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The Journal of Immunology, 1999, 163: 4349-4359.
Copyright © 1999 by The American Association of Immunologists

Role of IL-12 in Intrathymic Negative Selection

Björn R. Lúdvíksson1, Rolf O. Ehrhardt and Warren Strober

Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytokines are central regulatory elements in peripheral lymphocyte differentiation, but their role in T cell ontogeny is poorly defined. In the present study, we evaluated the role of IL-12 in thymocyte selection more directly by determining its role in two models of in vivo negative selection. In initial studies we demonstrated that abundant intrathymic IL-12 synthesis occurs during OVA peptide-induced negative selection of thymocytes in neonatal OVA-TCR transgenic mice, and such synthesis is associated with increased IL-12R ß2-chain expression as well as STAT4 intracellular signaling. In further studies, we showed that this form of negative selection was occurring at the {alpha}ßTCRlowCD4lowCD8low stage and was prevented by the coadministration of anti-IL-12. In addition, the IL-12-dependent thymocyte depletion was occurring through an intrathymic apoptosis mechanism, also prevented by administration of anti-IL-12. Finally, we showed that IL-12 p40-/- mice displayed aberrant negative selection of double positive CD4+CD8+ thymocytes when injected with anti-CD3 mAb. These studies suggest that intact intrathymic IL-12 production is necessary for the negative selection of thymocytes occurring in relation to a high "self" Ag load, possible through its ability to induce the thymocyte maturation and cytokine production necessary for such selection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is now well established that various cytokines, particularly IL-4, IL-7, and TGF-ß, play a central role in thymocyte development (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Recently, evidence has emerged that IL-12 should be added to this list. First, in thymic organ culture studies it was shown that the addition of IL-12 to the culture stimulated double negative (CD4-D8-) thymocyte proliferation and led to a significant reduction in the number of single positive (SP)2 thymocytes (6). Second, in recent studies of IL-2-/- mice, it was demonstrated that the dysregulated maturation of autoreactive Th1-like thymocytes occurring in such mice requires the presence of IL-12 (11). This latter observation also links IL-12 to the development of the various autoimmune phenomena that characterizes IL-2 deficiency (11, 12, 13, 14).

IL-12 is a heterodimeric cytokine synthesized by APCs, including monocytes/macrophages and dendritic cells of various types (15, 16), usually as a result of interactions between CD40 on the APCs and CD40 ligand on the interacting T cells (17). Thus, in the thymus, stromal cells bearing CD40 are a potential source of IL-12, particularly in the light of the fact that thymocytes up-regulate CD40 ligand during thymocyte selection (18). In addition, in previous studies, thymic APCs and stromal cells have been found to be a potential source of IL-12 (6). In the peripheral lymphoid system, IL-12 has a variety of effects; it acts as a growth factor for activated T cells, enhances cytolytic T cell activity, and perhaps most importantly, it induces the differentiation of naive T cells into Th1 lymphocytes capable of producing IFN-{gamma} (19, 20, 21, 22). Its effects in the thymus have not yet been fully explored, although it is known that thymocytes produce IFN-{gamma} under some circumstances and thus have been stimulated by IL-12 (23, 24, 25).

Recent evidence has indicated that negative selection of thymocytes can occur at several different stages of their development depending on Ag dose and affinity for its receptor. Thus, deletion of CD4+CD8+ (double positive; DP) thymocytes early in their development is induced by anti-CD3 treatment of mice and by exogenous administration of high doses of Ags to transgenic (Tg) mice bearing a Tg TCR for that Ag (26, 27, 28, 29), whereas superantigen-mediated deletion and low-dose endogenous Ag deletion occurs in part at other stages (30, 31, 32, 33, 34). Negative selection at these different stages of development may be mediated by different mechanisms. This is supported by studies showing that negative selection of DP thymocytes during the early stages of development is particularly dependent on signaling by members of the TNFR family of ligands. This is evident from the fact that mice deficient in their expression of CD30 or TNFR manifest some forms of faulty selection; furthermore, anti-CD3-induced negative selection of DP TCR to thymocytes is associated with secretion of IFN-{gamma} and TNF-{alpha} (29).

Given the role of IL-12 in peripheral T cell production of IFN-{gamma} and TNF-{alpha}, and the above-mentioned role of these cytokines in negative selection, we evaluated the role of IL-12 in thymic selection during the early stages of DP thymocyte development. Utilizing in vivo models of negative selection in both IL-12 p40-deficient and TCR Tg mice, we obtained evidence that IL-12 synthesis and signaling is in fact an important feature of certain types of intrathymic negative selection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

OVA-TCR Tg mice in a BALB/c background, clone D011.10 that recognized the 323–339 peptide fragment of OVA in the context of I-Ad, and cytochrome c-TCR Tg mice in a B10.A background that recognized the 88–104 peptide fragment of cytochrome c in the context of I-Ek, were bred under normal conditions. Mice of both sexes were used. The IL-12 p40-deficient mice in a BALB/c background and syngenic control BALB/c mice were purchased from Taconic Farms (Germantown, NY). Female mice 4–5 wk of age were used.

Immunization

Newborn neonatal mice were injected with the following: group 1, cytochrome c peptide 88–104 (42.5 µg) i.p. on days 1 and 3 and coadministered i.p. with 0.1 mg of rat IgG Ab on days 1, 2, and 3; group 2, the 323–339 peptide fragment of OVA (OVAp; 42.5 µg) i.p. injections on days 1 and 3 and coadministered i.p with 0.1 mg of rat IgG Ab on days 1, 2, and 3; and group 3, OVAp (42.5 µg) i.p. injections on days 1 and 3 and coadministered i.p. with 0.1 mg of anti-IL-12 Ab on days 1, 2, and 3. Adult (4–7 wk old) OVA-TCR Tg mice were immunized i.v. with 0.5 ml of 450 µM solution of either OVA or OVAp, and sacrificed 20 h after the injection. Another group of mice were immunized at days 1, 2, and 3 and evaluated 24 h after the last injection. Similarly, cytochrome c-TCR Tg mice were injected with the 88–104 peptide fragment of cytochrome c. Four control mice were injected i.v. with 0.5 ml of sterile PBS. For anti-CD3-induced in vivo negative selection of DP thymocytes, IL-12 p40+/+ and p40-/- mice were injected i.v. with 100 µg of anti-CD3 (NA/LE; PharMingen, San Diego, CA) or isotype control IgG in a sterile solution of PBS.

