HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grundström, S. Articles by Sundstedt, A. Search for Related Content PubMed PubMed Citation Articles by Grundström, S. Articles by Sundstedt, A.

IL-2 Unresponsiveness in Anergic CD4⁺ T Cells Is Due to Defective Signaling Through the Common -Chain of the IL-2 Receptor¹

Susanna Grundström^2,*, Mikael Dohlsten and Anette Sundstedt

^* Active Biotech Research Center, Lund, Sweden; and Department of Cell and Molecular Biology, Section for Tumor Immunology, Lund University, Lund, Sweden

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Repeated administration of the superantigen staphylococcal enterotoxin A to mice transduces a state of anergy in the CD4⁺ T cell compartment, characterized by inhibition of IL-2 production and clonal expansion in vivo. In contrast to what has been reported on anergic T cell clones in vitro, culture of in vivo anergized CD4⁺ T cells in the presence of exogenous IL-2 did not overcome the block in responsiveness. In this study, we demonstrate that CD4⁺ T cells from mice anergized with staphylococcal enterotoxin A also exhibit a reduced proliferative capacity in response to IL-7 and IL-15, cytokines that share a common -chain with the IL-2R. Flow-cytometric analysis revealed only modest changes in the expression of the different IL-2R chains. In a number of experiments, our results also provide evidence that excludes a major role of the IL-2R -chain in this system. According to these results, the inability of anergic cells to respond to IL-2 is not mainly due to a down-regulation of the high affinity IL-2R, but to a perturbation in intracellular signaling. Our study confirmed that the activation and tyrosine phosphorylation of Janus-associated kinase 3 and STAT5 were considerably weaker after anergy induction. Moreover, anergic CD4⁺ T cells showed significantly reduced DNA-binding ability to STAT5-specific elements. Taken together, we suggest that the observed IL-2 unresponsiveness in anergic CD4⁺ T cells could be due to a defect in signaling through the common -chain of the IL-2R.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
An efficient immune system depends on the establishment of tolerance to tissue-specific self Ags, while retaining reactivity to foreign pathogens. Physical elimination of autoreactive T cells during thymocyte development is the primary mechanism used by the immune system to establish self-tolerance (1). However, not all self peptides are present in the thymus. T cell tolerance to peripheral Ags is achieved in many ways, including ignorance of tissue-specific Ags (2), induction of clonal deletion and anergy (3), down-modulation of TCR (4), and possibly by evoking a T cell suppressor response (5).

T cell anergy is a functionally defined state of hyporesponsiveness in which T cells neither proliferate nor produce IL-2 following subsequent TCR ligation. Anergy was first described in mouse T cell clones, and shown to be due to a block in Ag receptor-generated signals as a result of Ag recognition in the absence of costimulation and IL-2 (6). Thus, functional responses to Ag (signal 1) require second signals provided by costimulators and/or growth factors, and signal 1 alone leads to anergy. It is well established that the anergic state induced in Th1 clones in vitro can be reversed by the addition of IL-2 (7). The reversal was evident both at the level of cytokine production and transcriptional activation of the IL-2 gene (7, 8). The finding that IL-2 could reverse the unresponsive state has led to the questioning of whether the anergy observed in the T cell clones represents a true physiological state. However, anergy has also been demonstrated in several in vivo models, including the injection of superantigens (9, 10, 11) and adoptive transfer of T cells from TCR-transgenic mice (12, 13). Jenkins and coworkers (14) recently provided direct evidence that functionally impaired TCR-transgenic CD4⁺ T cells persist in vivo following induction of peripheral tolerance. These studies confirm that anergy may function as a peripheral tolerance mechanism in vivo.

Restimulation of the T cells anergized by superantigen in vivo with anti-CD3 resulted in marginal levels of IL-2 and a block in proliferation (11). However, in contrast to what was evident in the anergized T cell clones, addition of IL-2 only partially reversed the anergy, suggesting that IL-2R signaling was affected as well (10, 15). We have previously shown that both CD4⁺ and CD8⁺ T cells exhibit a reduced capacity to proliferate in response to the superantigen staphylococcal enterotoxin (SEA)³ in vitro after repeated SEA stimulations in vivo (16, 17). Addition of IL-2 restored the proliferative capacity in the CD8⁺ compartment (18), and the ability of CD8⁺ T cells to synthesize TNF and IFN- was also partly restored in the presence of IL-2, which indicated that the reduced response was due mainly to lack of IL-2. Interestingly, this was not observed in the CD4⁺ T cell compartment, whih was resistant to stimulation with IL-2 (17). In addition, the CD4⁺ T cells failed to produce IL-2, which is a hallmark of anergy. The IL-2R expression was not significantly altered, which favored the proposal of a signal transduction defect.

The high affinity receptor of IL-2 consists of a -, ß-, and a -chain, while the intermediate receptor consists of the ß- and the -chain only (19). Normally, there is a constitutive expression of the -chain on resting T cells, low expression of the ß-chain that is enhanced by stimulation, while the -chain is absent on resting cells, but inducible upon activation (20). The growth signal of IL-2 is transduced by either the high or intermediate affinity IL-2R complex. Experiments in transgenic mice expressing the human IL-2R ß-chain have revealed that proliferation of CD4⁺ T cells is much more strictly regulated than that of CD8⁺ T cells (21). Although the CD4⁺ T cells expressed the transgenic human IL-2R ß-chain as well as the endogenous -chain on their surface and bound IL-2, no proliferation was observed. By contrast, CD8⁺ T cells and T cells proliferated in response to IL-2, suggesting that CD4⁺ T cells may require another triggering signal to respond to IL-2. Indeed, when the CD4⁺ T cells were stimulated through the TCR, the cells proliferated to IL-2. On the other hand, CD8⁺ T cells, NK cells, and T cells can respond to IL-2 even in the absence of additional stimulations (21). The observed differential responsiveness to IL-2 may reflect different functional roles in immunological responses, and suggests the existence of distinct triggering signals in CD4⁺ vs CD8⁺ T cells that are of physiological importance.

