, seven NF-B inhibitor proteins were known: IB
, IB
, IB, IB, Bcl-3, p100, and p105 (reviewed in Ref. 17). This family expanded with the identification of IBNS (7) and IB
(18, 19, 20). IBNS and IB show more sequence homology to each other and to Bcl-3 than to IB and, like Bcl-3, these proteins appear to be nuclear rather than cytoplasmic. IB (21), Bcl-3 (22, 23), and IBNS (24, 25) have been shown to regulate promoters of cytokines controlled by NF-B and to be involved in inflammatory responses. Their mechanisms of action are different from that of IB because their expression is transcriptionally regulated and more restricted in cell expression patterns (7, 18, 19, 20, 21, 26, 27) unlike the more ubiquitous NF-B and other IB family members. These IBNS, Bcl-3, and IB properties can narrow the action of NF-B providing an additional layer of specificity control. For example, ligands such as TNF or IL-1 bind distinct receptors on a cell but use the same NF-B signaling pathways to induce different sets of genes. Such differential gene expression can be explained by the fact that, in addition to activating NF-B, receptor triggering may induce a particular IB family member, which differentially controls NF-B binding at specific gene promoters. We show in this article for the first time, using targeted gene disruption, that IBNS has such a function in regulating cytokine production during T cell activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BNS knockout (KO) mice
An IBNS BAC clone was obtained from Incyte Genomics and fragments generated by PCR from the BAC clone used to generate a KO construct in the pPNTloxPneo vector. 129/sv W4 embryonic stem (ES) cells were transfected and rES cell lines identified by Southern analysis. Potential founders were crossed to C57BL/6 mice and positive offspring bred to generate homozygous KO animals. For cognate Ag stimulation, KO mice were crossed to N15TCRtg+/+ RAG-2–/– H-2b mice (28). F1 and F2 offspring of the IBNS KO x N15TCRtg+/+ RAG-2–/– H-2b mice were intercrossed and N15TCRtg+/– RAG-2–/– IBNS+/+, IBNS+/–, or IBNS–/– or N15TCRtg+/–RAG-2+/– IBNS+/+, IBNS+/–, or IBNS–/– animals used as indicated. No difference has been observed between IBNS+/+ or IBNS+/– mice. Indicated biochemical experiments were performed using extracts from N15TCRtg+/+RAG-2–/–H-2b animals. Animals were used under protocols reviewed and approved by the Dana-Farber Cancer Institute Animal Care and Use Committee.
Abs and flow cytometric analysis
mAbs used: R-PE- or PE-Cy5-anti-mouse CD4 (H129.19), FITC- or PE-Cy5-anti-mouse CD8 (53-6.7), PE-Cy5-anti-TCR C (H57-597), PE-anti-CD25 (PC61), FITC- or PE-anti-V 5.1, 5.2 (MR9.4), PE-anti-B220 (RA3-6B2), PE-anti-NK1.1 (PK136), biotin-anti-pan NK (DX5) or PE-Cy5-streptavidin (BD Pharmingen). For T cell V repertoire analysis, a V TCR screening panel (BD Pharmingen) was used. In vitro stimulations were performed with plate-bound purified anti-CD3 mAb 145-2C11 and anti-CD28 clone 37.51 (eBioscience).
Proliferation assays
Total thymocytes, lymph node (LN) T cells prepared as I-Ab-negative cells by magnetic bead separation, or FACS-sorted CD4+ or CD8+ LN T cells from IBNS KO or WT littermate mice were used for proliferation assays. Thymocytes (1 x 106/well) or LN T cells (2 x 105/well) were cultured for 48 h in complete RPMI 1640 medium alone, plate-bound anti-CD3 (5 µg/ml), anti-CD3 (5 µg/ml) plus anti-CD28 (10 µg/ml), Con A (2 µg/ml) plus irradiated wild-type (WT) spleen cells (2 x 105/well), or PMA (50 ng/ml) plus ionomycin (200 ng/ml). Where indicated recombinant human (rh) IL-2 (50 U/ml) was added. [3H]Thymidine (37 Bq/well) (PerkinElmer) was added for the last 18 h of culture, and incorporated radioactivity determined.
For peptide-induced proliferation of N15TCRtg+/– H-2b IBNS+/– or –/– T cells, total thymocytes (2 x 105/well) or I-Ab-negative LN T cells (2 x 105/well) were incubated with irradiated (3 Krad) N15TCRtg+/– H-2b IBNS+/+ or IBNS+/– spleen cells (1 x 105/well) preloaded with VSV8 peptide for 2 h. After 24 h, 37 Bq/well [3H]thymidine were added and incorporated radioactivity measured 18 h later.