Immunoprecipitation and Western blot analysis

OVA-TCR Tg thymocytes and Th1 pigeon cytochrome c (PCC)-TCR Tg cell line (positive control; 1 x 107 cells/lane) lysates were immunoprecipitated for the STAT4 protein and separated by electrophoresis on 4–12% Tris-glycine gel. After transfer to nitrocellulose, blots were probed with phosphotyrosine HRP-labeled antisera (1:2000). To control for loading, blots were then stripped and reprobed with STAT4 antisera (0.1 µg/ml). Protein G plus agarose (cat. no. sc2002), STAT4 (cat. no. sc-486), and P-Tyr (cat. no. sc-508) Abs were used as recommended by the manufacturer (Santa Cruz Biotechnology, Santa Cruz, CA). A positive control for phosphorylated STAT4 was a whole cell lysate obtained from the A.E7 a PCC-TCR-CD4+ Tg T cell line that recognizes the 88–104 peptide fragment of cytochrome c in the context of I-Ek derived from a PCC-TCR Tg mice with a B10.A background (35).

Nuclear extract preparation and EMSA

Nuclear extracts were prepared essentially as described previously (36), with the following modification. After washing with Tris buffered saline and being pelleted by centrifugation at 1500 x g for 5 min, the pellet was resuspended in 400 µl cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF) by gentle pipetting and incubated on ice for 15 min. Then 25 µl of 10% Nonidet P-40 (Fluka, Bucks, Switzerland) was added and the suspension vigorously vortex mixed for 10 s. After centrifugation, the nuclear pellet was resuspended in 50 µl of ice-cold buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF) and the suspension vigorously rocked for 15 min at 4°C on a shaking platform. The amount of nuclear extracts was quantified by Bio-Rad Protein Assay as recommended by the manufacturer (Bio-Rad Laboratories, Hercules, CA). Mobility shift assays were performed in a total volume of 20 µl in buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.5 mM MgCl, 0.1% Nonidet P-40, 1 mg/ml BSA, and 10% glycerol). Each reaction, containing 1 µg of poly(dI-dC) and 10 fmol of 32P end-labeled probe, was initiated by the addition of 5 µg of nuclear extract and allowed to incubate at 37°C for 30 min before electrophoretic analysis on a 5% polyacrylamide gel in 0.25x Tris-borate/EDTA buffer. The following oligonuclotide probes were purchased from Santa Cruz Biotechnology: STAT4, 5'-GAGCCTGATTTCCCCGAAATGATGAGC-3'; and mutant STAT4, 5'-GAGCCTGATTTCTTTGAAATGATGAGC-3'.

RT-PCR analysis

Total cellular RNA was isolated from whole thymuses using RNA STAT60 total RNA/mRNA isolation reagent (Tel-Test, Friendswood, TX) and was quantitated by optical density determination. RNA (1 µg) was converted to cDNA with oligo(dT) primer, and SuperScript II RT (Life Technologies, Grand Island, NY). PCR was performed by amplifying the cDNA in PCR SuperMix (Life Technologies) with specific primer pairs (PCR conditions were strictly defined for each primer pair). The following sequences (sense (s) and antisense (as)) were used as the primers: ß2 microglobulin (s), 5'-TGACCGGCTTGTATGCTATC-3' and (as), 5'-CATTGTGAGCCAGGATATAG-3'; IL-12 p35 (s), 5'-CATCATGAAGACATCACACGG-3' and (as), 5'-AGAATGATCTGCTGATGGTTG-3'; IL-12 p40 (s), 5'-CAGTACACCTGCCACAAAGGA-3' and (as), 5'-GTGTGACCTTCTCTGCAGACA-3'. The RT-PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide.

Northern blot analysis

Total cellular RNA was isolated from whole thymic tissues by standard technique as indicated above. Ten micrograms of total RNA was loaded in each lane, electrophoresed, and transferred to nitrocellulose membranes, which were then sequentially probed with full-length murine probes specific for IL-12Rß2. For loading control, membranes were stripped and reprobed with murine probes specific for GAPDH. A positive control consisted of mRNA isolated from the A.E7 Tg cell line described above.

Injection and generation of anti-IL-12

Immunized 4- to 7-wk-old mice were injected with 1 mg of anti-IL-12 Ab i.p. 1 h before immunization, and mice that were immunized at days 1, 2, and 3 with OVA/OVAp or PBS were injected with anti-IL-12 at days 1 and 3. Anti-IL-12 Abs were obtained from ascites fluids of mice subjected to C17.8 hybridoma cells (kindly donated by G. Trinchieri, Wistar Institute, Philadelphia, PA) by i.p. injection of 4 x 106 cells/animal. Ascites were collected after 2 wk and the Abs purified using E-Z-SEP Ascites IgG kit (Pharmacia Biotech, Piscataway, NJ). Control mice were injected i.p. with 1 mg of rat-IgG at the same schedule as for anti-IL-12.

Flow cytometry

Thymocytes were washed 2x in FACS buffer (National Institutes of Health Media Unit), resuspended at 1 x 107 cells/ml in FACS buffer, and transferred to FACS tubes (Becton Dickinson, Franklin Lakes, NJ). To prevent nonspecific FcR-mediated binding of Abs, 50–100 µg/ml of Fc Block (01241D; PharMingen, San Diego, CA) was added to each tube 3 min before staining. Cells were stained with 1 µg/ml for 30 min on ice with FITC-conjugated CD3, FITC-conjugated CD4a, FITC-conjugated/TCR, PE-conjugated CD4b, PE-conjugated heat shock Ag (HSA), PE-conjugated CD8, biotin-conjugated CD8, and biotin-conjugated CD69, all purchased from PharMingen.