In this study, we have addressed the question of differential IL-2 responsiveness in CD4⁺ T cells activated or anergized by SEA in vivo. Data suggest that induction of IL-2 unresponsiveness in the CD4⁺ compartment includes a block in IL-2R signaling. Such as many other cytokine receptors, the IL-2R does not possess any enzymatic activity (22), but generates proliferative signals in T cells by ligand-induced heterodimerization of the IL-2Rß- and the IL-2R -chain. This triggers a signaling cascade known as the JAK/STAT pathway, which is commonly used by many different cytokines and IFNs (23, 24). We provide evidence that the anergic CD4⁺ T cells exhibit reduced activation of JAK3 and STAT5, suggesting that this decrease in activation could be the result of a signaling defect related to the common -chain (_c-chain). Accordingly, the anergic T cells failed to respond to IL-7 and IL-15, which utilize the same _c-chain. In conclusion, these results suggest that in vivo anergized CD4⁺ T cells not only are defective in their ability to synthesize IL-2, but also express a perturbed responsiveness for signals through the IL-2R. In parallel, it was recently proposed that anergic T cells might act as suppressor cells by competing for the membrane of the APC and for locally produced IL-2 (25).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals and treatment

TCR Vß3 transgenic mice expressing a rearranged genomic clone of the 2B4 ß-chain gene under the influence of an inserted Ig heavy chain enhancer (26) were generously provided by Dr. M. Davis (Stanford, CA). All animals were bled and typed for transgene expression by FACS analysis. CD4⁺ T cells of the TCR Vß3 mice were >95% TCR Vß3⁺. rSEA was expressed in Escherichia coli and purified to homogeneity, as described previously (27). Ten micrograms of SEA in 0.2 ml PBS with 1% normal syngeneic serum or PBS alone was injected i.v. at 4-day intervals. At different time points after the last SEA injection, the mice were sacrificed, spleens were removed, and further analyses were made on single cell suspensions.

Reagents

Mouse rIL-2 was obtained from Boehringer Mannheim (Mannheim, Germany), mouse rIL-7 was obtained from PharMingen (San Diego, CA), and simian rIL-15 was obtained from Genzyme Diagnostics (Cambridge, MA). IL-2-F42A and IL-2-F42K contain point mutations in the binding site to IL-2R -chain that substitute phenylalanine for alanine and lysine, respectively, at the 42nd amino acid residue, as previously described (28, 29). A DNA sequence coding for human IL-2 was introduced in a previously reported C215Fab-SEA E. coli expression vector, coding for secreted Fab-IL-2 products (30). Fab-conjugated wild-type IL-2 was used as a control. PMA and ionomycin were obtained from Sigma-Aldrich (St. Louis, MO). The mAbs directed to murine CD4, TCR Vß3, CD8, CD25, CD122, and the _c-chain of the IL-2R were purchased from PharMingen. Polyclonal Abs to JAK1 and JAK3 were from Upstate Biotechnology (Lake Placid, NY). Polyclonal Abs to STAT5, STAT5A, STAT5B, and STAT3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and mAbs to STAT5 and STAT3 were purchased from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine Abs directed to phospho-STAT3 and phospho-STAT5 were obtained from New England Biolabs (Beverly, MA) and Zymed Laboratories (San Francisco, CA), respectively. Anti-phospho-STAT5-specific mAb and polyclonal STAT5A, STAT5B, and STAT3 Abs used for EMSA were from Zymed Laboratories.

Cell separation

Splenocytes were prepared from mice injected i.v. with SEA or PBS at different times before analysis. Purified CD4⁺ T cells (>95% CD4⁺, as determined by FACS analysis) and CD8⁺ T cells (>85% CD8⁺) were obtained by positive selection using magnetic beads coated with anti-CD4 mAb or anti-CD8 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions.

Assay for DNA synthesis

Purified CD4⁺ T cells were plated into 96-well tissue culture plates using 2 x 10⁵ cells/0.2 ml R10 (RPMI 1640 supplemented with glutamine, HEPES, gentamicin, 2-ME, sodium pyruvate, and 10% FCS) medium and were analyzed for uptake of [³H]thymidine in the presence or absence of SEA, IL-2, IL-2-F42A, IL-2-F42K, or PMA and ionomycin at indicated concentrations. In the presence of SEA, irradiated Raji cells were used as APCs. After different times of culture, [³H]thymidine was added to the cultures, which were harvested 4 h later. The cells were harvested onto glass fiber filters, [³H]thymidine incorporation was measured in a scintillation counter, and the results were expressed as mean cpm from triplicate cultures. SDs were routinely less than 10% of the mean.

Assay for IL-2

The production of IL-2 in culture supernatants was determined using a specific ELISA from PharMingen, according to the instructions of the manufacturer.

Analysis by flow cytometry

Flow-cytometric analysis was performed according to standard settings on a FACSort flow cytometer (Becton Dickinson, Mountain View, CA).

Preparation of cellular extracts

Whole cell extracts for immunoprecipitations and Western blots were made from 7 to 12 x 10⁶ purified CD4⁺ T cells. In individual experiments, the same cell numbers from each group were used for extraction. Before stimulation with IL-2, the cells were starved in R1 medium for 2 h in 37°C. After stimulation for the indicated periods of time in the presence of 20 U/ml IL-2 or medium, the cells were rapidly pelleted and the reactions were stopped by lysing the cells in 500 µl of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 1% Nonidet P-40 containing the following inhibitors: 5 mM p-nitrophenylphosphate, 10 mM ß-glycerophosphate, 1 mM Na₃VO₄, 1 mM PMSF, 5 mM NaF, and a protease inhibitor mixture (Boehringer Mannheim)). The cells were lysed on ice for 30 min with occasional mixing. The extracts were spun for 10 min in an Eppendorf centrifuge at 4°C to pellet cellular debris. The supernatants were removed and frozen instantly in liquid nitrogen and stored at -70°C.

Immunoprecipitation and Western blotting

Protein A-Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden) beads were preincubated overnight at 4°C rotating with the indicated Abs (3–5 µl/10 x 10⁶ cells). The cell lysates were precleared for 1 h using 50 µl/sample of protein A-Sepharose alone. Ab-conjugated protein A was then added to each sample and incubated for 2 h rotating at 4°C. The supernatants were precleared once more, and protein A conjugated with a different Ab was added to the samples for sequential immunoprecipitations. The immunoprecipitated proteins were pelleted and washed three times in lysis buffer. The proteins were then extracted by boiling the pelleted proteins in 2x SDS sample buffer for 5 min. After boiling, the samples were centrifuged for 5 min at 4°C and the supernatants were subjected to analysis by SDS-PAGE using 4–12% Bis-Tris gels (Novex, San Diego, CA). The separated proteins were electrophoretically transferred to nitrocellulose membranes by semidry blotting. The membranes were blocked for 1 h at room temperature in blocking solution (3% skimmed milk powder in PBS) and subsequently probed with indicated Abs (1 µg/ml) in blocking solution overnight at 4°C. Blots were washed three times in 0.05% Tween-20 in PBS. The membranes were incubated with anti-rabbit or anti-mouse Ig-peroxidase-linked species-specific F(ab')₂ fragments (Amersham Life Science, Buckinghamshire, U.K.) in blocking buffer for 1 h. Blots were then washed twice with 0.05% Tween 20 in PBS and once with PBS. The immune complexes were visualized using enhanced chemiluminescence detection (Amersham Life Science).