For MLRs, LN T cells purified as described above (2 x 105/well) were cultured with irradiated spleen cells (2 x 105/well) from syngeneic WT mice or allogeneic BALB/c mice for 48 h and [3H]thymidine incorporation determined as above.
Measurement of cytokine production
Cytokine production was induced as above except 5 x 105 LN T cells were used/well. After 24 h, supernatants were collected for multiplex cytokine analysis (Luminex) as described (29) but using an anti-Mouse MultiCytokine Reporter. Concentrations were calculated using Bio-Plex Manager 3.0 software with a five-parameter curve-fitting algorithm applied for standard curve calculations.
NK cell function and cytokine production
Splenic NK cells (purity >75% as determined by anti-mouse CD49b/Pan-NK DX5 (BD Biosciences) staining) were enriched using a SpinSep Mouse NK Cell Enrichment kit (StemCell Technologies). NK cytotoxicity was analyzed by a 51Cr-release assay (30) with mouse YAC-1 targets. Enriched NK cells at 5 x 105 cells/well in complete DMEM medium supplemented with 500 U/ml rat IL-2 were stimulated with 1 µg/ml phytohemagglutinin for 3 days. Supernatants were analyzed for IFN- using the Mouse IFN- OptEIA set (BD Biosciences). The sensitivity of the assay was 31.3–2000 pg/ml and results are shown as the mean from duplicate wells.
Adoptive transfer of bone marrow (BM) cells
BM cells from 2- to 3 mo-old WT or IBNS KO mice were depleted of T and B cells by CD4–, CD8–, and B220-negative separation using magnetic beads. A total of 2 x 106 cells were injected i.v. into irradiated (700 rad) B6 Ly5.1 mice. Recipient mice were sacrificed at 6 wk and thymus, LN, spleen, and peripheral blood cells analyzed for donor-derived Ly5.2+ cells. Donor-derived Ly5.1– IAb– LN T cells were prepared by magnetic bead negative selection and then sorted for Ly5.2+, CD4+, and CD8+ T cells and proliferation assays performed as described above.
Transfection analysis
Transfections were performed using an Amaxa Nucleofector II and the Nucleofector kit with modifications for use with EL4 cells. A total of 100 µl of solution T are added to 1.4 x 106 cells and plasmids (1 µg each expression plasmid, 0.1 µg of pRLnull transfection efficiency control and pcDNA3.1 to total 5 µg of DNA) added subsequently. After transfection (program O-17), cells were cultured 16–18 h before determination of luciferase activity using the Dual-Luciferase Reporter Assay System (Promega).
Plasmids
Plasmids for transfections: pmoIL-2–2kLuc, empty vector control and pUC00Luc (31) (all provided by S. Miyatake, Tokyo Metropolitan Institute of Medical Science, Tokyo); transfection efficiency control plasmid, pRL null (Promega), pCDM8-p50, pcDNA3.1-c-Rel, pcDNA3.1-IBNS, pcDNA3.1 (Invitrogen Life Technologies), pmoIL-2–2kLuc-mNF-B, and pmoIL-2–2kLuc-mCD28RE. The mutations of the NF-B and CD28RE sites were performed with the QuickChange II Site-Directed Mutagenesis kit (Stratagene). The antisense primers for mutation in the NF-B and CD28RE sites were 5'-GGGCTAACCCGACCAAGAGGCTTTTCACCTAAATCCATTCAG-3' and 5'-CAGTGTATGGGGGTTTAAAGCCATTCCAGAGAGTCATCAGA-3', respectively. The boldface letters in the primers indicate the residues mutated. Mutations were confirmed by DNA sequence analysis.
DNA-binding assays
Nuclear extracts (32) were prepared from N15TCRtg+/+RAG-2–/–H-2b thymocytes or LN T cells (see Fig. 9, C and E) or from N15TCRtg+/– RAG-2–/– IBNS+/+, +/–, or –/– mice (Fig. 9D). Mice (4–11 wk) were untreated or injected i.v. with 24 µg of VSV8. DNA probes containing the IL-2 promoter NF-B and CD28RE region were prepared by PCR using 5‘Bio-AAACTGCCACCTAAGTGTGG3' and 5'-Bio-TTCCTCTTCTGATGACTCTC-3' biotinylated primers (IDT) with pmoIL-2–2kLuc, pmoIL-2–2kLuc mNF-B and pmoIL-2–2kLuc mCD28RE as templates, respectively.