To detect the expression of the IL-12R ß2-chain, 1 x 106 cells/200 µ1 thymocytes were stained with hamster IgG anti-mouse IL-12Rß2 mAb (0.5 µg) or isotype control hamster IgG (both provided by Dr. R. O. Ehrhardt, Protein Design Labs., Palo Alto, CA), incubated on ice for 30 min, washed twice in FACS buffer, and incubated on ice for 30 min with biotinated goat anti-hamster IgG polyclonal Ab (1:400 dilution) (Jackson ImmunoResearch, West Grove, PA). Cells stained with biotin-conjugated Abs were subsequently washed once in FACS buffer and incubated on ice for 30 min with streptavidin-PE or streptavidin Cy-Chrome (PharMingen) at 5 µg/ml. Finally, the stained thymocytes were washed twice in FACS buffer, resuspended at 1 x 106 cells/200 µ1, and examined on a FACScan Analyzer with Lysis II Software (Becton Dickinson, Mountain View, CA). Nonviable cells were excluded by forward angle scatter or by propidium iodide uptake.

In situ staining for apoptosis

Thymic tissue obtained from mice was immediately immersed in OCT compound (Tissue Tek; Miles, Elkart, IN) and rapidly frozen in dry ice. Six- to eight-micrometer sections were cut, air-dried, and fixed in 4% paraformaldehyde (in PBS, pH 7.4). For direct labeling of degraded DNA, the Boehringer Mannheim in situ cell death detection kit, fluorescein/alkaline phosphatase was utilized as per the manufacturer’s instructions (cat. no. 1684809; Boehringer Mannheim Biochemica, Indianapolis, IN). In brief, after fixation, cells were incubated in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate), residues of dioxigenin-nucleotide were added to the DNA by terminal deoxynucleotidyl transferase, and newly synthesized nucleotide polymers were then detected by antidioxigenin fluorescein Ab conjugated with alkaline phosphatase and the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate.

In situ staining for IL-12

Thymic cryosections (7 µm) were air dried and fixed in cold acetone for 3 min at room temperature. Next, samples were incubated in 0.6% H2O2 in methanol for 30 min to block endogenous peroxidase. Samples were then rehydrated in PBS plus 0.01% Triton X-100 for 15 min, blocked with 100% normal rabbit serum for 3 h, and incubated with the primary Ab (rat anti-mouse IL-12 p70; 1:100 dilution in 10% normal rabbit serum) overnight at 4°C in a dark, humid chamber. Sections were then washed for an additional 15 min in PBS. Finally, sections were incubated with the secondary Ab (FITC-labeled rabbit anti-rat IgG; 1:100 dilution) and washed in PBS for 10 min. Samples incubated with isotype-matched control Abs and without primary Ab served as negative controls.

Statistical analysis

Descriptive statistics and testing for significance of differences were assessed by Student’s t test using the Microsoft Excel statistical analysis computer program.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intrathymic IL-12 production during Ag-induced thymocyte depletion in TCR Tg mice

In initial studies, we assessed intrathymic synthesis of mRNA of the p35 and p40 chains of the IL-12 p70 heterodimer during Ag-induced thymocyte depletion in a previously described model of negative selection (26, 27, 29). Accordingly, we injected neonatal OVA-TCR Tg mice with OVAp or control peptide (PCC peptide fragment 88–104; PCCp) and extracted mRNA from whole thymic tissue 16 h later for RT-PCR analysis with p35 and p40 chain probes. As shown in Fig. 1GoA, the p35 chain mRNA was constitutively expressed and only minimally increased after OVAp injection. In contrast, as shown in Fig. 1GoB, the p40 chain in mRNA expression was greatly increased after OVAp injection compared with p40 mRNA expression after control PCCp injection. To demonstrate that this increased mRNA expression was associated with translation into secreted p70 heterodimeric IL-12, we administered OVAp and control PCCp to OVA-TCR Tg neonatal mice and stained frozen thymic tissue sections obtained from such mice 20 h later with FITC-anti-IL-12 Ab directed against the p70 heterodimer. As shown in Fig. 2Go, there was abundant IL-12 staining in the thymic tissue area from Tg mice administered OVAp but virtually no IL-12 staining in thymic tissue from Tg mice administered PCCp control peptide. The staining was located mainly in scattered cells located within the cortex and cortico-medullary junction area. Thus, these data indicate that OVAp injection leads to enhanced synthesis and secretion of IL-12.



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  		FIGURE 1. IL-12 p35 and p40 mRNA expression after OVAp administration. Neonatal OVA-TCR Tg mice were injected with either OVAp or control peptide (PCCp); 16 h later mRNA from neonatal thymocytes was isolated and transcription of p35 (A) and p40 (B) was evaluated by RT-PCR. Results obtained with two OVAp and PCCp injected mice are shown.

 


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  		FIGURE 2. Detection of IL-12 synthesis in thymic tissue by in situ staining with anti-IL-12 mAb (p70). Thymic tissue was stained with fluorescent Ab against the IL-12 heterodimer (p70) from OVA-TCR Tg mice that had been injected with OVAp (A) or PCCp (B) 20 h before thymic isolation. Results are representative of four different experiments.