Immunocomplex protein kinase assay

Precleared cellular extracts were incubated with polyclonal Ab to JAK1 or JAK3 (Upstate Biotechnology) for 3 h at 4°C with gentle rotation. Protein A-Sepharose was added, and the extracts were incubated for an additional hour. Immune complexes were washed twice in ice-cold lysis buffer and then twice in ice-cold kinase buffer (20 mM HEPES, pH 7.6, 50 mM NaCl, 6 mM MnCl₂, 5 mM MgCl₂, 1 mM DTT, 1 mM EDTA, 1 mM p-nitrophenylphosphate, 10 mM ß-glycerophosphate, and 0.1 mM Na₃VO₄), and then assayed for enzyme activity in the context of autophosphorylation. The beads were pelleted and resuspended in 30 µl of kinase buffer containing 1 µM ATP and 1 µCi [-³²P]ATP. Incubations were conducted for 20 min at 30°C, then reactions were stopped by addition of 4x SDS sample buffer. Autophosphorylation was analyzed by 4–12% SDS-PAGE, followed by autoradiography of the dried gel.

Preparation of nuclear extracts

Nuclear extracts for gel-shift and supershift analyses were made from 7 to 12 x 10⁶ purified CD4⁺ T cells. After in vitro stimulation for the indicated periods of time in the presence of 20 U/ml IL-2 or medium, the cells were washed twice with ice-cold PBS and the cell pellet was resuspended in in 400 µl of cold buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and the following inhibitors: 1 mM DTT and a protease inhibitor mixture). The T cells were allowed to swell on ice for 15 min, followed by the addition of 25 µl 10% solution of Nonidet P-40, and the tube was vortex mixed vigorously for 10 s. The homogenate was centrifuged and the nuclear pellet was resuspended in 50 µl of ice-cold buffer C (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and the following inhibitors: 1 mM DTT and a protease inhibitor mixture), and the tube was agitated for 30 min at 4°C on a shaking platform. The nuclear extracts were centrifuged for 5 min at 4°C, and the supernatants were frozen in aliquots at -70°C. Protein concentrations of the extracts were measured by the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Electrophoretic mobility shift assay (EMSA)

The STAT5-binding consensus oligonucleotide used was derived from the bovine ß-casein promoter and contained the following sequence: 5'-AGATTTCTAGGAATTCAAATC-3'. The probe was end labeled with [-³²P]ATP using T4 polynucleotide kinase (Promega), according to instructions from the manufacturer, and purified on 6% DNA retardation gels (Novex) in 0.5x TBE. Binding reactions were performed with the same amount of protein in each reaction (2–2.5 µg) in binding buffer (100 mM KCl, 20 mM Tris, pH 7.5, 20 mM HEPES, 1 mM DTT, 1 mM EDTA, and 20% glycerol) and 2 µg poly(dI-dC) (Pharmacia Biotech). The reactions were incubated at 37°C for 30 min with 30,000 cpm double-stranded ³²P-labeled oligonucleotides. The samples were electrophoresed on 4–12% TBE gels in 1x TBE. The gels were dried under vacuum and exposed to autoradiography at -70°C. For supershift analyses, the nuclear extracts were incubated on ice with specific Abs for 20–25 min prior to the addition of labeled oligonucleotide.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proliferation and IL-2 production are abrogated in CD4⁺ T cells after repeated SEA treatments in vivo

In this study, we used TCR Vß3 transgenic mice as a sensitive model (31) to investigate intracellular signaling events in CD4⁺ T cells activated or anergized by SEA in vivo. In these mice, all T cells express a TCR Vß-chain specific for SEA. Three SEA injections were administrated to the mice with a 4-day interval to induce anergy or one injection to induce T cell activation. Spleens were removed at different time points after the last SEA injection, whereupon CD8⁺ or CD4⁺ T cells were purified and used for further analysis. Administration of SEA in vivo resulted in an initial hyperactivation, and induction of IL-2 production and proliferation in the CD4⁺ subset in response to challenge with SEA-coated APC in vitro (Fig. 1A). The proliferative response of these cells was comparable with the proliferation of CD4⁺ T cells from untreated mice (data not shown) (17). However, the kinetics of in vivo SEA-treated compared with untreated T cells is different; in vivo activated T cells peak at 24 h compared with untreated cells having their maximal response at 72 h (17). The CD8⁺ T cells also proliferated in response to SEA, but were more dependent on exogenously provided IL-2 for their proliferation (Fig. 1A). Repeated stimulations with SEA inhibited proliferation (Fig. 1A) and blocked IL-2 production (Fig. 1B) by the CD4⁺ T cells. Interestingly, addition of exogenous IL-2 did not restore the proliferative response in the CD4⁺ T cell compartment, while the CD8⁺ T cells maintained the ability to respond to IL-2 (Fig. 1A), suggesting the existence of a possible intrinsic difference in IL-2 signaling between CD4⁺ and CD8⁺ T cells. Because repeated stimulations with SEA also induce deletion of some responsive T cells, one explanation for T cells not responding to SEA in vitro could be that they may be TCR Vß3 negative rather than anergic. However, FACS analysis of the CD4⁺ T cells revealed that Vß3 expression was similar both quantitatively and qualitatively comparing the different treatment protocols (data not shown) (17).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Reduced proliferation and IL-2 production in anergic CD4+ T cells. TCR Vß3 transgenic mice were injected i.v. one or three times with 10 µg of SEA at 4-day intervals. PBS-treated mice were used as controls. Spleens were removed 48 h after the last SEA injection, and purified CD4+ and CD8+ T cells were prepared for in vitro cultures. A, Proliferation of T cells after 1 day of culture in medium, SEA (1 µg/ml), or SEA in combination with rIL-2 (20 U/ml). Irradiated Raji cells were used as APC. B, IL-2 protein levels in culture supernatants. SDs were less than 10% of the mean. One of three similar experiments is shown.