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Real-time PCR
RNA isolated by TRIzol (Invitrogen Life Technologies) from purified WT or IBNS KO LN T cells was used for real-time PCR with the Applied Biosystems Taq Man Universal PCR Master Mix. Taq Man Gene Expression Premade probes were used for IL-2, -actin, Fbxo17, Bcl2l1, and Sh2d1a. Probes were custom ordered for Cpd and sequences are available upon request.
Microarray analysis
I-Ab-negative LN T cells prepared using magnetic beads were incubated in anti-CD3- (5 µg/ml) plus anti-CD28- (10 µg/ml) coated plates for 0 or 6 h. Four independently prepared sets of total RNA (TRIzol) were hybridized to standard Affymetrix GeneChip MouseGenome 430A 2.0 arrays in the Beth Israel Deaconess Medical Center Genomics Center (Beth Israel Hospital, Boston, MA). The quality of each array was tested using the Affy package developed by Gauiter (33) considering variation in the percentage of presence calls, background, RNA degradation and scaling factors. High-quality arrays were normalized and analyzed using the four different methods listed in Table I. All signature genes were obtained on the basis of presence calls, fold change (FC) and unpaired Student t test (p-value) as shown in Table I. To reduce false-positive results, the final signature was obtained by taking into consideration the genes that are identified as differentially expressed by at least three methods. The unsupervised two-dimensional clustering was performed on these signature genes using the DCHIP clustering utility (34).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BNS KO mice
To examine the role of IBNS in thymocyte development, we created mice with a targeted disruption in the IBNS gene. The 4.8-kb deleted region contains exons I through V and 127 bp of the 153 bp exon VI, removing 208 of the 327 aa of IBNS as well as 2.8 kb upstream of exon I (Fig. 1A). Southern blot genotyping of tail DNA is shown in Fig. 1B. IBNS KO mice appear grossly normal and breed well; WT, heterozygous, and KO offspring are born in the expected Mendelian ratios and homozygous KOs appear healthy for more than 2 years.
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BNS from the thymus (7), we began analysis on the thymus and T cells. Surprisingly, there are no significant differences in the thymus, LN, or spleen in terms of cell numbers or distribution of major subpopulations (Fig. 1C). In addition, the distribution of Vs expressed on peripheral CD4+ and CD8+ T cells is similar between WT littermates and KO animals as analyzed with 15 different anti-V Abs (data not shown). Thus, no overt T cell developmental or selection phenotype was observed.
IBNS KO T cells are impaired in proliferation
We tested proliferation of the IBNS KO T cells and thymocytes as induced with plate-bound anti-CD3, anti-CD3 plus anti-CD28, Con A, or PMA plus ionomycin. IBNS KO thymocytes proliferate less in response to Con A than WT (Fig. 2A). However, PMA plus ionomycin induces nearly equivalent proliferation in WT and IBNS KO thymocytes. IBNS KO thymocytes (Fig. 2B) as well as CD4+ and CD8+ LN T cells (Fig. 2C) exhibit reduced proliferation to anti-CD3 and anti-CD3 plus anti-CD28. The proliferation in KO thymocytes is more severely reduced than in the mature KO T cells. We previously demonstrated that IBNS is induced by TCR cross-linking and not by agents that do not act directly through TCR ligation (7). The fact that PMA plus ionomycin bypasses the IBNS deficiency is consistent with this combination acting downstream of the TCR beyond the point at which IBNS is required. Con A, by cross-linking CD3 glycans (35), likely acts via the TCR complex and, perhaps, additional surface receptors and therefore, is impaired like the anti-CD3 ± anti-CD28 Ab cross-linking stimulation.
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BNS KO T cells exhibit reduced responses to cognate and allogeneic Ags
To examine IBNS KO T cell responses to cognate Ag, IBNS KO mice were crossed with N15TCRtg+/+RAG-2–/–H-2b mice (hereafter termed N15TCRtg+/+ mice) (28). N15TCRtg+/+ mice express a TCR recognizing a vesicular stomatitis nucleoprotein octapeptide (VSV8) in the context of H-2Kb (36). Thymocytes and LN T cells purified from N15TCRtg+/–RAG-2+/–IBNS–/– and N15TCRtg+/–RAG-2+/–IBNS+/+ littermate animals were tested for proliferation in response to peptide-loaded, irradiated IBNS+/+ spleen cells (Fig. 3A). The N15TCRtg+/– IBNS KO T cells require VSV8 at 
2 logs higher concentration for proliferation equivalent to WT T cells.