 
IL-12R ß2-chain is expressed on thymocytes during Ag-induced thymocyte depletion

The IL-12 receptor has recently been shown to be composed of two chains, a ß1-chain that is more or less constitutively expressed and a ß2-chain that is expressed only on activated T cells (21, 37, 38, 39). We therefore determined whether the up-regulation of intrathymic IL-12 production during Ag-induced thymocyte depletion was associated with thymocyte expression of the IL-12R ß2-chain using flow cytometric analysis in association with an Ab specific for murine IL-12R ß2-chain. Accordingly, thymocytes were isolated and stained with FITC-conjugated anti-CD4, Cy-Chrome-conjugated anti-CD8, and PE-labeled anti-IL-12R ß2-chain mAbs as indicated in Materials and Methods. As shown in Fig. 3Go, DP thymocyte expression of the IL-12R ß2-chain was increased 24 h after OVAp injection into OVA-TCR Tg neonates, whereas its expression was not increased after injection of control peptide (PCCp). In addition, the expression of the IL-12R ß2-chain was particularly abundant among the CD4lowCD8low subpopulation of thymocytes. In parallel studies, we observed significant mRNA expression of the IL-12 R ß2-chain by Northern blot analysis in thymocytes 24 h after OVAp-injection into OVA Tg neonatal mice, but not in thymocytes after PCCp injection (data not shown). Thus, these data provide evidence that during OVAp-induced thymocyte depletion in OVA-TCR Tg neonatal mice, thymocytes subject to negative selection manifest enhanced expression of a functional IL-12R.



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  		FIGURE 3. Flow cytometric analysis of IL-12R ß2-chain expression on CD4+CD8+ thymocytes of neonatal OVA-TCR Tg mice after OVAp or control peptide PCCp administration. Left, PCCp (42.5 µg) administration; right, OVAp (42.5 µg) administration. The expression of the IL-12R ß2-chain (gray) or isotype-specific control Ab (black line) was analyzed on the following three gated groups: top, CD4lowCD8low DP thymocytes; middle: CD4highCD8high DP thymocytes; and bottom, CD4+CD8- SP thymocytes. Results are representative of three experiments.

 
IL-12-dependent phosphorylation of STAT4 during thymocyte depletion

To further demonstrate that up-regulation of IL-12 synthesis during Ag-induced thymocyte depletion was having an effect on thymocyte function, we took advantage of the fact that the signaling pathway of IL-12 has recently been shown to be dependent on the phosphorylation and nuclear localization of STAT4 (40, 41, 42). We therefore performed immunoprecipitation studies on cellular and nuclear extracts from thymic tissues obtained from neonatal OVA Tg mice after administration of OVAp or PBS in which blots of anti-STAT4 precipitants were probed with labeled antiphosphotyrosine Ab (see Materials and Methods). As shown in Fig. 4Go, A–C, significant phosphorylation of STAT4 was demonstrated in neonatal thymocytes as early as 6 h after injection of neonatal OVA-TCR Tg mice with OVA peptide, but not in thymocytes obtained from PBS-injected OVA-TCR Tg mice. In addition, nuclear extracts of thymocytes isolated from neonatal mice administered OVAp bound to tagged STAT4 oligonucleotides, indicating nuclear translocation of activated STAT4 in such thymocytes. These experiments thus demonstrate that thymocytes expressing functional IL-12R are subject to IL-12-mediated intracellular signaling.



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  		FIGURE 4. STAT4 phosphorylation after OVAp administration to neonatal OVA-TCR Tg mice. A and B, Twenty-four hours after OVAp or PBS injection into neonatal OVA-TCR Tg mice, thymocyte whole cell lysates were immunoprecipitated with anti-STAT4 antisera. After transfer to nitrocellulose, the blot was probed with antiphosphotyrosine (A) and then stripped and reprobed with anti-STAT4 antisera (B). C, Time course of STAT4 phosphorylation after OVAp administration. D, EMSA to detect nuclear localization of activated STAT4 using a STAT4-oligonucleotide probe and mutant STAT4-oligonucleotide probe (the latter to control for nonspecific binding). Positive control for the presence of activated STAT4 consisted of whole cell lysate from IL-12 treated PCC-TCR Tg Th1 cell line (AE.7).

 
Anti-IL-12 coadministration blocks Ag-induced thymocyte depletion in TCR Tg mice

To learn whether the enhanced IL-12 secretion occurring during Ag-induced thymocyte depletion as noted above is necessary for such depletion, we determined whether the depletion is affected by coadministration of anti-IL-12. As shown in Table IGo, injection of OVAp into neonatal OVA-TCR Tg mice led to significant reduction in total thymocyte number (PCCp injection (control peptide): 4.69 x 107 ± 1.8, vs OVAp injected: 1.8 ± 0.49 x 107 thymocytes). However, coadministration of anti-IL-12 prevented such Ag-induced thymocyte depletion (OVAp injection + anti-IL-12: 4.58 ± 1.74 x 107). In addition, as shown in Table IGo, coadministration of anti-IL-12 prevented OVAp-induced thymocyte depletion in 4- to 7-wk-old OVA-TCR Tg mice and prevented PCCp-induced thymocyte depletion in 4- to 7-wk-old PCCp-TCR Tg mice.


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  		Table I. Total thymocyte numbers in TCR Tg mice after various treatments1

 
OVAp administration to neonatal OVA-TCR Tg mice induces depletion of CD4+CD8+ thymocytes and is blocked by anti-IL-12 coadministration

We next sought to demonstrate that the blockade of thymocyte depletion by anti-IL-12 demonstrated above is an Ag-specific intrathymic phenomenon. For this we took advantage of the fact that intrathymic negative selection requires engagement of the TCRs on DP thymocytes and induces preferential deletion of these cells (43, 44, 45, 46). Thus, if anti-IL-12 is preventing Ag-induced thymocyte depletion by affecting negative selection, it should have a preferential effect on DP thymocytes. Accordingly, we administered OVAp with or without anti-IL-12 to neonatal OVA-TCR Tg mice and determined the phenotype of the residual thymocytes. As shown in Fig. 5Go, OVAp injection led to depletion of neonatal DP thymocytes, and anti-IL-12 administration blocked such depletion. Interestingly, however, although such depletion resulted in a consistent decrease in SP CD4+, it did not lead to a decrease in SP CD8+ thymocytes. This may reflect the fact that the OVAp is recognized in the context of a MHC class II molecule (I-Ad), and thus by thymocytes expressing CD4; this in turn leads to negative selection of DP thymocytes, and perhaps in a lesser extent to SP CD4+ thymocytes, but not to SP CD8+ thymocytes. Similar findings were obtained in 4- to 7-wk-old mice (data not shown).



