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The Journal of Immunology, 2001, 166: 5594-5603.
Copyright © 2001 by The American Association of Immunologists

Neonatal Exposure to Antigen Induces a Defective CD40 Ligand Expression that Undermines Both IL-12 Production by APC and IL-2 Receptor Up-Regulation on Splenic T Cells and Perpetuates IFN-{gamma}-Dependent T Cell Anergy1

Booki Min2, Kevin L. Legge3, J. Jeremiah Bell, Randal K. Gregg, Lequn Li, Jacque C. Caprio and Habib Zaghouani4

Department of Microbiology, University of Tennessee, Knoxville, TN 37996


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell deletion and/or inactivation were considered the leading mechanisms for neonatal tolerance. However, recent investigations have indicated that immunity develops at the neonatal stage but evolves to guide later T cell responses to display defective and/or biased effector functions. Although neonatal-induced T cell modulation provides a useful approach to suppress autoimmunity, the mechanism underlying the biased function of the T cells remains unclear. In prior studies, we found that exposure of newborn mice to Ig-PLP1, a chimera expressing the encephalitogenic proteolipid protein (PLP) sequence 139–151, induced deviated Th2 lymph node cells producing IL-4 instead of IL-2 and anergic splenic T cells that failed to proliferate or produce IFN-{gamma} yet secreted significant amounts of IL-2. However, if assisted with IFN-{gamma} or IL-12, these anergic splenic T cells regained full responsiveness. The consequence of such biased/defective T cells responses was protection of the mice against experimental allergic encephalomyelitis. In this study, investigations were performed to delineate the mechanism underlying the novel form of IFN-{gamma}-dependent splenic anergy. Our findings indicate that CD40 ligand expression on these splenic T cells is defective, leading to noneffective cooperation between T lymphocytes and APCs and a lack of IL-12 production. More striking, this cellular system revealed a requirement for IL-2R expression for CD40 ligand-initiated, IL-12-driven progression of T cells into IFN-{gamma} production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Evidence recently has accumulated indicating that neonatal exposure to soluble Ag can prime rather than ablate immunity (1, 2, 3). However, after several weeks, reexposure to Ag drives a response that differs markedly from controls that are not given Ag neonatally (4, 5, 6, 7). In early studies, T cell deletion as well as inactivation were considered the main portrayers of neonatal tolerance (8, 9, 10). However, careful examination of the response in various organs detected immunity that developed in the spleen rather than the lymph node and manifested effector functions tolerant toward the Ag (4, 7, 11). In the last few years, it has become clear that exposure to Ag at birth engenders a neonatal immunity that dictates the type of response to a subsequent encounter with Ag. A number of factors have been defined that could control neonatal immunity. These include the type of APCs (6), the adjuvant into which the Ag is emulsified (4), the dose of Ag (7), and the availability of Ag in vivo (12). Our own studies that used Igs for delivery of antigenic peptides have indicated that upon reexposure to Ag, immunity can develop in both the lymph node and the spleen of animals given an Ig peptide on the day of birth (5, 13, 14). Indeed, Ig-PLP1, a chimera expressing the encephalitogenic proteolipid protein (PLP)5 sequence 139–151 (15, 16), given to newborns within 24 h of birth, confers protection against autoimmunity, as such mice resisted induction of experimental allergic encephalomyelitis (EAE) later in life (5). This mechanism of suppressed autoimmunity appears to involve deviated lymph node T cells producing IL-4 in place of IL-2 and splenic T cells that fail to proliferate or produce IFN-{gamma} in response to Ag while still secreting significant amounts of IL-2 (5). The latter unresponsive splenic T cells can regain full proliferative and cytokine responsiveness if supplied with IFN-{gamma} or IL-12 (5). This neonatal-induced splenic phenotype then was termed IFN-{gamma}/IL-12-dependent anergy. Prior studies have reported that neonatal exposure to Ag causes apoptosis of Th1 cells, leading the response to default toward Th2 (2). However, because in this system the splenic cells produce IL-2 upon Ag stimulation and regain full Th1 phenotype when assisted with cytokines, the observation provides an additional means by which the neonatal system restrains Th1 responses and favors Th2 deviation. In this study, investigations were performed to delineate the mechanism operating this novel form of neonatal-induced IFN-{gamma}/IL-12-dependent splenic T cell anergy. The results indicate that defective expression of CD40 ligand (CD40L) underlies such unresponsiveness by sustaining a lack of IL-12 production by APCs. Moreover, the studies reveal a dependence on IL-2R expression in order for IL-12 to drive progression of the T cells into IFN-{gamma} production. A model is provided that discusses the control that IL-2R and CD40L exert on IL-12-driven IFN-{gamma} production.


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

SJL/J (H-2s) mice were purchased from Harlan Sprague Dawley (Frederick, MD). For generation of newborn mice, breeding sets of one male and three females were caged together, and when pregnancy was visible, the females were separated and caged individually. Offspring were weaned when they reached 3 wk of age. All experimental procedures were conducted according to the guidelines of the institutional animal care committee.

Peptides and Ig-PLP chimeras

All peptides used in these studies were purchased from Research Genetics (Huntsville, AL) and purified by HPLC to >90% purity. The encephalitogenic PLP1 (HSLGKWLGHPDKF) and PLP2 (NTWTTCQSIAFPSK) peptides encompass PLP sequences 139–151 (17) and 178–191 (18), respectively. Both peptides are presented to T cells in association with I-As MHC class II molecules and induce EAE in SJL/J mice (17, 18). For construction of Ig-PLP1, the complementarity determining region 3 of the 91A3 antiarsonate Ab heavy chain variable region was deleted and replaced with nucleotide sequence encoding PLP1 peptide. This chimeric heavy chain then was cotransfected into the non-Ig producing SP2/0 myeloma cell line with the parental 91A3 light chain to generate a complete Ig-PLP1 chimera (15). Ig-W, the parental IgG2b not encompassing PLP1 peptide, has been described elsewhere (19) and was used as negative control.