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BNS KO T cells were tested for recognition of allogeneic MHC in MLR. The LN T cells of the IBNS KO animals do not respond to syngeneic splenocytes, but are activated by splenocytes from MHC H-2d mice, although to a significantly reduced extent compared with LN T cells from WT littermates (Fig. 3B).
Microarray analysis of IBNS KO T cells
The genetic consequences of IBNS deletion on T cell activation were determined using four independent microarray analyses. Unstimulated KO and WT T cells segregate completely and are discriminated by eight genes including IBNS (AY078069)(Fig. 4A and Table II "Genes expressed differentially between WT and IBNS KO LN T cells at 0 h"). This low number is not surprising because we observe normal development of T cells in the IBNS KO mouse and because IBNS expression is not constitutive but rather induced upon TCR triggering (7). In addition, the expression levels of Hcst, Fbxo17, and Sbsn are more similar in the IBNS KO and WT T cells after activation. Expression of Tyrobp in the IBNS KO is decreased 2-fold at both zero and 6 h while the level of IBNS expression is 5-fold down at 0 h and 10.9-fold down at 6 h. Expression of IBNS in the unstimulated WT LN T cells may result from manipulations conducted during cell separation.
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BNS KO and WT T cells yielded a final signature containing 30 genes (Fig. 4B and Table II "Genes expressed differentially between WT and IBNS KO LN T cells at 6 h postactivation with anti-CD3 + anti-CD28"). Only IBNS, Hcst, and Fbxo17 were present in the final signature of both resting and stimulated T cells. The 30 genes in the six hour signature are distributed over 15 different chromosomes with six located on chromosome 7; IBNS maps to 7A3 as do Hcst, Fbxo17, and Map4k1 (chromosome locations from National Center for Biotechnology Information). Pep4 at 7B1 and Ifitm2 at 7F5 are more distant. The significance of the dysregulation of these genes and their location on the same chromosome as IBNS is undetermined. IBNS may play a role in regulating these genes but we cannot exclude the possibility that the IBNS gene disruption alters transcription in some global manner around that position on chromosome 7.
Tyrobp and Hcst encode adapter proteins that participate in forming activating receptors on NK cells (reviewed in Ref. 37). Given their altered expression, we tested the cytotoxic capacity of IBNS KO NK cells. IBNS KO NK cells killed Yac-1 targets as efficiently as NK cells from IBNS WT littermates (Fig. 5A). Furthermore, flow cytometric analysis demonstrated equivalent levels of NKG2D on the surface of IBNS KO and WT NK cells (data not shown). Thus, a 2-fold reduction in Hcst mRNA does not affect expression of NKG2D or the killing function of NK cells. In addition, numbers of NK cells are equivalent between the WT and KO mice. IBNS KO NK cells are defective in IFN- production, however, when cultured in vitro (infra vide and Fig. 5B).
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BNS KO T cells (Fig. 4C). Furthermore, as discussed below, the IL-2 deficiency is confirmed at the protein level in our cytokine assays (Fig. 6, A and B).
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BNS KO thymocytes and T cells are impaired in cytokine production
Because primary T cell proliferation depends on cytokine production, particularly IL-2, we used cytokine multiplex analysis to measure cytokine production by IBNS KO and WT littermate T cells and thymocytes in response to plate-bound anti-CD3, anti-CD3 plus anti-CD28, Con A and PMA plus ionomycin. Upon TCR cross-linking, IL-2 and IFN- production are reduced in IBNS KO animals (Fig. 6, A and B). IL-2 production is reduced in total thymocytes (Fig. 6A), CD4+ (data not shown) and CD8+ LN-derived IBNS KO T cells (Fig. 6B). IFN- production is reduced in IBNS KO CD8+ T cells and thymocytes undergoing similar stimulation. Under these conditions, IBNS KO and WT CD4+ T cells do not produce detectable IFN-. With PMA plus ionomycin, IL-2 production was almost equal between IBNS KO and WT thymocytes and T cells (data not shown) indicating that KO cells can produce more IL-2 with stimuli bypassing the TCR. This observation parallels the effect of PMA plus ionomycin on proliferation as described above. IL-2 production by N15TCRtg+/–RAG-2+/–IBNS–/– and N15TCRtg+/–RAG-2+/–IBNS+/+ LN T cells stimulated with VSV8-loaded APCs shows that cognate peptide stimulation of IBNS KO T cells results in much lower IL-2 production compared with WT T cells (Fig. 7).