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  		FIGURE 5. Thymocyte depletion occurring in neonatal OVA-TCR Tg mice injected i.p. with: group 1, PCCp (42.5 µg) on days 1 and 3 and coadministered i.p. 0.1 mg of rat IgG Ab on days 1, 2, and 3 (filled bars); group 2, OVAp (42.5 µg) on days 1 and 3 and coadministered i.p. 0.1 mg of rat IgG Ab on days 1, 2, and 3 (striped bars); and group 3: OVAp (42.5 µg) on days 1 and 3 and coadministered i.p. 0.1 mg of anti-IL-12 Ab on days 1, 2, and 3 (open bars). Results are representative of eight different experiments. *, p < 0.01; **, p < 0.001.

 
Administration of OVAp to neonatal OVA-TCR Tg mice induces a specific depletion of Vß8.2low and KJ1-26Id-bearing thymocytes and such depletion is blocked by anti-IL-12 coadministration

In parallel studies, we determined the effect of administration of OVAp to neonatal OVA-TCR Tg mice alone or in the presence of anti-IL-12 on the depletion of DP Vß8.2low thymocytes because this is the subpopulation of thymocytes at risk for negative selection (27, 43). We found that neonatal OVA-TCR Tg mice injected with control peptide (PCCp) contained thymuses in which the majority of DP thymocytes bear the Vß8.2low phenotype, whereas those injected with OVAp contained thymuses in which this DP thymocyte population was depleted (mean fluorescent intensity of PCCp-control-injected neonatal mice 94.8 ± 10.1 vs OVAp-injected mice 215 ± 57.5; p = 0.002). Evidence that the depletion of the DP Vß8.2+ thymocyte subpopulation was dependent on IL-12 was inherent in the further observation that coadministration of anti-IL-12 mAb prevented such depletion (mean Vß8.2 fluorescent intensity of OVAp-injected and coadministered anti-IL-12 neonatal mice: 99 ± 12.6).

The same specificity of depletion was observed in relation to cells bearing the Id (KJ1–26) of the OVA-TCR transgene. Thus, administration of OVAp to neonatal OVA-TCR Tg mice led to preferential depletion of KJ1–26+ DP thymocytes, and such

depletion was blocked by the administration of anti-IL-12. Furthermore, as shown in Fig. 6Go, the subpopulation of thymocytes most subject to such depletion was the KJ1–26low CD4lowCD8low thymocytes, i.e., the subpopulation in the "R2" gate that also expresses the highest levels of IL-12R. This specific depletion of KJ1–26lowCD4lowCD8low thymocytes after OVAp injection was even more compelling when the total number of thymocytes within this subpopulation was taken into account: total number of KJ1–26lowCD4lowCD8low neonatal thymocytes after PCCp injection 3.4 x 105 vs OVAp injection 0.6 x 105; p < 0.001). Finally, as also shown in Fig. 6Go, thymocytes reaching the CD4highCD8high stage, i.e., the cells in the "R3" gate, all express high levels of KJ1–26 whether they had been subject to OVAp-induced negative selection or not. However, as a result of OVAp-induced depletion of KJ1–26lowCD4lowCD8low thymocytes, there was a 3-fold reduction of thymocytes reaching this stage. Taken together, these studies demonstrate that OVAp injection into OVA-TCR Tg neonatal mice induces Ag-specific depletion of TCRlowCD4lowCD8low thymocytes, and that such depletion can be prevented by the coadministration of anti-IL-12. Thus, they support the notion that IL-12 is necessary for Ag-specific intrathymic negative selection.



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  		FIGURE 6. Flow cytometric analysis of thymocytes from OVA-TCR Tg neonatal mice during OVAp negative selection. A, Neonatal mice administered PCCp and coadministered rat IgG. B, Neonatal mice administered OVAp and coadministered rat IgG. C, Neonatal mice administered OVAp and coadministered anti-IL-12 mAb. Left, R2 (CD4lowCD8low ) and R3 (CD4highCD8high) gates conforming cell for determination of KJ1–26 expression. Middle, Percentage of KJ1–26low thymocytes in the R2 gate. Right, Expression of KJ1–26 in the R3 gate. Results are representative of four separate experiments.

 
Anti-IL-12 administration to OVA-TCR/Tg mice inhibits Ag-induced intrathymic apoptosis

In the next set of studies, we examined the role of IL-12 in intrathymic negative selection more directly by assessing the effect of anti-IL-12 on OVAp-induced apoptosis in the thymus of OVA-TCR Tg mice. Accordingly, thymic tissues from OVA-TCR Tg mice that had been injected i.p. with either control peptide (PCCp) + rat IgG, OVAp + rat IgG, or OVAp + anti-IL-12 mAb were examined 16 h after injection by the in situ TUNEL technique to detect and quantify the number of apoptotic cell nuclei present in the thymic tissue. As shown in Fig. 7Go, A–C, thymic tissue from OVAp-injected mice manifested a much larger number of apoptotic nuclei than did tissue from PCCp-injected mice. In contrast, thymic tissue of OVAp + anti-IL-12-injected mice displayed similar numbers of apoptotic nuclei as PCCp-injected mice. The apoptotic cells were predominantly located within the cortex and cortico-medullary junction, the same sites positive for in situ IL-12 staining. These results were corroborated by more quantitative studies in which the thymus tissue of neonatal OVA-TCR Tg mice in each of the above mouse groups was dispersed into single cell suspensions, and the thymocytes thus obtained were analyzed for the presence of apoptotic nuclei using the flow cytometric TUNEL method and cell cycle analysis. Thus, as shown in Fig. 6Go, D–F, although 30.8% of thymocytes from OVAp-injected mice were apoptotic, only 3.3% of OVAp-injected and anti-IL-12-injected mice were apoptotic. In addition, the apo-ptotic thymocytes were primarily concentrated within the PIlow (cell cycle: sub G) population of cells, which is also characteristic of apoptotic cells. Taken together, these studies support the hypothesis that IL-12 secretion is necessary for Ag-induced thymocyte apoptosis.