Abs and cytokines

Recombinant murine IFN-{gamma}, IL-12 (p70), and human IL-15 were purchased from BD PharMingen (San Diego, CA). Purified anti-mouse IL-2 (S4B6, rat IgG2a) and anti-mouse CD25 (PC61, rat IgG1) also were purchased from BD PharMingen. Hybridoma 3/23 cells (anti-CD40; Ref. 20) were kindly provided by Dr. Michael Cancro (University of Pennsylvania, Philadelphia, PA) and anti-CD40 Ab was affinity-purified over a mouse anti-rat {kappa} light chain (MAR18.5) column. PE-labeled anti-CD25 (3C7), FITC-labeled anti-CD4 (RM4-5), and biotin-labeled anti-CD40L (MR1) were purchased from BD PharMingen. The following Abs were used to perform cytokine detection by ELISA and were purchased from BD PharMingen. The capture anti-cytokine Abs were rat anti-mouse IL-2 (JES6-1A12), rat anti-mouse IFN-{gamma} (R4-6A2), and rat anti-mouse IL-12 (9A5). The biotinylated anti-cytokine Abs were rat anti-mouse IL-2 (JES6-5H4), rat anti-mouse IFN-{gamma} (XMG1.2), and rat anti-mouse IL-12 (C17.8). Rat IgG was purchased from Sigma (St. Louis, MO).

Neonatal tolerization with Ig-PLP1 and immunization of adult mice with PLP1 peptide

Newborn mice were injected i.p. with 100 µg of Ig-PLP1 or Ig-W in 100 µl of saline within 24 h after birth, and when the mice reached 7 wk of age, they were immunized s.c. with 100 µg of PLP1 peptide emulsified in 200 µl of PBS/CFA (v/v). This tolerization and immunization regimen was found to be optimal for induction of lymph node deviation and splenic IFN-{gamma}-dependent anergy (5, 13, 14). Ten days later, the mice were sacrificed and their spleens were removed for analysis of proliferation, cytokine production, and IL-2R expression.

Induction of EAE

EAE was induced by s.c. injection in the foot pads and at the base of the tail and limbs with a 200-µl IFA/PBS (v/v) solution containing 100 µg of free PLP1 peptide and 200 µg of Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). Six hours later, 5 x 109 inactivated Bordetella pertussis were given i.v. After 48 h, another 5 x 109 inactivated B. pertussis were given to the mice. In some experiments the mice also were given i.p. 200 µg/mouse of anti-CD40 Ab or rat IgG on the day of disease induction and 3 days later. Mice were scored daily for clinical signs as follows: 0, no clinical signs; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb paralysis; and 5, moribund or death.

Cell purification

T cells. After lysis of RBC, splenic T cells were purified by passage on a nylon wool column. Cell purity was {approx}90% as determined by FACS analysis.

T cell-depleted APCs. Splenic cells were treated with anti-CD3 (2C11, 1 µg/ml) and incubated with rabbit complement for 45 min. at 37°C. APCs then were separated on a dense BSA (Intergen, Purchase, NY) gradient as described previously (21).

Dendritic cells (DCs). DCs were purified from SJL/J spleen according to standard collagenase and differential adherence method (21). Briefly, SJL/J splenic cells were dissociated in the presence of collagenase (Life Technologies, Grand Island, NY), separated on a dense BSA gradient, and further isolated by differential adherence on 60-mm culture dishes. Cell purity was >90% as determined by FACS analysis with 33D1 Ab.

Proliferation assay

Spleen cells were incubated in 96-well flat-bottom plates at 10 x 105 cells/100 µl/well with 100 µl of stimulator for 3 day. Subsequently, 1 µCi [3H]thymidine was added per well, and the culture was continued for an additional 14.5 h. The cells then were harvested onto glass fiber filters, and incorporated [3H]thymidine was counted by the trace 96 program and an Inotech beta counter (Inotech, Wohlen, Switzerland). The stimulators, PLP1 and PLP2 peptides, were used at 15 µg/ml, a concentration that was defined previously as optimal for in vitro T cell stimulation (5, 15). A control of medium with no stimulator was included for each mouse and used as background.

ELISA

Spleen cells were incubated in 96-well round-bottom plates at 10 x 105 cells/100 µl/well with 100 µl of stimulator for 24 h. Cytokine production was measured by ELISA according to BD PharMingen’s instructions with 100 µl of culture supernatant (5). The OD405 was measured on a SpectraMAX 340 counter (Molecular Devices, Sunnyvale, CA) with SoftMAX PRO version 1.2.0 software (Molecular Devices). Graded amounts of recombinant mouse IL-2, IFN-{gamma}, and IL-12 (BD PharMingen) were included in all experiments to construct standard curves. The concentration of cytokines in culture supernatants was estimated by extrapolation from the linear portion of the standard curve.

Restoration of T cell responses

For restoration of splenic responses, the stimulation was conducted in the presence of an optimal dose (10 U) of mouse rIL-12 (BD PharMingen) as described previously (5). Proliferation and cytokine production were measured as described above. For inhibition of IL-12-mediated restoration of proliferation, the stimulation was conducted in the presence of 10 µg/ml anti-mouse IL-2 (S4B6) or anti-mouse CD25 (PC61) Abs (BD PharMingen). Rat IgGs were used as an isotype control. Proliferation and cytokine production were measured as described above.

Flow cytometry analyses

Staining for CD4 and CD25. Splenic cells from mice tolerized at birth with either Ig-PLP1 or Ig-W and immunized with PLP1 peptide at the age of 7 wk were in vitro stimulated with PLP1 peptide (15 µg/ml) for 24 h. Subsequently, erythrocytes were lysed, the cells washed with FACS buffer (0.5% BSA and 1 mM sodium-azide in 1x PBS), and incubated with anti-FcR mAb (2.4G2) for 30 min at 4°C. The cells then were labeled with FITC-anti-CD4 and PE-anti-CD25 for 30 min according to BD PharMingen’s instruction and immediately fixed with 2% formaldehyde.

Staining for CD4 and CD40L. Staining was conducted as described previously (22). Briefly, the spleen cells were mixed with PLP1 peptide and biotinylated anti-CD40L Ab or hamster IgG (1 µg/ml) was immediately added because of the rapid down-regulation of CD40L (23). After 8 h, the erythrocytes were lysed and the cells were labeled with FITC-anti-CD4 Ab.