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BNS KO T cells are capable of responding to IL-2. Exogenous IL-2 reduces the disparity in proliferation between IBNS KO and WT LN T cells (Fig. 6C). Proliferation of both IBNS KO and WT T cells is increased by IL-2 addition, but that of the IBNS KO cells is more strongly enhanced. By flow cytometry, no obvious difference in expression of CD25 was observed in IBNS KO mice (data not shown).
To test whether the observed defects are intrinsic to IBNS KO T cells, we examined IL-2 production in thymocytes and T cells from BM chimeric animals created by injecting IBNS KO or WT BM into irradiated B6Ly5.1 recipients. Repopulation of thymus and LN 6 wk after injection was equivalent for IBNS KO and WT (Fig. 8A). However, LN T cells from the IBNS KO BM chimeras exhibited proliferation defects in vitro, which were largely restored by addition of exogenous IL-2 (Fig. 8B). Thus, the T lineage phenotype observed in IBNS KO animals is cell autonomous and not dependent on the environment in which the T cells mature.
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BNS transfection increases IL-2 promoter transcription
The IL-2 promoter is controlled by a number of transcription factors including NF-B family members (38, 39, 40, 41). The major NF-B contributor to induction of IL-2 is c-Rel (42). Indeed, the c-Rel and IBNS KO mice have similar phenotypes in that the immune systems appear normal but the T cells make reduced IL-2 upon activation (8, 9). Because the IBNS protein has no DNA-binding domain, its affect must be mediated through interaction with a DNA-binding protein and the similar phenotypes of the c-Rel and IBNS KO mice suggest that c-Rel and IBNS might interact to regulate IL-2.
To examine the role of IBNS and c-Rel in control of the IL-2 promoter, we used transfection assays with a plasmid in which the IL-2 promoter drives a luciferase reporter (Fig. 9A). EL4 cells were transfected with the IL-2 promoter vector only or in combination with IBNS- or c-Rel-expressing constructs. Because we previously determined that IBNS binds p50 (7), we also tested the effect of p50 transfection either alone or in combination with IBNS and c-Rel. IL-2 promoter transcription is increased upon cotransfection with either IBNS or c-Rel (Fig. 9B, upper panel). Cotransfection of IBNS plus c-Rel activates transcription from the IL-2 promoter in an additive fashion. Cotransfection of p50 did not result in altered transcription of the IL-2 promoter either alone or with IBNS or c-Rel (data not shown).
A canonical and a noncanonical NF-B site, the latter termed a CD28 response element (CD28RE), lie within 300 bp of the IL-2 promoter (43, 44, 45). Specificities of the IBNS and c-Rel activities were investigated using individual NF-B and CD28RE mutants (Fig. 9A). Mutation of the NF-B site disrupts the effect of IBNS on the IL-2 promoter (Fig. 9B, bottom panel); c-Rel regulation of the NF-B-mutated promoter is intact. Conversely, mutation of the CD28RE blocks activation of transcription by c-Rel while induction of transcription by IBNS is not affected (Fig. 9B, middle panel). Thus, as expected, c-Rel acts on the CD28RE element. IBNS, however, acts through the NF-B site.
IBNS association with the IL-2 promoter
To detect binding of transcription factors to the IL-2 promoter, a biotinylated DNA-binding assay was used. A 109-bp probe (bp –134 to –242) (numbering according to Ref. 46) was added to nuclear lysates extracted from thymus and LN cells and DNA-bound proteins detected by Western blotting after DNA precipitation. Samples were prepared from N15TCRtg+/– IBNS KO mice and N15TCRtg+/– WT littermates after i.v. injection with VSV8 peptide for various times. The IBNS/DNA pulldown matches the specificity demonstrated in transfection assays; one hour following VSV8 injection, nuclear IBNS can be precipitated with WT or CD28RE mutant (mCD28RE) but not with NF-B mutant (mNF-B) DNA (Fig. 9C). Note that although the level of IBNS in the total nuclear lysate is below detectable levels, significant amounts of IBNS are pulled down with IL-2 promoter DNA and this is dependent on the NF-B site. p50 shows the same specificity of IL-2 binding as IBNS; hence, there is no binding to the mNF-B DNA. However, p50 is present in the lysates in the absence of VSV8 stimulation and binds to the IL-2 promoter DNA 1 h after VSV8 stimulation as well as in lysates from unstimulated T cells (Fig. 9C). Two hours postinjection, the amount of p50 pulled down by the IL-2 promoter is barely detectable even though significant p50 is present in the lysate (Fig. 9D). Similarly, at 2 h c-Rel is present in the lysates of both WT and IBNS KO LN T cells, but is bound to the IL-2 promoter only in control, not activated, lysates (Fig. 9D). However, c-Rel binds to the IL-2 promoter in lysates prepared 30 min after VSV8 treatment (Fig. 9E); this binding is decreased or completely gone (Fig. 9E) after 1 h as described further below. Although there appears to be slightly less p50 in the IBNS KO than in the WT (Fig. 9D), the DNA binding patterns appear nearly identical between the IBNS KO and WT lysates. Likewise, the binding pattern exhibited by c-Rel appears very similar in IBNS KO vs WT lysates (Fig. 9D). As expected, the IBNS KO lysate has no IBNS protein (Fig. 9D).