To further address the defective IL-2 responsiveness in the anergic CD4⁺ T cells, we used a less complicated model in which the T cells were cultured in IL-2 alone after in vivo treatment with SEA. The system was set up in the absence of APC and SEA to avoid disturbance from signals generated by TCR triggering. IL-2, IL-7, and IL-15 share a _c-chain of their ligand receptor (24). IL-2 and IL-15 also share a common ß-chain, while the -chain of the IL-2R is unique (32). These cytokines exhibit pleiotropic and redundant functions, including being potent inducers of T cell proliferation (24). When purified CD4⁺ T cells from SEA-treated mice were cultured in the presence of these cytokines, poor induction of proliferation was observed in the anergic T cells to IL-2, IL-7, as well as IL-15 (Fig. 2, A–C). The overall proliferative response of CD4⁺ T cells to IL-7 and IL-15 was weaker than the response to IL-2, which might be due to IL-2 binding with higher affinity to the ß-receptor than IL-7 and IL-15. Interestingly, anergized CD4⁺ cells maintained the ability to proliferate in response to IL-2 up to 12 h after in vivo treatment, but at later time points the proliferation was dramatically repressed (Fig. 2A). The observed block in proliferation of anergic CD4⁺ T cells to IL-2, IL-7, as well as IL-15 implied the existence of a defect in the signaling through the _c-chain of these cytokine receptors.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Reduced proliferative capacity of anergic CD4+ T cells to IL-2, IL-7, and IL-15. TCR Vß3 transgenic mice were treated one or three times with 10 µg SEA at 4-day intervals. PBS-treated mice were used as controls. Spleens were removed at different time points after the last SEA injection, and purified CD4+ T cells were prepared for in vitro cultures. Proliferation of CD4+ T cells after 1 day of culture in A, IL-2 (20 U/ml); B, IL-7 (0.02 µg/ml); or C, IL-15 (0.1 µg/ml). SDs were less than 10% of the mean. One of two similar experiments is shown.

Previous studies have demonstrated that the capacity of T cells to proliferate in response to IL-2 correlates with IL-2R -chain expression (20, 33). To investigate whether a down-regulation of the high affinity receptor could be the explanation for the observed defective IL-2 responsiveness, CD4⁺ T cells were analyzed for expression of the different receptor chains by FACS analysis. The -chain was constitutively expressed and was not modulated by the different treatments (Fig. 3). In contrast, the ß-chain was up-regulated after SEA injection in both activated and anergic CD4⁺ T cells, in particular after 24 h (Fig. 3). Strong induction of -chain expression was observed 12 h after treatment. At 24 h, the expression was down-regulated in both groups, but a more pronounced decrease was observed in anergic CD4⁺ T cells (Fig. 3). Interestingly, the proliferative capacity of the activated T cells to IL-2 was as strong at 24 h after SEA injection compared with the early time point (Fig. 2A), suggesting that a down-regulation of the high affinity receptor may not be solely responsible for the impaired proliferation of anergized CD4⁺ T cells.

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Kinetics of IL-2R chain expression on activated and anergic CD4+ T cells. TCR Vß3 transgenic mice were injected one or three times i.v. with 10 µg of SEA at 4-day intervals. Spleens were removed 12 and 24 h after the last SEA injection, and single spleen suspensions were prepared. These were double stained for CD4 and IL-2R (CD25)-, IL-2Rß (CD122)-, or IL-2R -chain expression and analyzed by FACS. Cells were gated for CD4 expression, and the log fluorescence intensities of the indicated markers are presented in the histograms. The dotted line represents the PBS-treated control group, the bold line shows one SEA injection, and the filled line three SEA injections. One of three similar experiments is shown.

Differential expression of IL-2R -chain is not the main explanation for the defective proliferation of anergic CD4⁺ T cells

To investigate whether the level of IL-2R -chain expression was crucial for the anergic phenotype, we correlated the time point for equal expression of this chain between activated and anergized CD4⁺ T cells. We found that 29 h after the last SEA injection, the activated CD4⁺ T cells have the same surface expression of the IL-2R -chain as the anergized T cells have 24 h after the last SEA injection (Fig. 4A). The proliferative capacity of activated and anergized cells expressing the same levels of IL-2R -chain was still different (Fig. 4B). This result implies that the inability of anergized T cells to proliferate to the same extent as activated T cells is not only dependent on the expression of the IL-2R -chain.

View larger version (9K): [in this window] [in a new window]   FIGURE 4. Equal IL-2R -chain expression cannot restore IL-2 responsiveness in anergic CD4+ T cells. TCR Vß3 transgenic mice were injected one or three times i.v. with 10 µg SEA at 4-day intervals. PBS-treated mice were used as control. Spleens were removed 24 h (3x SEA) and 29 h (1x SEA) after the last SEA injection. A, Single spleen suspensions were prepared and double stained for CD4 and IL-2R (CD25)-chain expression and analyzed by FACS. Cells were gated for CD4 expression, and the log fluorescence intensity of the indicated marker is presented in the histogram. The bold line shows one SEA injection, and the filled line three SEA injections. B, Purified CD4+ T cells were prepared and put into culture for 1 day in medium or rIL-2 (20 U/ml). SDs were less than 10% of the mean. One of two similar experiments is shown.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. High concentrations of IL-2 cannot compensate for the defect in IL-2 responsiveness in anergic CD4+ T cells. TCR Vß3 transgenic mice were injected one or three times i.v. with 10 µg SEA at 4-day intervals. PBS-treated mice were used as control. Spleens were removed 24 h after the last SEA injection, and purified CD4+ T cells were prepared for in vitro cultures. T cells were cultured for 24 h in medium or stimulated with different concentrations of IL-2 (1, 10, 100, 300, 1000 U/ml). After 24 h, [3H]thymidine was added to the cultures, which were harvested 4 h later, and the 3H incorporation was measured in a beta counter. SDs were less than 10% of the mean. One of two similar experiments is shown.

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Similar decrease in proliferation of both activated and anergic CD4+ T cells in response to IL-2R -chain mutants. TCR Vß3 transgenic mice were injected one or three times i.v. with 10 µg SEA at 4-day intervals. PBS-treated mice were used as control. Spleens were removed 24 h after the last SEA injection, and purified CD4+ T cells were prepared for in vitro cultures. T cells were cultured for 24 h in medium or stimulated with IL-2 (10-9 M), Fab-IL-2 (10-9 M), IL-2-F42A (10-9 M), IL-2-F42K (10-9 M), or PMA (P, 5 ng/ml) in combination with ionomycin (I, 250 ng/ml) and IL-2 (20 U/ml). After 24 h, [3H]thymidine was added to the cultures, which were harvested 4 h later, and the 3H incorporation was measured in a beta counter. SDs were less than 10% of the mean. One of four similar experiments is shown.