Detection of intracellular IL-12. Purified DCs (5 x 106 cells) were incubated with 40 µg/ml of anti-CD40 Ab or rat IgG. After 12 h, 10 µg/ml of brefeldin A (Epicentre, Madison, WI) was added and the culture continued for an additional 6 h. The cells then were stained with 33D1 mAb, fixed with paraformaldehyde, and permeabilized with 0.5% saponin in FACS buffer. Intracellular IL-12 was stained with biotinylated anti-IL-12 Ab. Bound 33D1 was revealed with biotinylated anti-rat IgG2b/FITC-streptavidin and bound biotinylated anti-IL-12 Ab was detected with PE-streptavidin. Isotype-matched controls were included in all experiments. All of the data were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) with CellQuest software (Becton Dickinson).

Detection of IL-2R{alpha}-chain mRNA by RT-PCR

Total RNA was isolated from splenic cells with TRIzol (Life Technologies) according to manufacture’s instructions. RT-PCR was conducted by using the Access RT-PCR system (Promega, Madison, WI) and consisted of a first-strand cDNA synthesis step followed by 40 PCR amplification cycles (94°C, 30 s denaturation; 68°C, 1 min annealing; and 72°C, 2 min polymerization). The primers used were; 5'-CAGACATGCAGAAGCCAACAC-3' and 5'-GGTGAGCCCGCTCAGGAGGA- 3' for CD25, and 5'-GTGGGGCGCCCCAGGCACCA-3' and 5'-CTCCTTAATGTCACGCACGAT-3' for {beta}-actin (24). All oligonucleotides were purchased from Life Technologies).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Neonatal exposure to Ig-PLP1 primes a subset of IL-2 producing nonproliferative T cells

Previous studies showed that neonatal exposure to Ig-PLP1 conferred resistance to EAE induction by a mechanism that involved deviated lymph node T cells and splenic T lymphocytes that produced IL-2 but were defective in proliferation and IFN-{gamma} production (5, 13). Specifically, mice that were injected with Ig-PLP1 in saline on the day of birth and subsequently challenged with free PLP1 in CFA at 7 wk developed lymph node T cells that produced IL-4 instead of the usual IL-2 in response to PLP1 peptide. However, the splenic T cells failed to proliferate or produce IFN-{gamma} in response to PLP1 stimulation yet still secreted significant amounts of IL-2 (Ref. 5 and Fig. 1Go). As shown in Fig. 1Go, a and b, the addition of exogenous IFN-{gamma} or the IFN-{gamma} inducer IL-12 restored splenic T cell proliferation as well as IFN-{gamma} production. Such restoration was Ag specific, as stimulation with PLP2 peptide, instead of PLP1, did not reinstate responsiveness. Because the cells produce significant levels of IL-2 and stimulation of the T cells in the presence of an excess of exogenous IL-2 (100 U/ml) was unable to restore proliferation and IFN-{gamma} production, we termed this form of anergy as IFN-{gamma}-dependent anergy (5). Interestingly, during restoration of the T cells by IL-12 or IFN-{gamma}, culture levels of IL-2 diminished rather than increased (Fig. 1Goc). This raised the possibility that during cytokine-mediated restoration, the T cells consumed or reabsorbed some of their own IL-2 to proliferate. Experiments performed to test this premise confirmed that IL-2 is required for IL-12-mediated restoration of splenic T cell responsiveness. Accordingly, neutralization of IL-2 by anti-IL-2 Ab during stimulation with PLP1 peptide in the presence of IL-12 inhibited restoration of both proliferation (Fig. 2Goa) and IFN-{gamma} production (Fig. 2Gob). Isotype control rat IgG Abs in place of the anti-IL-2 failed to inhibit the restoration of responses. These results indicated that the splenic T cells required IL-2 to proliferate and suggested that the proliferative unresponsiveness of these cells might be attributable to a lack of the functional IL-2R necessary for reabsorption of IL-2, as has been demonstrated previously in IL-2R{alpha}-/- mice (25).



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  		FIGURE 1. The anergic splenic T cells of Ig-PLP1-tolerized mice are restorable by exogenous IFN-{gamma} or IL-12. Groups of newborn mice (4–6 per group) were injected i.p. with 100 µg of Ig-PLP1 in saline on the day of birth and immunized s.c. with PLP1 in CFA when they were 7 wk of age. Ten days later, spleen cells (1 x 106 cells/well) were in vitro stimulated with 15 µg/ml PLP1 or PLP2 peptide in the presence or absence of 10 U/ml of cytokine and proliferation (a), IFN-{gamma} (b), and IL-2 (c) production were measured as described in Materials and Methods. The indicated values (cpm and pg/ml) represent the mean ± SD of individually tested mice and are representative of three experiments.

 


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  		FIGURE 2. Requirement of IL-2 for restoration of splenic T cell proliferation and IFN-{gamma} production. A group of eight newborn mice was injected i.p. with 100 µg of Ig-PLP1 in saline on the day of birth and challenged with PLP1 in CFA at the age of 7 wk. Ten days later, their splenic T cells were tested for IL-12-mediated restoration of proliferation (a) and IFN-{gamma} production (b) in the presence of neutralizing anti-IL-2 Ab (S4B6). The spleen cells (1 x 106 cells/well) were incubated with PLP1 (PLP1), PLP1 + rIL-12 (PLP1 + rIL-12), PLP1 + rIL-12 + anti-IL-2 Ab (PLP1 + rIL-12 + anti-IL-2), or PLP1 + rIL-12 + rat IgG (PLP1 + rIL-12 + rat IgG), and IFN-{gamma} production and proliferation were measured after 1 and 3 day(s) of stimulation, respectively. PLP1 peptide was used at 15 µg/ml, rIL-12 at 10 U/ml, anti-IL-2 Ab at 10 µg/ml, and rat IgG at 10 µg/ml. The data represent the mean ± SD of eight individually tested mice and are representative of two experiments.

 
Defective up-regulation of IL-2R{alpha}-chain expression on anergic splenic T cells

The IL-2R consists of {alpha}-, {beta}-, and {gamma}-chains (26, 27). In the murine system, the {beta}- and {gamma}-chains are constitutively expressed and fail to bind IL-2 (28). The {alpha}-chain or CD25, which is inducibly expressed on activated T cells, has a high affinity for IL-2 and plays a critical role in the formation of a functional IL-2R (26, 27, 29).