A comparison of the kinetics of p50, c-Rel, and IBNS expression in LN T cells and IL-2 promoter binding after TCR triggering is provided in Fig. 9E. Lysates were prepared 0, 0.5, 1, or 2 h after in vivo VSV8 treatment of N15TCRtg+/+ mice. p50 and c-Rel in the lysate increase 0.5 h after VSV8 activation of T cells while IBNS is not detected until 2 h. The amount of p50 and c-Rel bound to the IL-2 promoter increases in parallel with their expression level from 0 to 0.5 h but subsequently the amount bound by the IL-2 promoter DNA decreases even though the level present in the lysate remains constant (Fig. 9E). IBNS, in contrast, is pulled down with the IL-2 promoter DNA even though below detectable levels in the lysate. Furthermore, the amount of IBNS pulled down with the IL-2 promoter DNA continues to increase up to at least 2 h post-TCR activation.
What are the effects of these different IL-2 promoter DNA/protein combinations on IL-2 transcription? Real-time PCR analysis shows that IL-2 message levels are equal between WT and IBNS KO LN T cells at 0.5 and 1 h after anti-CD3 plus anti-CD28 stimulation in vitro, but by 2 and 4 h transcription is 3- to 5-fold lower in the IBNS KO T cells (Fig. 10). These data indicate that IBNS is required for IL-2 production, although the kinetics of the IBNS effect appear somewhat slower than those observed in Fig. 9E. This may be due to the fact that T cell activation in vitro is less efficient than cognate Ag T cell activation in vivo.
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BNS exhibits no DNA binding domain and as little or no c-Rel or p50 is present on the IL-2 promoter DNA at the later times where IBNS is pulled down by the DNA (Fig. 9, D and E), other DNA-binding proteins likely interact with IBNS to form a complex on the IL-2 promoter DNA. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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BNS showed a correlation between thymocyte negative selection and induction of IBNS (7). We concluded that IBNS played a role in thymic negative selection and created a targeted gene disruption to further characterize this gene. We anticipated greater numbers of thymocytes and T cells in IBNS KO animals compared with WT littermates and that the IBNS KO animals might suffer the effects of self-reactive T cells because these would not be deleted if negative selection was disrupted. However, this was not the case. Analysis of V expression in the IBNS KO T cell population indicated no effect of the disruption of IBNS on negative selection and IBNS KO animals show no ongoing autoimmune processes or obvious developmental abnormalities in the immune system. Thus, if IBNS does play a role in the process of negative selection, its absence must be compensated for by other mechanisms.
However, the proliferation of IBNS KO thymocytes and LN T cells in response to plate-bound anti-CD3, anti-CD3 plus anti-CD28 as well as Con A is significantly reduced. The response to alloantigen and to cognate peptide Ag is also diminished in IBNS KO T cells. Even at high cognate peptide concentrations, the level of proliferation in the IBNS KO LN T cells never reaches the level seen in WT littermate LN T cells (Fig. 3).
As IBNS alters the activity of the transcription factor, NF-B, we used microarray analysis to assess the effect of deletion of this factor on global gene transcription in T cells. Because we observed differences in activation and proliferation in vitro, we isolated RNA from IBNS KO and WT littermate LN T cells activated by plate-bound anti-CD3 plus anti-CD28. Without stimulation, eight genes are differentially expressed in the IBNS KO T cells; these include two unknown cDNAs, an F-box protein, three proteins with known roles in the immune system, IBNS itself and suprabasin, a protein of unknown function expressed in keratinocytes (47). All genes except Fbxo17 are decreased in the IBNS KO T cells.