Anergy in CD4⁺ T cells cannot be reversed by long-term culturing in IL-2

To be able to compare the acute response to IL-2 of anergized CD4⁺ T cells with a possible long-lasting effect that could reverse the unresponsive state, splenocytes from mice anergized (3x SEA) with SEA or untreated controls were put into in vitro culture in the presence of IL-2 for 4 days. The viable CD4⁺ T cells were then restimulated in vitro with either SEA or SEA + IL-2. According to this experiment, the anergic state induced in this model is persistent and cannot be reversed by long-term culturing in IL-2 (Fig. 7).

View larger version (13K): [in this window] [in a new window]   FIGURE 7. Long-term culturing in IL-2 cannot restore IL-2 responsiveness in anergic CD4+ T cells. TCR Vß3 transgenic mice were injected three times i.v. with 10 µg SEA at 4-day intervals. PBS-treated mice were used as control. Spleens were removed 24 h after the last SEA injection and put into bulk culture in the presence of rIL-2 (20 U/ml) for 4 days. Viable CD4+ T cells were purified and prepared for in vitro cultures. T cells were cultured for 24 h in medium or stimulated with SEA (1 µg/ml) or SEA in combination with rIL-2 (20 U/ml). Irradiated Raji cells were used as APC. SDs were routinely less than 10% of the mean. One of two similar experiments is shown.

Binding of IL-2 to its receptor induces sequential phosphorylations of tyrosine residues on the receptor chains, the JAK kinases, and finally the STAT proteins (22, 23, 24). It is well established that in response to IL-2, JAK1 and JAK3 are stimulated to activate STAT3 and STAT5 (35). To investigate whether there was a difference in the expression or degree of activation of these proteins, CD4⁺ T cells from mice activated (1x SEA) or anergized (3x SEA) by SEA were stimulated in vitro with IL-2 for different times and analyzed for the expression and phosphorylation status of the STAT proteins. A peak in STAT activation was evident at 10–15 min, and tyrosine phosphorylation was almost absent within 1 h (Fig. 8A). The primary STAT protein being activated in this system was STAT5, while surprisingly no tyrosine phosphorylation of STAT3 was observed (Fig. 8A). Furthermore, the phosphorylation of STAT5 was considerably weaker in the anergic CD4⁺ T cells, particularly at later time points (Fig. 8A). To investigate a possible difference in the activation between the two STAT5 isoforms, immunoprecipitations with Abs specific for STAT5A and STAT5B were made. Both STAT5A and STAT5B became activated upon IL-2 stimulation (Fig. 8B). However, STAT5A was phosphorylated to some greater extent than STAT5B. More importantly, reduced levels of both phosphorylated STAT5A and STAT5B were observed in the anergic CD4⁺ T cells (Fig. 8B). Thus, this suggests that inhibition of STAT5A and B phosphorylation contribute to the reduced activation of total STAT5.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Reduced tyrosine phosphorylation of STAT5 in anergic CD4+ T cells. One or three i.v. injections of 10 µg of SEA to TCR Vß3 transgenic mice were administrated with 4-day intervals. Spleens were removed 24 h after the last SEA injection, and single spleen suspensions were prepared. Purified CD4+ T cells were incubated with medium or IL-2 (20 U/ml) in vitro for indicated periods of time. Whole cell extracts were analyzed by immunoprecipitations and Western blotting for the expression of tyrosine-phosphorylated STAT5 and STAT3 (A) and STAT5A and STAT5B (B). The STAT proteins are 92–95 kDa in size. One representative experiment of six performed is shown.

It has been proposed that JAK3 associates with the _c-chain of the IL-2R and thereby prevents the induction of anergy, while JAK1 associates with the IL-2R ß-chain and propagates a mitotic signal (36, 37). The -chain of the IL-2R participates in ligand binding, but not in signal transduction. To investigate whether JAK kinase activity was reduced in parallel to STAT5, we used specific immunocomplex protein kinase assays measuring autophosphorylation. Analysis of JAK3 activity in response to IL-2 in purified CD4⁺ T cells revealed that a significant response was rapidly induced in activated cells with a peak in activation at about 5 min (Fig. 9). In contrast, there was only a minor activation-induced increase of JAK3 activity in the anergic cells (Fig. 9). There was no difference in the protein expression of JAK3 in activated compared with anergized T cells (Fig. 9). Furthermore, there were no signs of IL-2-induced activation of JAK1, which could only be detected at the protein level by Western blotting (data not shown). These findings imply that JAK3, which associates with the _c-chain of the IL-2R, is the essential JAK kinase being activated in this model. Thus, the defective STAT5 activation in anergized CD4⁺ T cells may be the result of deficient JAK3 activation.

View larger version (34K): [in this window] [in a new window]   FIGURE 9. Reduced kinase activity of JAK3 in anergic CD4+ T cells. TCR Vß3 TG mice were injected i.v. one or three times with 10 µg of SEA at 4-day intervals. Spleens were removed at 24 h after the last SEA injection, and protein extracts from purified CD4+ T cells were prepared after stimulation with IL-2 (20 U/ml) for indicated periods of time. The protein extracts were assayed for JAK3 activity using an immunocomplex protein kinase assay analyzing autophosphorylation. Radioactive proteins were transferred to a chromatography filter and after drying, [32P]ATP incorporation was determined by autoradiography. The total JAK protein expression was assayed by Western blotting. The JAK proteins are 125 kDa in size. One representative experiment of three is shown.

To evaluate whether the parallel decline in proliferation and decreased phosphorylation of STAT5 reflected a change in binding to specific promoter regions, we performed gel-shift analysis using a bovine ß-casein promoter sequence encoding a STAT-binding consensus motif (38). Upon phosphorylation, STAT proteins are able to dimerize and to be translocated from the cytoplasm to the nucleus, where these protein complexes exert their function as transcription factors (22, 23, 24). The STAT proteins and their possible coactivators are critical and responsible for the specificity of the pathway and decide which genes will be transcriptionally active. Spleens were removed at 24 h after the last SEA injection, and nuclear extracts were prepared from purified CD4⁺ T cells after in vitro stimulation with IL-2. Pronounced binding activity to the ß-casein promoter element was observed in the activated T cells in response to IL-2 (Fig. 10A). In contrast, the anergic CD4⁺ T cells contained only low levels of DNA binding, especially after longer incubations with IL-2 (Fig. 10A). Extracts of CD4⁺ T cells from PBS-treated mice contained no ß-casein binding (data not shown). Supershift analysis using different STAT Abs was performed to confirm the identity of the gel-shift bands. A STAT5A-specific Ab shifted the majority of the binding activity, but there was also a great shift in the presence of a STAT5B-specific Ab (Fig. 10B). The STAT5A/B-specific supershift Ab completely shifted the band, while no shifts were observed using a STAT3 Ab or control IgG (Fig. 10B). The supershift pattern was similar but significantly weaker in the anergized CD4⁺ compartment compared with the activated T cells. These results confirm a reduced activation of the JAK3/STAT5 pathway in the anergic CD4⁺ T cells, possibly giving rise to decreased transcription of genes essential for proliferation.