If the proliferative unresponsiveness of the splenic T cells was attributable to an inability of the cells to use IL-2, it was possible that a defect in expression of functional IL-2R was responsible for such a phenotype. To investigate this issue, both RT-PCR and cell surface staining analysis were performed to assess IL-2R{alpha}-chain mRNA and protein expression upon stimulation with Ag. As can be seen in Fig. 3Goa, proliferative T cells from mice neonatally injected with the control Ig-W chimera and immunized as adults with PLP1 peptide expressed, as expected, significant IL-2R{alpha}-chain mRNA. Furthermore, upon stimulation with PLP1 peptide, significant up-regulation of IL-2R{alpha}-chain mRNA was observed. Evaluation of the band intensity with the NIH Image analysis program (National Institutes of Health, Bethesda, MD) revealed that the IL-2R{alpha}-chain DNA intensity was 21% of that of the {beta}-actin band for T cells of Ig-W recipient mice and increased to 53% when the cells were stimulated with PLP1 peptide. In contrast, T cells from Ig-PLP1-tolerized mice had only marginal IL-2R{alpha}-chain mRNA, which was not up-regulated by stimulation with PLP1 peptide (Fig. 3Goa). Only the addition of IL-12 during T cell stimulation led to significant up-regulation of IL-2R{alpha}-chain mRNA (Fig. 3Goa). The percentage of {beta}-actin band intensity of IL-2R{alpha}-chain was <1% when the T cells were unstimulated and 2% after PLP1 peptide stimulation, but increased to 24% when the cells were stimulated with PLP1 peptide in the presence of IL-12. Moreover, when graded amounts of mRNA were used in RT-PCR, DNA amplification increased proportional to mRNA input, indicating a specific correlation between the amount of mRNA and the reverse transcription and amplification (data not shown). These results indicate that the level of IL-2R mRNA is minimal in the Ig-PLP1 vs Ig-W-tolerized splenic T cells. However, the lower level of IL-2R mRNA could be attributable to a reduced number of PLP1-specific T cells or to a defective up-regulation of the receptor. To address this issue, we performed cell surface staining for IL-2R that evaluates receptor intensity at the single-cell level. The results of this experiment, presented in Fig. 3Gob, indicate that T cells of Ig-PLP1-tolerized mice are defective in up-regulation of IL-2R. Indeed, when splenic T cells from control Ig-W-tolerized mice were stimulated with PLP1, increase of surface expression of IL-2R{alpha}-chain relative to the isotype control occurred. However, IL-2R expression intensity did not shift, relative to the isotype control, when the T cells were from Ig-PLP1-tolerized mice. Furthermore, stimulation with PLP1 in the presence of IL-12 was able to restore surface IL-2R{alpha}-chain expression (see Fig. 11Goe). These results indicate that the anergic splenic T cells from Ig-PLP1-tolerized mice fail to up-regulate IL-2R{alpha}-chain expression unless exogenous IL-12 is provided. This observation is consistent with a previous report showing that IL-12 can up-regulate IL-2R{alpha}-chain expression (30). Overall, the splenic T cells are possibly unable to proliferate and progress into IFN-{gamma}-producing cells because of a lack of up-regulation of functional IL-2R.



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  		FIGURE 3. Defective up-regulation of IL-2R{alpha}-chain mRNA and protein on the anergic splenic T cells. a, Splenic cells (5 x 106 cells/ml) from mice that were tolerized with 100 µg of either Ig-W or Ig-PLP1 at birth and immunized with 100 µg of PLP1 peptide in CFA at 7 wk of age were stimulated with 15 µg/ml PLP1 peptide in the presence or absence of IL-12 (10 U/ml) for 24 h. Total RNA was extracted from 5 x 106 cells, and 100 ng was subjected to first-strand synthesis by reverse transcription and DNA amplification by PCR with IL-2R {alpha}-chain (CD25) and {beta}-actin-specific primers. Amplified DNA then was run on a 1% agarose gel. The illustrated bands correspond to the expected m.w. of CD25 and {beta}-actin. +, Addition of peptide or IL-12; -, absence of PLP1 or IL-12. b, Splenic cells (1 x 106) from mice neonatally tolerized with Ig-PLP1 or Ig-W and immunized with PLP1 at adult life were in vitro stimulated with PLP1 peptide for 24 h and double-stained with anti-CD4 + anti-CD25 Ab or anti-CD4 + rat IgG2b and analyzed by FACS. CD4+ T cells were gated and their number is plotted against the fluorescent intensity of bound anti-CD25 Ab. The dotted line represents the histogram obtained by staining T cells from Ig-W-tolerized mice with anti-CD4 and the isotype control for anti-CD25 Ab.

 


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  		FIGURE 11. Requirement of IL-2R expression for IL-12-driven differentiation of the splenic T cells. Splenic cells (1 x 106 cells/well) from the mice tolerized with Ig-PLP1 at birth and immunized with PLP1 peptide at 7 wk of age were in vitro stimulated with 15 µg/ml PLP1 peptide without exogenous cytokine or in the presence of 10 U/ml of IL-12 or 10 ng/ml of IL-15. Proliferation (a) and production of IFN-{gamma} (b) and IL-2 (c) then were measured as described in Materials and Methods. Parallel experiments were performed to assess CD40L (d) and CD25 (e) expression on the T cells. Shown are the percentage of CD40L+ and CD25+ CD4 T cells as determined by first gating on the CD4+ population. The data are representative of two experiments.

 
To further demonstrate that IL-2R expression is required for restoration of splenic responses, peptide-driven, IL-12-dependent reactivation of the T cells was assayed in the presence of anti-IL-2R{alpha}-chain (anti-CD25) Ab. As can be seen in Fig. 4Go, blockade of IL-2R prevented the absorption of IL-2 by T cells and inhibited both proliferation and IFN-{gamma} production. In contrast, the addition of the rat IgG isotype control, instead of anti-CD25 Ab, allowed for IL-2 absorption and did not inhibit restoration of proliferation or IFN-{gamma} production (Fig. 4Go).