At 6 h activation, 30 genes are significantly different in expression between the WT and IBNS KO T cells (Table II "Genes expressed differentially between WT and IBNS KO LN T cells at 6 h postactivation with anti-CD3 + anti-CD28"). The gene with the greatest fold change is IBNS itself, which is 10.91-fold decreased in the IBNS KO T cells. Cpd at –5.5, Igl-V1 at –3.55 and Sh2d1a at –3.49 exhibit the next largest differences in expression. Twenty genes exhibit FC of –1.69 to –2.59 including five unknowns, genes induced by IFN-, proteases, cell surface molecules, zinc finger proteins, signaling molecules and transcription factors. Genes of particular interest are discussed further below.
In the activated microarray samples, six genes exhibit FCs of 2.02 to 2.3, including an IL-1 decoy receptor (Il1r2), a LIM-domain containing protein involved in gene regulation, cell growth, development, and differentiation (Csrp1), two Ig-like cell surface receptors (Igsf3 and Gp49a), an F-box-containing protein involved in ubiquitylation (Fbxo17) and GM-CSF. Igsf3 (CD101) has been implicated as the type 1 diabetes locus, Idd10, (48) and triggering of CD101 decreases activation of T cells (49). Gp49a is an inhibitory cell surface molecule that suppresses IFN- responses of T and NK cells (50). The Il1r2 gene is a negative regulator of T cell activation and thus IFN- and IL-2 production (51). IBNS KO thymocytes and T cells produce much less IFN- upon TCR triggering by anti-CD3 or anti-CD3 plus anti-CD28 (Fig. 6). Although it is possible that IBNS directly acts to control the IFN- promoter, an alternative explanation is that the increases in Il1r2, Igsf3,and Gp49a, all of which are inhibitory in terms of T cell activation, are the cause of the lower IFN- production in the IBNS KO. GM-CSF mRNA is also increased in the IBNS KO T cells. As opposed to Il1r2, Igsf3, and Gp49a, which appear to inhibit T cell activation, GM-CSF plays a proinflammatory role in the immune system. Although an increase in Il1r2, Igsf3, and Gp49a should therefore decrease T cell activation, an increase in GM-CSF should have the opposite effect. As there is no evidence for excessive inflammation in these mice, perhaps the pro- and anti-inflammatory changes cancel each other out.
Cpd encodes carboxypeptidase D, an integral membrane protein expressed in many tissues that may play a role in a secretory pathway contributing to peptide hormone processing (52). Although the lower expression level in IBNS KO T cells was confirmed by RT-PCR, the relevance to T cell function remains undetermined. Sh2d1a encodes a protein, SLAM-associated protein (SAP; where SLAM is signaling lymphocyte activation molecule or CD150), involved in regulation of Th2-type cytokines and NK cytotoxicity (reviewed in Ref. 53). SAP–/– CD4+ T cells produce less IL-4 and IL-13 but more IFN- and SAP–/– NK cells exhibit reduced cytotoxicity. Such deficits are not recapitulated in our IBNS KO mice as they have normal NK cell cytotoxicity, express reduced levels of IFN- and IBNS KO CD4+ T cells produce as much IL-4 and IL-13 as WT control CD4+ T cells (data not shown). Thus, the reduced SAP mRNA levels do not account for the IBNS KO phenotype.
Expression levels of two IFN- responsive genes, Irf8 and Ifitm2, are reduced in IBNS KO T cells. Irf8 encodes a transcription factor implicated in the immune response (reviewed in Ref. 54) and Ifitm2 is a transmembrane protein with a role identified in germ cell specification (55). Other genes of note in this category include the enzyme Ppic encoding cyclophilin C, the anti-apoptotic gene Bcl2l1, Tnfsf11 encoding RANKL or TRANCE, transcription factors including zinc finger proteins (Plagl1, Zfpm1) and the transcription factor Rora known to be involved in lymphocyte development (56). All of these genes with the exception of Plagl1 have known roles in the immune system. In addition, the signal molecule-encoding Map4k1 and Ksr genes are decreased in the IBNS KO.
Il2 is present among the 20 genes with –1.69 to –2.59 FCs; this is confirmed by cytokine and RT-PCR analyses. IBNS KO T cells as well as NK cells show a clear deficiency in IFN- production. However, decreased IFN- production was not apparent in the microarray analysis, probably due to the 6-h time point chosen, which may be too short to detect such differences.