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  		FIGURE 7. Detection of thymic apoptosis in OVA-TCR Tg mice. A–C, In situ detection of apoptotic cells (dark brown) in sections of thymic tissue from OVA-TCR Tg mice using the TUNEL technique. A, Thymic tissue from mice that had been injected i.v. with PCCp (control peptide) 16 h before isolation. B, Thymic tissue from mice that had been injected i.v. with OVAp 16 h before isolation. C, Thymic tissue from mice that had been injected i.v. with OVAp and coadministered 1 mg of anti-IL-12 Ab 16 h before isolation. D–F, Flow cytometric analysis of apoptotic neonatal OVA-TCR Tg thymocytes using TUNEL technique; apoptosis measured by TdT-induced incorporation of labeled nucleotides to DNA strand breaks on the x-axis and by cell cycle analysis (PI) on the y-axis. D, Mice injected with PBS; E, mice injected with OVAp; F, mice injected with OVAp and coadministered anti-IL-12. Results are representative of four different experiments.

 
Defective anti-CD3-induced negative selection of DP thymocytes in IL-12 p40-/- mice

To further evaluate the role of IL-12 in thymic negative selection, we determined its role in the negative selection of DP thymocytes in IL-12 p40-/- mice. In a first set of studies, we administered the superantigen staphylococcal enterotoxin B (SEB; 50 µg) to neonatal p40+/+ and p40-/- mice i.p. three times a week for 6 wk, recognizing that such superantigen administration causes depletion of Vß8.2-bearing thymocytes at a later stage of development (in the SP thymocyte stage) (18, 29). As shown in Fig. 8Go, SEB administration leads to depletion of Vß8.2+ SP thymocytes in p40+/+ neonates, but not in Vß8.2+ DP thymocytes. In addition, such administration also led to depletion of Vß8.2+ SP thymocytes in p40-/- mice.



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  		FIGURE 8. Depletion of Vß8.2+ neonatal thymocytes after in vivo SEB injection into either IL-12 p40+/+ or IL-12 p40-/- mice. Results expressed as a mean ± 1 SD; data obtained from two experiments using three to five neonates per group at each time.

 
In a second set of studies, we administered anti-CD3 (100 µg) to p40+/+ and p40-/- mice i.v., recognizing that such injection induces depletion of thymocytes at an early stage of thymocyte development, i.e., the DP stage also seen in Ag-induced depletion in TCR Tg mice, as studied above. As shown in Fig. 9Go, injection of anti-CD3 induced significant loss of DP thymocytes in IL-12 p40+/+ mice, whereas, in similarly treated p40-/- mice, such anti-CD3-induced thymocyte depletion was minimal (p < 0.001). Furthermore, although in p40+/+ mice this anti-CD3-induced thymocyte depletion was associated with increased number of apoptotic cells in their early (CD4lowCD8low ) stage of development, such apoptosis was not observed in p40-/- mice (data not shown). These studies support the idea that IL-12 is essential for negative selection of thymocytes at a relatively early stage of development but are less involved in negative selection of thymocytes at later stages.



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  		FIGURE 9. Total number of thymocytes after in vivo anti-CD3 administration to IL-12 p40+/+ (A) and IL-12 p40-/- (B) 1- to 3-wk-old mice. Results shown are mean values from five mice in each treatment group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although previous studies have provided ample evidence that a variety of cytokines are involved in thymocyte precursor proliferation and differentiation (2, 3, 4, 5, 8, 10, 11), they have not clearly addressed the issue of whether cytokines are also involved in thymocyte selection. One view of the role of cytokines in thymocyte development is based on the observation that DP thymocytes (i.e., thymocytes subjected to negative selection) are resistant to cytokine-induced proliferation, and thus holds that thymocyte responsiveness is suspended after the differentiation of double negative into DP thymocytes and is only restored when cells become positively selected SP thymocytes (7). These data therefore suggest that cytokines have, at best, a minor role in the selection process. Another view is based primarily on our own studies of thymocyte development in the IL-2-/- mouse, which showed that such mice are subject to dysregulated thymocyte development driven by intrathymic IL-12 production (11). These studies thus suggest that DP and SP thymocytes are directly influenced by cytokines during thymocyte selection and, in addition, that IL-12 is an important factor in the selection process.