View larger version (37K): [in this window] [in a new window]   FIGURE 10. Reduced DNA-binding activity of STAT5 in the anergic CD4+ T cells. TCR Vß3 transgenic mice were injected i.v. one or three times with 10 µg of SEA at 4-day intervals. Spleens were removed at 24 h after the last SEA injection, and nuclear extracts were prepared from purified CD4+ T cells stimulated with IL-2 (20 U/ml) for the indicated times. A, EMSA of nuclear extracts (2.5 µg/lane) and [-32P]ATP-labeled oligonucleotide specific for a STAT-binding motif. B, Supershift analysis of the DNA-binding complexes. Nuclear extracts from CD4+ T cells stimulated with IL-2 (20 U/ml) for 15 min were incubated in the absence or presence of antisera directed against STAT5A, STAT5B, STAT5A/B, STAT3, or the irrelevant transcription factor Fra1. One of five similar experiments is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The concept of T cell anergy is both complicated and controversial. In the past, a number of different protocols for induction of anergy both in vitro and in vivo have been used trying to elucidate the mechanisms underlying this state and the functional relevance of T cell anergy (39). One intriguing aspect of anergy is its ability to be reversed by stimulation of the T cell clones with IL-2 (7). A possible explanation could be that the clones abnormally express levels of the high affinity IL-2R on their surface. In this line, it was demonstrated that activating Abs against the _c-chain could antagonize anergy induction in human T cells (36). The ability to reverse anergy raised the question of the in vivo relevance of this state. It was argued that it would be dangerous to keep these anergic cells around, where they might be reactivated through IL-2 produced by T cells specific for foreign Ags, leading to autoreactivity. However, in vivo studies with superantigens (9, 10, 11) and adoptive transfer of T cells from TCR-transgenic mice (12, 13) have demonstrated that anergy can be induced in vivo. Interestingly, at least one functional difference seems to exist between T cells anergized in vitro vs in vivo. As stated above, T cells rendered anergic in vitro will proliferate when cultured with exogenous IL-2 (7), while we and others have found that anergic T cells from in vivo superantigen treatment cannot utilize exogenous IL-2 (9, 11, 17). The inability to induce high expression levels of IL-2R -chain may contribute to this phenomenon (40). However, the significance of the IL-2 utilization discrepancy is not yet completely clear, but may reflect different degrees of functional anergy or deficiencies of in vitro systems. Recently, Pape et al. provided evidence that functionally impaired CD4⁺ T cells persist in vivo following induction of peripheral tolerance in an adoptive transfer model (14). Interestingly, the clonal expansion defect of the unresponsive T cells was not corrected by T cell growth factors provided by bystander T cells responding in the same lymph node (14), suggesting that this form of tolerance would not be easily broken in vivo. By analogy, T cells anergized by superantigen in vivo in this study were resistant to growth factors such as IL-2, IL-7, and IL-15 (Fig. 2).

It has been postulated that peripheral tolerance is a multistep mechanism ranging from the most superficial form of tolerance resulting in autoimmunity and the most severe form resulting in deletion of tolerant T cells. This concept was first presented by Arnold and coworkers, who demonstrated that, depending on the amount of transgene expressed, different levels of T cell tolerance in vivo existed, ranging from TCR/CD8 down-regulation through anergy to deletion (41, 42). In conjunction with this, in a model in which T cell anergy was induced by T-T presentation of a specific Ag in the absence of professional APC, distinct anergic phenotypes were induced depending on the Ag dose with which T cells were incubated (43). A low Ag dose induced hyporesponsiveness in T cells without immune suppression, while higher Ag doses resulted in additional immunoregulatory functions (43). Incubation with a high Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2 (43). In this line, others have shown that anergic T cell clones can act as suppressor cells both in vitro and in vivo (39, 44). In vivo, anergic T cells could prolong the time of graft survival substantially and also possessed the ability to inhibit responses of autoreactive and polyclonal T cells (44, 45). It has not been established how the anergic T cells accomplish these suppressive effects, although there are several proposed mechanisms by which these regulatory T cells may act. Secretion of antiinflammatory cytokines such as IL-4, IL-10, and TGF-ß is one possibility. There is evidence that IL-10 plays an important role in the maintenance of T cell anergy (46) and of IL-10-producing T cells that persist after induction of anergy by superantigen that exerts immunoregulatory functions in vivo (16). Another possibility is that cell-cell contact is essential for creating an immunosuppressive environment. Anergic T cells have been shown to suppress the function of bystander T cells recognizing another epitope, but close contact between APC, responder T cell, and anergic T cell was required and also the presence of the specific Ag of the anergic T cell (45). The anergic T cells could compete for the APC surface and local growth factors like IL-2 or they might deliver inhibitory signals to APC or a nearby responsive T cell (39, 44, 45). Thus, multiple levels of anergy seem to exist, resulting in T cells that can contribute in a persistent and active manner or in a passive way to the regulation of the immune system (43).

In this study, we demonstrate that repeated injections of the superantigen induced a state of anergy in the CD4⁺ T cell compartment characterized by a failure to both produce and respond to IL-2. This may represent one of the nonexclusive levels of anergy. The defective IL-2 responsiveness could relate to deficient expression of the IL-2R (47) and/or a failure to transmit an activation signal intracellularly (10, 15). It has been suggested that diminished proliferation is an indirect mechanism due to defective IL-2R -chain expression that results in loss of the high affinity receptor (20, 47). Impaired IL-2 responsiveness could also involve selective changes in signal transduction and gene regulation in T lymphocytes (10, 15). In a number of experiments, our results provide evidence that exclude a major role of the IL-2R -chain in this system. At a time point when the surface expression of the IL-2R -chain was equal between activated and anergized T cells, the difference in proliferation was still significant. Furthermore, unphysiological concentrations of IL-2 could not compensate for the reduced proliferation of the anergic T cells due to possible signaling through the ß-intermediate receptor. In the presence of IL-2 mutants defective in their affinity for the -chain of the IL-2R, the relative difference in the proliferative capacity between activated and anergic CD4⁺ T cells was similar to that observed using wild-type IL-2. A decrease in the response of both groups was recorded, which verifies the importance of the -chain for the overall response, although it does not appear to be the sole explanation for the IL-2 hyporesponsiveness in the anergic T cells. A moderate down-regulation of the -chain was observed in both groups at 24 h after the last SEA injection. However, no difference in capacity to proliferate was evident in the activated T cells at 12 and 24 h, which further strengthens the conclusion that down-regulation of the -chain is inferior.