Thus, disruption of IBNS results in altered expression of cytokines, transcription factors, kinases, isomerase and peptidase enzymes, and ligands for TNF (Table II "Genes expressed differentially between WT and IBNS KO LN T cells at 6 h postactivation with anti-CD3 + anti-CD28"). Which of these, aside from the II2 gene, are direct targets of IBNS remains to be determined but this analysis serves as a rich source of further investigation into the role of IBNS and NF-B in regulation of T cell activation.
IBNS suppresses LPS-induced IL-6 production in colonic lamina proprial macrophages (24). Independent analysis of an IBNS KO (25) showed that IBNS inhibited NF-B induction of IL-6 and IL-12p40. The phenotype was similar to our IBNS KO with the exception of spontaneous development of chronic colitis in their mice. Kuwata et al., also observed normal T and B cell development and concluded that T cells in their IBNS KO proliferated normally; however their assays were performed in the presence of exogenous IL-2 thus minimizing the proliferation difference between the IBNS KO and WT cells (Fig. 6C). They concluded that IBNS acts to inhibit NF-B activity on IL-6 and IL-12p40 promoters because, in their KO mice, macrophages, and dendritic cells produce more of these cytokines. Our data with the IL-2 promoter are not consistent with a comparable inhibitory mechanism of action for IBNS in T cells. In contrast, IL-2 and IFN- production from T lymphocytes is lower in our IBNS KO animals suggesting that IBNS mediates distinct activities in a variety of cell types through regulation of different promoters.
It has been shown in intestinal macrophages that IBNS binds to p50 homodimers and reduces expression of IL-6 (24). Because IBNS coimmunoprecipitates with p50 both in our EL-4 transfectants and from lysates isolated from ex vivo cells (data not shown), one possibility was that IBNS bound p50 homodimers removing these from the IL-2 promoter and allowing c-Rel to enter this site. Instead, however, c-Rel binds to the IL-2 promoter DNA within 0.5 h after TCR triggering and then decreases (Fig. 9E and data not shown). Similarly, p50 binds the IL-2 promoter at earlier time points but is reduced or has disappeared by 2 h while IBNS binding increases from 1 to 2 h. Thus binding of IBNS to the IL-2 promoter appears to be independent of p50 and c-Rel at later time points. Given that c-Rel has been shown to play a role in chromatin remodeling and opening of the IL-2 promoter for transcription (57), one possibility is that p50 and c-Rel open the IL-2 promoter for transcription and, subsequently, IBNS along with other proteins then maintains transcription from the promoter thus extending IL-2 production in activated T cells. If so, transcription from the IL-2 promoter should initially be identical in WT and IBNS KO T cells upon TCR activation but should diverge at the point where IBNS plays a role in this process. Such kinetics are observed in comparing real-time PCR analysis of IL-2 transcription between WT and IBNS KO T cells (Fig. 10).
Because IBNS has no DNA binding domain, it must interact with other protein components to associate with IL-2 promoter DNA. Proteins known to affect IL-2 expression are histone deacetylase (Ref. 58 and reviewed in Ref. 59) and HMG-Y (60, 61). In addition, Creb-binding protein and p300 affect NF-B-driven transcription (reviewed in Ref. 59). The finding that IBNS interacts with proteins bound at the IL-2 promoter adds further complexity to the analysis of the proteins altering transcription of this important cytokine. Identification of IBNS-binding partners present at the IL-2 promoter is a subject of future investigation.
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1 This work was supported by National Institutes of Health Grants AI19807 (to E.L.R.) and AI51779 (to L.K.C.). 
2 Current address: Beth Israel Deaconess Medical Center Genomic Center, Department of Medicine, Harvard Medical School, Boston, MA 02115.
3 Current address: Department of Molecular and Cell Biology, Division of Immunology and Cancer Research Laboratory, University of California, Berkeley, CA 94720.
4 Current address: Center for Vascular Biology, University of Connecticut Health Center, Farmington, CT 06030.
5 Address correspondence and reprint requests to Dr. Linda K. Clayton, Dana Farber Cancer Institute, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: linda_clayton{at}dfci.harvard.edu
6 Abbreviations used in this paper: pMHC, peptide-MHC complex; DP, double positive; DN, double negative; LN, lymph node; WT, wild type; KO, knockout; BM, bone marrow; FC, fold change; rh, recombinant human; SAP, SLAM-associated protein.
Received for publication April 12, 2007. Accepted for publication May 18, 2007.
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, p < 0.05 comparing the pro-IL-2, mCD28, or mNF-
, p < 0.001 comparing + c-Rel, + I