To determine the role of IL-12 in thymocyte selection suggested by the previous studies of the IL-2-/- mouse more directly, we took advantage of the fact that the effect of IL-12 on such development can be studied in the TCR Tg mouse undergoing thymocyte depletion (negative selection) as a result of the administration of exogenous Ag specific for the TCR transgene. In pursuing this approach, we were cognizant of the fact that Ag-induced thymocyte depletion in TCR Tg mice would be construed as an Ag-nonspecific phenomena arising from secondary stress effects resulting from massive peripheral responses to the injected Ag. That this is the mechanism behind IL-12-mediated intrathymic negative selection demonstration in these studies is unlikely for several reasons. First, Kishimoto and Sprent (47) found that anti-TCR-induced thymocyte depletion of DP thymocytes was the predominant population depleted in adult mice (8 wk old), whereas in neonates (1 wk old) such anti-TCR-induced thymocyte depletion was also observed within the SP cell compartment, both groups of mice in our study manifested evidence of IL-12-mediated negative selection. In confirmation of this fact, neonatal OVA-TCR Tg splenocytes in the mice used in our studies did not proliferate or produce IFN-{gamma} when stimulated in vitro with OVAp (data not shown). Second, the IL-12-mediated negative selection demonstrated in our studies was limited to DP thymocytes bearing the idiotypic Vß8.2 transgene (i.e., KJ1–26-bearing cells) and was associated with a relative increase in DP thymocytes bearing other Vß TCR. Thus, the depletion was not a nonspecific phenomenon, as one would expect if it were due to stress effects. Finally, the depletion caused by Ag administration was focused on thymocytes in a particular stage of maturation, i.e., intermediate stage TCRlow DP thymocytes having a CD4lowCD8low phenotype. Such thymocytes previously have been identified as the target cells undergoing negative selection due to endogenous "self" peptides. Although the above considerations make it likely that the model of negative selection used in these studies, i.e., Ag-induced depletion of thymocytes in TCR Tg neonatal mice is a physiologic model, one cannot claim that this model represents all forms of negative selection. The fact is that intrathymic negative selection is a complex phenomenon affecting not only DP thymocytes as in this study, but also thymocytes at an earlier and later stage of thymic development. These different forms of negative selection may relate to the dose and the affinity of the inducing Ag and in this regard it seems likely that the negative selection studied here (and involving IL-12) is mainly due to Ags present at high doses, which serves to maintain intrathymic homeostasis following administration of high doses of exogenous Ags (see further discussion below).

The evidence generated in these studies that IL-12 is involved in at least one form of negative selection of thymocytes as discussed above is several fold. First, we found enhanced IL-12 production, both on the mRNA and protein levels in thymic tissue of neonatal OVA-TCR Tg mice after Ag-induced (OVA-induced) thymocyte depletion, and such IL-12 production was accompanied by evidence of IL-12 signaling, i.e., enhanced expression of IL-12R ß2-chain and STAT4 activation. Second, Ag-induced thymocyte depletion was greatly inhibited by coadministration of anti-IL-12 and such coadministration specifically blocked depletion of those thymocytes, most likely to be undergoing selection, i.e., TCRlowCD4lowCD8low thymocytes (27, 43, 48). Third, in data not shown, we demonstrated that administration of OVA or OVA peptide along with anti-IL-12 prevented the up-regulation of CD69 and down-regulation of HSA thymocyte surface Ags, phenotypic changes that normally accompany Ag-induced thymocyte depletion (49, 50). Finally, administration of OVA or OVA peptide to OVA-TCR Tg mice induced massive intrathymic apoptosis that was completely blocked by the coadministration of anti-IL-12.

The above findings strongly supporting a role for IL-12 in intrathymic negative selection in TCR Tg neonatal mice were both verified and qualified by additional studies in IL-12 p40-deficient mice. Thus, in studies of negative selection of thymocytes in such IL-12 p40-deficient mice, we found that although anti-CD3-induced depletion was strongly reduced in an IL-12-deficient environment, superantigen (SEB)-induced thymocyte depletion was only marginally reduced. Depletion induced by anti-CD3 administration mimics depletion due to high-dose Ag exposure, so that the reduced depletion by this agent indicates that the effect of anti-IL-12 on Ag-induced depletion in a TCR Tg mouse is applicable to a lymphoid system with a normal distribution of T cell specificities. On the other hand, depletion due to superantigen (SEB) has been considered a form of depletion that affects relatively mature DP or early SP thymocytes. Thus, the findings in IL-12 p40-/- mice relating to superantigen-induced depletion suggest that IL-12 does not play a significant role in negative selection of these more mature thymocytes. Our findings of defective anti-CD3-induced negative selection of immature thymocytes in IL-12 p40-deficient mice suggest that IL-12 might be working through a TNFR/Fas-dependent death pathway in these mice. However, the fact that this pathway plays a role in SEB-induced negative selection of thymocytes and, as shown here, this pathway is more or less intact in IL-12 p40-deficient mice makes this hypothesis less likely (51, 52). It should be noted that the above data are seemingly at odds with recent studies by Magram et al. (53) that show that IL-12 p40-/- mice (mice that are identical with those used in the present study) appear to have normal thymocyte development. Among the factors that can be cited to explain this apparent discrepancy is the fact that in the studies of IL-12 p40-/- mice previously reported, the mice may not have been challenged with Ags that have revealed a defect in negative selection; in addition, it is possible that IL-12 p40-/- mice have a normal thymic phenotype because they still have intact positive selection. Finally, the lack of development of Th1 cell clones capable of causing autoimmunity in IL-12 p40-/- mice in the face of a defect in negative selection can be attributed to the fact that, in the absence of IL-12, potential autoreactive T cells developing in the thymus could not develop.

IL-12 could be acting in several ways to affect negative selection of thymocytes. Perhaps the most likely is that IL-12 is acting directly on thymocytes to promote the latter’s growth and differentiation into cells capable of undergoing intracellular signaling events leading to cell death. This notion is supported by the known role of IL-12 as a T cell differentiation factor, not only for mature peripheral T cells, but for thymocyte precursor cells as well (6, 11, 16, 22, 54). It should be noted, however, that IL-12 is not a "universal" differentiation factor, in that, in the case of peripheral T cells, it induces naive T cells to develop into Th1 T cells and not into Th2 T cells. Indeed, Th2 development is accompanied by the down-regulation of the ß2-chain of the IL-12R on nascent Th2 T cells and thus the failure to induce Th1 T cell-specific Janus kinase (Jak)/STAT phosphorylation in the latter cells (42). This specificity of IL-12-induced T cell differentiation bears on the fact that intrathymic positive and negative selection has been shown to involve distinct intracellular signaling pathways (55, 56), because it is conceivable that although IL-12 secretion in the thymus induces specific signaling pathways leading to some forms of negative selection, it does not induce signaling pathways to all forms of negative selection or those necessary for positive selection.