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  		FIGURE 4. Blockade of IL-12-restored IL-2R{alpha}-chain reinstates T cell anergy. Splenic cells (1 x 106/well) from mice that were tolerized with Ig-PLP1/saline on the day of birth and challenged with PLP1/CFA at the age of 7 wk were stimulated with 15 µg/ml PLP1 peptide + 10 U/ml IL-12 in the presence of anti-CD25 Ab (+PLP1 +IL-12 + anti-CD25) or control rat IgG (+PLP1 +IL-12 + rat IgG) and assayed for proliferation (a), IFN{gamma} (b), and IL-2 (c) production. Cells stimulated with PLP1 alone (+PLP1) or PLP1 and IL-12 (+PLP1 + IL-12) were included for control purposes. Anti-CD25 Ab and rat IgG were used at 10 µg/ml. Proliferation was measured by [3H]thymidine incorporation, and cytokines were measured by ELISA. The indicated values (cpm and pg/ml) represent the mean ± SD of four individually tested mice. The results presented here are representative of two separate experiments.

 
The defect in IL-2R{alpha}-chain expression is long-lasting and parallels with the persistence of in vivo T cell tolerance

If IL-2R{alpha}-chain expression accounted for the anergic splenic T cell responses, it should parallel with the status of T cell unresponsiveness in vivo. To address this issue, mice were neonatally injected with Ig-PLP1, and each week starting at week 8, a group of mice was challenged with PLP1 in CFA. The splenic T cells then were tested for IL-2R{alpha}-chain expression, proliferation, and cytokine production. The results of these experiments show that the mice remained unable to mount splenic proliferative responses or produce IFN-{gamma} upon immunization with PLP1 peptide through week 20, although IL-2 production did occur (Fig. 5Go, a and b). The ability to mount normal splenic proliferative and cytokine responses returned by week 28 (Fig. 5Go, a and b). IL-2R{alpha}-chain expression followed a similar pattern. Weak expression was still evident at week 20, but had increased significantly by week 28 to a level of 7.9% among all CD4+ T cells (Fig. 5Goc). These results indicated that the defect in IL-2R{alpha}-chain expression is long-lasting and parallels the persistence of in vivo T cell tolerance and suggested that IL-2R{alpha}-chain plays a critical role in the maintenance of Ig-PLP1-mediated neonatal tolerance.



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  		FIGURE 5. Weak expression of IL-2R{alpha}-chain is long lasting and parallels with T cell tolerance. a, Groups of mice (eight per group) were injected i.p. at birth with 100 µg of Ig-PLP1 in saline and immunized at the indicated weeks with 100 µg of PLP1 in CFA. Ten days after immunization, the mice were sacrificed, and splenic cells were in vitro stimulated with PLP1 peptide (15 µg/ml) and tested for proliferation (a), IL-2 and IFN{gamma} production (b), and cell surface expression of IL-2R{alpha}-chain (c). Proliferation was measured by [3H]thymidine incorporation and cytokines were assessed by ELISA. Stimulation with PLP2 peptide was included as a negative control in all experiments and no significant response to PLP2 was found (data not shown). The indicated cpm and pg/ml values represent the mean ± SD of individually tested mice. For cell surface analysis of IL-2R{alpha}-chain, the cells were double-stained with anti-CD4 and either anti-CD25 or rat IgG2b and analyzed for CD25 expression by FACS. The histograms were generated by gating on CD4 expression and assessing fluorescent intensity of bound anti-CD25 Ab or rat IgG2b. The results shown in c are those obtained at 20 wk, where T cell tolerance was still apparent, and at 28 wk, where recovery from tolerance was significant.

 
The defect in proliferation and differentiation is intrinsic to T cells

Because IL-12 up-regulates IL-2R{alpha}-chain expression (Fig. 3Goa; Ref. 30) and restores the splenic T cell proliferation and IFN-{gamma} production (Fig. 1Go), one explanation for the observed defect in IL-2R{alpha}-chain up-regulation on splenic T cells is that the APCs of Ig-PLP1-tolerized mice are defective and cannot secrete the IL-12 necessary for up-regulation of IL-2R{alpha}-chain. Alternatively, the APCs may display normal IL-12 secretion, but the T cells might have an intrinsic defect and fail to provide the appropriate signal to trigger IL-12 secretion by the APCs. To investigate this issue, swap experiments were performed between T cells and APCs from Ig-PLP1- vs Ig-W-tolerized animals, and the function of each population was assessed. As can be seen in Fig. 6Goa, when the T cells originated from Ig-W-tolerized mice, significant proliferative responses were observed regardless of the source of APCs. Similarly, the production of IFN-{gamma} followed a similar pattern and was not affected by the origin of the APCs (Fig. 6Gob). However, when the T cells were derived from Ig-PLP1-tolerized mice, proliferation and IFN-{gamma} production were at background levels irrespective of the origin of the APCs. These results indicate that the APCs have minimal or no contribution in the proliferative and differentiation defect of the splenic T cells. Furthermore, ligation of CD40 by anti-CD40 Ab stimulates DCs derived from Ig-PLP1-tolerized mice to produce IL-12 to the same extent as DCs from mice that were tolerized with Ig-W (Fig. 6Goc). Therefore, the APCs of Ig-PLP1-tolerized mice display normal IL-12 secretion when provided the appropriate signal.



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  		FIGURE 6. Minimal contribution of APCs to splenic T cell unresponsiveness. Groups of newborn mice (four per group) were injected i.p. on the day of birth with either Ig-PLP1 or Ig-W in saline and were challenged with PLP1 peptide in CFA when they reached 7 wk of age. Ten days later, splenic T cells from both groups were isolated and assayed for proliferation (a) and IFN-{gamma} production (b) upon stimulation with PLP1 peptide and purified APCs from both groups. The source of T cells and APCs is indicated below each bar. T cells (1 x 105 cells/well) and APCs (2 x 105 cells/well) were incubated with 15 µg/ml PLP1 peptide, and IFN-{gamma} production was measured after 24 h by ELISA and proliferation was measured by [3H]thymidine incorporation after 3 days. In vitro stimulation with PLP2 peptide was included as a negative control, and no significant response to PLP2 was observed (data not shown). The data represent the mean ± SD of triplicate wells (invisible error bars represent small variations) and are representative of two experiments. c, DCs were isolated from adult mice that were neonatally tolerized with either Ig-PLP1 or Ig-W, incubated with 40 µg/ml of anti-CD40 (bold line) or rat IgG (thin line) for 24 h, and then stained for 33D1 and intracellular IL-12. Shown are histograms of intracellular IL-12 expression on 33D1+-gated cells. The shaded histogram represents staining with isotype control.