Signal transduction after IL-2R triggering is mediated by dimerization of the cytoplasmic domains of the ß- and the -chain to be able to transduce a biological response (36, 37, 48). The cytoplasmic domain of IL-2R constitutively associates with JAK3 (36, 38, 49). Defective _c-JAK3 association has been found in many XSCID patients who suffer from immunodepression due to nonsense mutations in the _c gene (50). In several systems, JAK1 is also predicted to be activated during IL-2 responses and associates specifically with the IL-2R ß-chain (49). Some studies have suggested that heterodimerization of the ß- and the -chain is necessary to bring JAK1 and JAK3 into close proximity and thereby promote cross-activation (37). However, others have shown an independent activation of the different tyrosine kinases. Several lines of evidence indicate that JAK1 is not essential for activation of JAK3 or cell growth signaling by IL-2 stimulation (38, 51). Taken together, these results suggest in some circumstances that activation of JAK3 alone is sufficient to enter the cell into mitosis after stimulation by IL-2. The usage of an autophosphorylation assay enabled us to conclude that there is no IL-2-induced kinase activity of JAK1 as opposed to the pronounced JAK3 activation (Fig. 9). The inducible activity of JAK3 was diminished, but not entirely abrogated, in the anergized CD4⁺ T cells, which is consistent with the block in proliferation. These results suggest that JAK1 is dispensable in this system, while JAK3 and STAT5 activation correlated well with growth promotion. Similarly, others have shown that IL-2 stimulated JAK3 to a significantly larger extent than JAK1 in human T lymphocytes (52).

The finding that JAK1 phosphorylation may not be required for IL-2-induced proliferation in SEA-treated CD4⁺ T cells is compatible with the lack of tyrosine phosphorylation of STAT3 (Fig. 8A). It is claimed that Tyr³³⁸, Tyr³⁹², and Tyr⁵¹⁰ of the cytoplasmic part of the IL-2R ß-chain serve as primary docking sites for STAT5 (24, 53, 54). Binding of STAT5 to these residues mediates the activation of both the STAT5A and the STAT5B isoforms (55). In contrast to data reflecting the selective association of JAK1 and JAK3 with the ß- and the -chain, respectively, it was recently shown that a JAK1-independent JAK3-IL-2Rß association is important for IL-2-induced STAT5 activation (56). When the dual ability of JAK3 to associate with the ß- and the -chain was impaired due to defects in the -chain, JAK3 was still able to associate with IL-2Rß (56). However, this led to a significant reduction in STAT5 activation. In this way, it is plausible that JAK3 normally stabilizes the receptor complex and facilitates downstream signaling.

IL-2-induced phosphorylation of IL-2Rß, JAK1, and STAT5 all require the presence of JAK3 (57). The importance of JAK3 in inducing cell proliferation has been shown using a dominant-negative JAK3 mutant lacking intrinsic kinase activity, in which it was hypothesized that JAK3 may indirectly be involved in the induction of the c-fos gene (58). Along with this, there is also a possibility that STAT5 activation is partly responsible for AP-1 induction through its docking and phosphorylation to the different tyrosine residues located in the IL-2R ß-chain (59). These data indicate that there could be a direct connection in the regulation between deficient IL-2 production and loss of IL-2 responsiveness.

Both STAT5A and STAT5B are activated by IL-2 in lymphocytes (60). Mice deficient in both genes have normal lymphoid development, but STAT5A/B mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression (61). These phenotypes are not seen in mice lacking STAT5A or STAT5B alone, suggesting that the STAT5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells. However, although STAT5 is suggested to play a crucial role in the biological effects of certain cytokines, little is known how STAT5 couples cytokine signals to the cell cycle machinery. STAT5 has been shown to participate in transcriptional regulation of various genes such as ß-casein, p21^WAF1, cytokine-inducible SH2-containing protein, oncostatin M, pim.1, c-fos, Id-1, and IL-2R -chain (62). Tyrosine phosphorylation of STAT5 is vital for its DNA-binding activity, while activation of STAT5 does not require ongoing protein synthesis (63). In this study, we show, using a ß-casein promoter element, that there was a strong reduction in the STAT5 DNA-binding activity of the nuclear extracts from the anergized compared with activated T cells. Several recent studies, using promoter sequences of the IL-2R and the cytokine-inducible SH2-containing protein genes among others, reveal convincing evidence that STAT5 dimers are able to create tetramer structures by interaction on two low affinity binding sites within the promoter, which enhances the transcriptional activity of respective genes (20, 64, 65). The biological significance of the STAT5 tetramers is reflected by the triggering of transcription that is absent or abrogated in the absence of these tetramers. Possibly, several genes important for the growth and proliferation of a T cell contain STAT5-binding motifs in tandem in their promoter sequences, which is crucial for the activation of the gene. There seems to be a biochemical difference between STAT5A and STAT5B in that only STAT5A have the ability to form tetramers (65). Also, the existence of other coactivators and transcriptional factors, e.g., Ets, with binding sites adjacent to STAT5 binding sites, could be important to the transcriptional activity of different genes. Lower abundance of tyrosine-phosphorylated STAT5 means lower ability to form active transcription complexes, and this is most likely what happens in the anergized CD4⁺ T cells. Supershift analyses reveal that there was a total shift of the DNA-binding complexes in the presence of STAT5A/B Ab, while no shifts were observed in the presence of STAT3 or irrelevant Abs. Anti-STAT5A supershifted the complex more readily than STAT5B in accordance with the higher degree of phosphorylation of STAT5A. More importantly, no qualitative difference in the composition of the nuclear extracts was found in anergized compared with activated CD4⁺ T cells. Thus, the difference was quantitative, probably as a consequence from the reduced activation of STAT5 factors.