The idea that IL-12 is necessary for negative selection because it drives thymocytes into a stage in which they become susceptible to signaling that leads to thymocyte death is supported by several pieces of evidence. First, it has been shown that anti-CD3 Ab-induced depletion of thymocytes (as noted above, a type of depletion likely to be similar to Ag-induced depletion in the TCR Tg mouse) is targeted on DP thymocytes that secrete IFN-{gamma}, suggesting that depletion caused by anti-CD3 administration occurs in an IL-12-driven thymocyte population (23, 57). Second, it has recently been shown that Jak3-deficient mice, i.e., mice that have intrinsic defects in signaling via cytokines utilizing the common {gamma}-chain of the IL-2, IL-4, IL-7, and IL-13 receptors, fail to delete self-reactive thymocytes (58). This suggests that cytokine receptor signaling is necessary for negative selection and thus that heretofore unexpected maturation of DP thymocytes, perhaps driven by IL-12, is necessary for such selection. Third, as already alluded to above, we have observed that thymuses of OVA-TCR Tg mice administered OVA contain increased numbers of thymocytes displaying the CD3highCD69highHSAlow phenotype, i.e, a phenotype associated with cells that have escaped negative selection (47, 49, 59), and that these cells do not occur in mice coadministered anti-IL-12. Thus, anti-IL-12 appears to be preventing the normal process of thymocyte negative selection under these circumstances. Finally, the probability that IL-12 is necessary for some forms of negative selection relates to the finding of IL-12-driven thymocyte dysregulation in IL-2-/- mice (11, 14). The key point here is that the thymocyte dysregulation found in IL-2-/- mice is associated with defective anti-CD3-induced thymocyte apoptosis (11). On this basis, one can say that under certain conditions, IL-12 drives thymocytes to a stage where they become sensitive to IL-2-dependent apoptosis. Thus, given the previous established role of IL-12 in thymocyte proliferation and differentiation (6) and IL-2 in activation-induced cell death, it is conceivable that within the thymic microenvironment both IL-2 and IL-12 are necessary to maintain intrathymic homeostasis.

As noted above, negative selection may occur in several thymocyte populations depending on Ag dose and Ag affinity for the TCR. In addition, the mechanism of cell death involved in the selection of these populations, or even in cells of the same population, may differ, as may the role of IL-12. Recently, two distinct mechanisms of negative selection of DP thymocytes have been described, one that is dependent on CD28 signaling (costimulation) and another that is dependent on APCs (independent of CD28), and both were found to be independent of the Fas-death pathway (45). Nevertheless, Fas is highly expressed on DP thymocytes and its cross-linking clearly induces their apoptosis (60). In addition, negative selection caused by administration of anti-CD3 or high-dose Ag-administration has been shown to be due to Fas pathway activation and cell death (52). Thus, although the role of Fas in negative selection remains controversial (61, 62, 63, 64, 65, 66, 67, 68, 69), one cannot rule it out for certain forms of negative selection. Finally, it has recently been shown that TNF receptor p55/p75 knock-out mice display defective negative selection of thymocytes associated with administration of high-dose self-Ag and anti-CD3 but normal negative selection in certain experimental forms of thymocyte deletion at a late stage of development (29). However, the authors questioned the role of TNF in endogenous in vivo negative selection (29). Whether the Fas or TNF pathways are indeed involved in "normal" negative selection or whether another death pathway is involved remains to be seen. The question remains whether IL-12 is inducing apoptotic cell death by driving maturing thymocytes to produce cytokines and/or express receptors, ultimately resulting in their self-elimination.

Another explanation for the role of IL-12 in negative selection and one that is not mutually exclusive to that already discussed above concerning thymocyte maturation, relates to the fact that the expression of several costimulatory molecules such as CD28, CD40, and CD54, which have been shown to be induced by IL-12, are now known to be necessary for at least some forms of negative selection of thymocytes (17, 44, 70). Of particular interest to the present discussion is the observation of Foy et al. (18) who demonstrated that the gp39 (CD40) and CD40 ligand (CD40L) interaction was sufficient for negative selection of endogenously expressed self-peptides. Because there is evidence that CD40-CD40L interaction and expression are regulated by IL-12, it is possible that IL-12 could be influencing negative selection through its affect on CD40-CD40L expression (17, 71). It should be pointed out, however, that in the present study we found no difference in the expression of CD40 or CD40L during Ag-induced thymocyte depletion before or after in vivo administration of anti-IL-12 Ab, suggesting that in this model IL-12 was not acting via its affect on the CD40-CD40L mechanism. This apparent discrepancy may relate to the additional finding of Foy et al. (18) that negative selection induced by high doses of exogenous Ag (as used in this study) is not blocked by anti-CD40L Ab, suggesting that Ag dose and affinity influence whether or not CD40/CD40L is important for negative selection.

To summarize, in these studies we provide evidence that the intrathymic cytokine milieu influences negative selection, at least in regard to selection operating during a high "self" Ag load. In particular, we show that IL-12 secretion is up-regulated during induction of negative selection and that such secretion is critical for the occurrence of apoptosis. We propose that the chief mechanism of the IL-12 effect is in the ability of this cytokine to influence the expression of surface molecules involved in the interactions necessary for thymocyte negative selection. This observation raises the possibility that deviant intrathymic IL-12 secretion could potentially allow autoreactive T cells to escape thymic negative selection and cause autoimmune disease.


   Acknowledgments
 
We thank Dr. A. Singer for helpful discussion and critical reading of the manuscript and B. R. Marshall and Sara Kaul for excellent editorial assistance. We also thank Dr. Markus Neurath for his technical assistance in the performance of IL-12 in situ staining.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Björn R. Lúdvíksson, 10 Center Drive, Building 10, R11N238, Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1890. E-mail address: Back

2 Abbreviations used in this paper: SP, single positive; DP, double positive; Tg, transgenic; SEB, staphylococcal enterotoxin B; PCC, pigeon cytochrome c; OVAp, peptide fragment 223–239 of OVA; PCCp, PPC peptide fragment 88–104. Back

Received for publication April 27, 1999. Accepted for publication August 9, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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