 
The anergic splenic T cells display defective expression of CD40L and require substitution with anti-CD40 Ab for restoration of IL-2R{alpha} expression, proliferation, and IFN-{gamma} production

CD40L (gp39) on activated T cells interacts with CD40 on APCs (31), induces IL-12 production by APCs (32, 33), and controls differentiation to the Th1 phenotype (34, 35). Because the APCs of Ig-PLP1-tolerized mice produced IL-12 upon ligation by anti-CD40 Ab, the splenic T cells might be unable to up-regulate CD40L expression upon interaction with the APCs, leading to inadequate ligation of CD40 and lack of IL-12 production by the APCs. To evaluate this hypothesis, CD40L expression on splenic T cells upon PLP1 peptide stimulation was measured. As can be seen in Fig. 7Go, the splenic T cells from Ig-PLP1-tolerized and PLP1-immunized mice show minimal expression of CD40L upon stimulation with PLP1 peptide (Fig. 7Goa). In contrast, 3.1% of the splenic CD4+ T cells from recipients of the control Ig-W instead of Ig-PLP1 expressed CD40L upon peptide stimulation (Fig. 7Gob). Similar deficiency was observed when the experiment was repeated with enriched CD4+ T cells and purified DCs as APCs. Indeed, CD40L was at background levels for T cells from Ig-PLP1-tolerized mice (Fig. 7Goc), but when the T cells were from the Ig-W group, 6.9% of the splenic CD4+ T cells showed significant expression of CD40 ligand (Fig. 7God). To further explore this possibility, we used anti-CD40 Ab to substitute for CD40L and tested for restoration of splenic T cell proliferation and differentiation into IFN-{gamma} production. As shown in Fig. 8Go, a and b, anti-CD40 Ab was able to substitute for CD40L and restore responsiveness. In contrast, the isotype control rat IgG did not restore either proliferation or IFN-{gamma} production. Anti-CD40-mediated restoration of splenic T cell responses was Ag-dependent and could not occur in the absence of PLP1 peptide (Fig. 8Go, a and b). IL-2R{alpha}-chain expression also was restored by anti-CD40 Ab but not by the control rat IgG (Fig. 8Goc). Furthermore, when anti-CD40 Ab was administered into Ig-PLP1-tolerized mice during the induction of EAE with PLP1 peptide, the severity of disease was restored, reaching a maximum of 4.2 ± 0.9 clinical score (2.5 ± 0.5 for rat IgG-administered group), and the mice never recovered from paralysis during the entire 70-day observation period (Fig. 8God). The restoration of splenic T cell proliferation by anti-CD40 Ab seemed to operate through IL-12 and IL-2R. This conclusion was drawn from the results illustrated in Fig. 9Go showing that neutralization of IL-12 abolished anti-CD40-mediated restoration of proliferation. Similarly, neutralization of IL-2 by anti-IL-2 Ab inhibited the restoration of T cell proliferation by anti-CD40 (Fig. 9Go). All together, these results indicate that the splenic T cells display an intrinsic defect that precludes their up-regulation of CD40L expression. Consequently, CD40 ligation does not occur, thereby impairing IL-12 production by the APCs. Thus, the splenic T cells are unable to up-regulate IL-2R{alpha}-chain and fail to proliferate and differentiate into IFN-{gamma}-producing cells.



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  		FIGURE 7. Defective expression of CD40L on the splenic anergic T cells. Whole splenic cells (1 x 106 cells/ml) from the mice tolerized at birth with either Ig-PLP1 (a) or Ig-W (b) and immunized with PLP1 in CFA at 7 wk of age were stimulated with 15 µg/ml PLP1 peptide for 8 h and double-stained with anti-CD40L and anti-CD4 Abs. In parallel, T cells were purified from Ig-PLP1 (c) or Ig-W (d) recipient mice, and 0.25 x 106 cells were stimulated for 8 h with 15 µg/ml peptide presented on 0.2 x 106 normal SJL DCs as APCs, and double-stained with the above Abs. Shown are the histograms of CD40L expression on CD4+-gated cells. Staining with isotype-matched control (hamster Ig) was included in all experiments and used as background (data not shown).

 


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  		FIGURE 8. Anti-CD40 substitutes for CD40L and restores proliferation, IFN-{gamma} production, IL-2R{alpha}-chain expression, and disease severity. Groups of mice (4 per group) were injected i.p. with Ig-PLP1 on the day of birth and immunized s.c. with PLP1 in CFA when they were 7 wk of age. Ten days later, spleen cells (1 x 106 cells/well) were in vitro stimulated with 15 µg/ml PLP1 peptide in the presence of the indicated amounts of anti-CD40 (•) or rat IgG ({circ}). Stimulation with anti-CD40 without PLP1 peptide ({diamond}) was also included as a control. Subsequently, proliferation (a) and IFN-{gamma} (b) production were measured as described in Materials and Methods. The data represent the mean ± SD of individually tested mice and are representative of four experiments. c, Total RNA was extracted from splenic cells (5 x 106 cells), and used to assess CD25 expression on these cells by RT-PCR subsequent to stimulation with PLP1 alone (lane 2), PLP1 + anti-CD40 (40 µg/ml; lane 3), or PLP1 + rat IgG (lane 4). Unstimulated cells (lane 1) were included for control purposes. d, Groups of mice (seven per group) tolerized with Ig-PLP1 at birth were subjected to EAE induction with PLP1 peptide and given i.p. 200 µg of either anti-CD40 (•) or rat IgG ({diamond}). Three days later, the mice were given an additional injection of anti-CD40 or rat IgG. Mice were scored daily for signs of paralysis, and the indicated data represent the mean clinical score of all tested mice.

 


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  		FIGURE 9. Requirement of IL-2 and IL-12 for restoration of splenic T cell proliferation. Splenic cells from mice (four per group) tolerized with Ig-PLP1 at birth and immunized with PLP1 at 7 wk of age were in vitro stimulated for 3 day with PLP1 peptide and anti-CD40 mAb in the presence of rat IgG (PLP1 + anti-CD40 + rat IgG), anti-IL-12 (PLP1 + anti-CD40 + anti-IL-12), or anti-IL-2 (PLP1 + anti-CD40 + anti-IL-2). A group stimulated with PLP1 peptide alone was included for control purposes. Subsequently, proliferation was measured by thymidine incorporation. PLP1 peptide was used at 15 µg/ml, anti-CD40 at 10 µg/ml, anti-IL-12 at 10 µg/ml, anti-IL-2 at 10 µg/ml, and rat IgG at 10 µg/ml. The data represent the mean ± SD of individually tested mice.

 
Up-regulation of CD40L on the splenic T cells drives IL-12 production by the APCs

To further investigate the role of CD40L in the splenic anergy, we tested whether restoration of its expression drives IL-12 production by APCs. To this end, enriched splenic T cells from Ig-PLP1-tolerized and peptide-immunized mice were incubated with peptide-loaded DCs along with IFN-{gamma}, which restores splenic responses (Fig. 1Goa), and CD40L expression and IL-12 production were assessed. Fig. 10Go shows that the number of splenic CD4+ T cells expressing CD40L increased from 0.4% in the absence of IFN-{gamma} (Fig. 10Goa) to 3.7% in IFN-{gamma} supplemented cultures (Fig. 10Gob). Furthermore, DCs presenting PLP1 peptide produced IL-12 when the T cells were from mice tolerized with the control Ig-W but were unable to do so when the T cells originated from mice that were tolerized with Ig-PLP1 (Fig. 10Go, c and d). However, when the culture was supplemented with IFN-{gamma}, IL-12 production was induced to the same level as that induced by anti-CD40 Ab (Fig. 10Goc). Such IFN-{gamma}-restored IL-12 induction required peptide presentation because exclusion of PLP1 from the culture ablated IL-12 production. Similarly, omission of the T cells from the culture led to a failure of IL-12 induction (data not shown), suggesting that IFN-{gamma} is not acting directly on APCs but rather requires T cells for promoting IL-12 production. All together, these results indicate that up-regulation of CD40L on the splenic T cells, triggered by addition of IFN-{gamma}, drives the synthesis of IL-12 by the DCs.



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  		FIGURE 10. Restoration of CD40L expression on the splenic T cells promotes IL-12 production by the APCs. T cells (0.25 x 106) purified from the mice tolerized with Ig-PLP1 at birth and immunized with PLP1 at 7 wk of age were incubated with purified 0.2 x 106 DCs from normal SJL mice, 15 µg/ml PLP1 peptide, and biotinylated anti-CD40L Ab (1 µg/ml) in the absence (a) or presence (b) of 10 U/ml of IFN-{gamma}. After 8 h, the cells were double-stained for CD4 and CD40L expression. Shown are histograms of CD40L expression on CD4-gated cells. Isotype-matched controls (hamster Ig) were included in all experiments and used as a background level (data not shown). To test for IL-12 production by DCs upon up-regulation of CD40L by IFN-{gamma} purified T cells (2 x 105 cells/well) from Ig-PLP1 (c) or Ig-W (d)-tolerized and PLP1-immunized mice were incubated with purified DCs (2 x 105 cells/well) from normal SJL mice in the presence of indicated stimuli. After 24 h, IL-12 production was measured from the culture supernatant by ELISA. PLP1 peptide was used at 15 µg/ml, anti-CD40L at 40 µg/ml, and IFN-{gamma} at 10 U/ml. The results are representative of two experiments.

 
Requirement for functional IL-2R expression for differentiation of splenic T lymphocytes into IFN-{gamma}-producing cells

The defect in CD40L expression by the splenic T cells provides these lymphocytes with the susceptibility to regulation at various points along the activation and differentiation pathway and offers an experimental model to understand IL-12-driven differentiation. Our data indicate that IL-2R{alpha} expression is crucial for IL-12-driven Th1 differentiation. Because IL-15 has been shown to partially rescue T cell proliferation in IL-2R{alpha}-/- mice (25), we questioned whether or not IL-15 could restore proliferation and IFN-{gamma} production by splenic anergic T cells by bypassing IL-2R. To address this point, the splenic T cells from Ig-PLP1-tolerized and peptide-immunized mice were stimulated with PLP1 in the presence of IL-15, and the cells were tested for proliferation, IL-2 and IFN-{gamma} production, and expression of CD40L and IL-2R{alpha}-chain. The results presented in Fig. 11Go indicate that IL-15 restored proliferation (Fig. 11Goa) but did not reinstate IFN-{gamma} production (Fig. 11Gob) or CD40L or IL-2R{alpha}-chain expression (Fig. 11Go, d and e). In addition, IL-2 was not reduced in the supernatant as occurs with IL-12 (Fig. 11Goc), further supporting the absence of a functional IL-2R. All together, these results indicate that a functional IL-2R is required for differentiation into IFN-{gamma}-producing cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The studies presented here provide evidence that IFN-{gamma}-dependent T cell anergy arises as a consequence of defective expression of CD40L on these lymphocytes. Moreover, they reveal a requirement for expression of a functional IL-2R for IL-12 to promote the progress of T cells from proliferation to IFN-{gamma} production.

Neonatal exposure of mice to Ig-PLP1, the chimera expressing amino acid sequence 139–151 of PLP, gives rise to a subset of splenic anergic T cells that are committed to the Th1 lineage upon reimmunization of the mice as adults with PLP1 peptide. These cells produce significant amounts of IL-2 but do not proliferate or produce IFN-{gamma} (Fig. 1Go). However, if assisted with IL-12 or IFN-{gamma} during Ag stimulation, the cells regain proliferative capacity and secrete IFN-{gamma}. Two separate but sequentially connected issues became targeted for investigation to delineate the mechanism responsible for the defective proliferation and progression into IFN-{gamma} production. The first issue was related to expression of functional IL-2R on the splenic T cells. These lymphocytes need to reabsorb their own IL-2 to proliferate and produce IFN-{gamma} on Ag stimulation (Fig. 2Go). However, because of a lack of expression of functional IL-2R, they were unable to respond to Ag stimulation unless assisted with IL-12 (Figs. 3Go and 4Go). Further evidence for the lack of IL-2R was provided by the finding that a minimal expression of IL-2R{alpha}-chain was long lasting, paralleled with the status of splenic unresponsiveness, and reappeared at week 28 when responsiveness was reestablished (Fig. 5Go). At this point, it is unclear whether such receptor reestablishment is the result of reexpression on previously anergic T cells or because of accumulation of newly migrant nontolerized T cells.

The defective up-regulation of IL-2R{alpha}-chain seems to be intrinsic to T cells, as APCs from Ig-PLP1-tolerized mice were able to present Ag to T cells from nontolerized animals, but the anergic T cells were not responsive when the Ag was presented with APCs from nontolerized animals (Fig. 6Goa). In addition, APCs from Ig-PLP1-tolerized animals were able to produce IL-12, the cytokine required for reactivation of the splenic T cells upon ligation of their CD40 with anti-CD40 Ab (Fig. 6Goc). This led us to speculate that the T cells may be defective in the expression of the CD40L required to stimulate the APCs for CD40-induced production of IL-12. The results presented in Fig. 7Go proved this postulate correct, as the T cells could not express CD40L upon stimulation with APCs. Furthermore, if the presentation was assisted with anti-CD40 Ab, which substitutes for CD40L, the proliferation, IFN-{gamma} production, IL-2R{alpha}-chain expression, and disease severity were restored (Fig. 8Go). In addition, neutralization of IL-2 or anti-CD40 Ab-induced IL-12 inhibited restoration of T cell proliferation (Fig. 9Go). When CD40L expression is restored on the T cells by Ag stimulation in the presence of IFN-{gamma}, IL-12 synthesis by APCs occurred (Fig. 10Go).

CD40-CD40L interaction has been shown to drive IL-12 production by APCs (32, 33) and promote differentiation to Th1 during priming of T cell responses (34, 35). However, the role of this interaction in effector functions during a subsequent encounter with Ag is still debatable (36, 37, 38). The studies illustrated here provide a clear demonstration that lymphocytes that have experienced the Ag at the neonatal period require CD40-CD40L interactions to proliferate and produce IFN-{gamma} during a subsequent encounter with the Ag.

It has been shown that T cells display a quantitative defect in the expression of CD40L at the neonatal stage (39). Consequently, depending on the type of APCs, such insufficient CD40L may drive variable IL-12 production. Under such circumstances, most neonatal T cells would be exposed to minimal IL-12 and default toward the Th2 phenotype (40, 41), leading to deviated responses upon reexposure to the Ag later in life (3, 4, 5, 7, 11). However, a few cells, although exhibiting low CD40L expression, may be exposed to suboptimal amounts of IL-12 and undergo Th1 differentiation. Because of the insufficient CD40L expression, these cells, when recalled with Ag at adult life, may not proliferate or progress into IFN-{gamma} production.

As stated above, the splenic T cells, although producing significant amounts of IL-2, are unable to up-regulate functional IL-2R and use such IL-2 for responsiveness. Because IL-2 plays a critical role in the induction of IL-12R expression (42) and IL-12 restores splenic T cells responses (Fig. 1Go), it is logical to envision a dual control of IFN-{gamma} production emanating from both IL-2R and IL-12R. Investigation of this issue was facilitated by IL-15, a cytokine defined to support division (43) and to maintain homeostasis (44) of memory T cells. IL-15 was able to restore proliferation but not IFN-{gamma} production of the splenic T cells (Fig. 11Go). Surprisingly, neither CD40L nor IL-2R was restored by IL-15. However, IL-12, which restored proliferation and IFN-{gamma} production, up-regulated both CD40L and IL-2R expression. Therefore, we concluded that IL-2R expression was required for IL-12-driven production of IFN-{gamma} and predict that CD40L controls splenic T cell responsiveness through IL-12R and IL-2R (42, 45).

The model presented in Fig. 12Go, which we propose for this form of T cell anergy, postulates that interaction of the TCR with MHC-peptide complex triggers up-regulation of CD40L that interacts with CD40 on APCs to initiate IL-12 production (36). However, the splenic anergic T cells are unable to up-regulate CD40L and cannot trigger IL-12 production by the APCs. IL-12, upon binding to IL-12R initiates up-regulation of IL-2R. IL-2R facilitates both IL-2 absorption and, in conjunction with IL-12R, triggers IFN-{gamma} production. However, the splenic T cells were unable to up-regulate functional IL-2R because IL-12R was not turned on because of the lack of IL-12. In contrast, this cytokine/cytokine receptor interplay also influences costimulatory molecules, as the results show that CD40-CD40L interaction and IL-2/IL-12 receptor cross-talk operate through regulation of these molecules (42, 46, 47).



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  		FIGURE 12. Proposed mechanism of splenic anergy.

 
Overall, Ag-induced CD40L deficiency, which has been suggested to support T cell unresponsiveness (39, 48, 49), provides a mechanism for both neonatal tolerance and the novel form of IFN-{gamma}/IL-12-dependent anergy. Moreover, although the lack of IL-2 promotes standard IL-2-dependent anergy (50), diminished CD40L expression leads to defective IL-2R up-regulation, which evolves into an IFN-{gamma}-dependent anergy. Finally, IL-2R seems to control T cell differentiation rather than growth or division as was recently demonstrated in a mouse model of controlled IL-2R expression (51).


   Acknowledgments
 
We thank Matthew Johnson for technical help and Dr. Michael Cancro (University of Pennsylvania, Philadelphia, PA) for providing 3/23 hybridoma.


   Footnotes
 
1 This work was supported by startup funds (to H.Z.) from the University of Tennessee (Knoxville, TN). Back

2 Current address: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N314, Bethesda, MD 20892-1892. Back

3 Current address: Beirne B. Carter Center for Immunology Research, University of Virginia, 400 Lane Road, MR-4 Building, P.O. Box 801386, Charlottesville, VA 22908-1386. Back

4 Address correspondence and reprint requests to Dr. Habib Zaghouani, University of Tennessee, Department of Microbiology, M409 Walters Life Sciences Building, Knoxville, TN 37996. Back

5 Abbreviation used in this paper: PLP, proteolipid protein; EAE, experimental allergic encephalomyelitis; CD40L, CD40 ligand; DC, dendritic cells. Back

Received for publication November 3, 2000. Accepted for publication February 20, 2001.


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 Introduction
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 Discussion
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