Several reports have shown that anergic T cells are hyperresponsive to stimulation with IL-2 (45). This is not true in this model, in which anergic CD4⁺ T cells were unable to respond to exogenously provided IL-2. One could speculate that the anergic T cells purposely bind IL-2 to its receptor to keep it away from adjacent T cells and thereby prevent their further activation. Concomitantly, the anergic T cells may be programmed not to be able to respond to the bound IL-2 stimulus. This could be one possible mechanism of many in the multistep regulation of T cell anergy. Although cellular responses to cytokines are tightly controlled, few molecules have been identified that are able to switch these signals off. The suppressors of cytokine-signaling (SOCS) proteins are a new family of negative regulators of cytokine signal transduction (66). The expression of SOCS proteins is induced by cytokine. Once expressed, SOCS down-regulate JAK/STAT pathways, and hence the biological response. Thus, it would be interesting to address whether the anergic T cells express elevated levels of SOCS proteins. Incidentally, IL-10 has been shown to induce expression of SOCS3 in monocytes (67).

In conclusion, we have demonstrated that in vivo anergized CD4⁺ T cells possess a defect in IL-2 responsiveness possibly due to an error located to the _c-chain of the IL-2R. This led to significantly less potent downstream signaling with reduced activation and tyrosine phosphorylation of JAK3 and STAT5. The differences in DNA binding of both STAT5 isoforms in anergized compared with activated CD4⁺ T cells are rather quantitative than qualitative. It is tempting to speculate that induction of IL-2 unresponsiveness in anergic T cells may represent an additional immunoregulatory mechanism.

	Acknowledgments

 
We thank Ulrika Tellström, Christa Edvardsson, Kristina Behm, and Jan Nilsson for excellent technical assistance. We also thank Dr. Annelie Sjöberg, Dr. Göran Forsberg, Dr. Morten Søgaard, and Karin Tsiobanelis for developing and supplying proteins essential to this work.

	Footnotes

 
¹ Part of this work was supported by grants from the Swedish Cancer Society.

² Address correspondence and reprint requests to Susanna Grundström, Active Biotech Research Center, Scheelevägen 22, S-223 63 Lund, Sweden. E-mail address:

³ Abbreviations used in this paper: SEA, staphylococcal enterotoxin A; JAK, Janus-associated kinase; _c-chain, common -chain; SOCS, suppressors of cytokine signaling.

Received for publication June 21, 1999. Accepted for publication November 11, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. King, L. B., J. D. Ashwell. 1994. Thymocyte and T cell apoptosis: is all death created equal?. Thymus 23:209.[Medline]
2. Miller, J. F., W. R. Heath. 1993. Self-ignorance in the peripheral T-cell pool. Immunol. Rev. 133:131.[Medline]
3. Goodnow, C. C.. 1997. Glimpses into the balance between immunity and self-tolerance. Ciba Found. Symp. 204:190.[Medline]
4. Miethke, T., C. Wahl, H. Gaus, K. Heeg, H. Wagner. 1994. Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: dose-response relationship. Eur. J. Immunol. 24:1893.[Medline]
5. Arnon, R., D. Teitelbaum. 1993. On the existence of suppressor cells. Int. Arch. Allergy Immunol. 100:2.[Medline]
6. Schwartz, R. H.. 1990. A cell culture model for T lymphocyte clonal anergy. Science 248:1349.[Abstract/Free Full Text]
7. Beverly, B., S. M. Kang, M. J. Lenardo, R. H. Schwartz. 1992. Reversal of in vitro T cell clonal anergy by IL-2 stimulation. Int. Immunol. 4:661.[Abstract/Free Full Text]
8. Kang, S. M., B. Beverly, A. C. Tran, K. Brorson, R. H. Schwartz, M. J. Lenardo. 1992. Transactivation by AP-1 is a molecular target of T cell clonal anergy. Science 257:1134.[Abstract/Free Full Text]
9. Rammensee, H. G., R. Kroschewski, B. Frangoulis. 1989. Clonal anergy induced in mature Vß6⁺ T lymphocytes on immunizing Mls-1b mice with Mls-1a expressing cells. Nature 339:541.[Medline]
10. Rellahan, B. L., L. A. Jones, A. M. Kruisbeek, A. M. Fry, L. A. Matis. 1990. In vivo induction of anergy in peripheral Vß8⁺ T cells by staphylococcal enterotoxin B. J. Exp. Med. 172:1091.[Abstract/Free Full Text]
11. Bhandoola, A., E. A. Cho, K. Yui, H. U. Saragovi, M. I. Greene, H. Quill. 1993. Reduced CD3-mediated protein tyrosine phosphorylation in anergic CD4⁺ and CD8⁺ T cells. J. Immunol. 151:2355.[Abstract]
12. Kearney, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[Medline]
13. Rocha, M., A. Kruger, N. Van Rooijen, V. Schirrmacher, V. Umansky. 1995. Liver endothelial cells participate in T-cell-dependent host resistance to lymphoma metastasis by production of nitric oxide in vivo. Int. J. Cancer 63:405.[Medline]
14. Pape, K. A., R. Merica, A. Mondino, A. Khoruts, M. K. Jenkins. 1998. Direct evidence that functionally impaired CD4⁺ T cells persist in vivo following induction of peripheral tolerance. J. Immunol. 160:4719.[Abstract/Free Full Text]
15. Baschieri, S., R. K. Lees, A. R. Lussow, H. R. MacDonald. 1993. Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines. Eur. J. Immunol. 23:2661.[Medline]
16. Sundstedt, A., I. Hoiden, A. Rosendahl, T. Kalland, N. Van Rooijen, M. Dohlsten. 1997. Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo. J. Immunol. 158:180.[Abstract]
17. Sundstedt, A., M. Dohlsten, G. Hedlund, I. Hoiden, M. Bjorklund, and T. Kalland. 1994. Superantigens anergize cytokine production but not cytotoxicity in vivo. [Published erratum appears in 1994, Immunology 82:504.] Immunology 82:117.
18. Sundstedt, A., I. Hoiden, J. Hansson, G. Hedlund, T. Kalland, M. Dohlsten. 1995. Superantigen-induced anergy in cytotoxic CD8⁺ T cells. J. Immunol. 154:6306.[Abstract]
19. Takeshita, T., H. Asao, K. Ohtani, N. Ishii, S. Kumaki, N. Tanaka, H. Munakata, M. Nakamura, K. Sugamura. 1992. Cloning of the chain of the human IL-2 receptor. Science 257:379.[Abstract/Free Full Text]
20. Meyer, W. K., P. Reichenbach, U. Schindler, E. Soldaini, M. Nabholz. 1997. Interaction of STAT5 dimers on two low affinity binding sites mediates interleukin 2 (IL-2) stimulation of IL-2 receptor gene transcription. J. Biol. Chem. 272:31821.[Abstract/Free Full Text]
21. Asano, M., Y. Ishida, H. Sabe, M. Kondo, K. Sugamura, T. Honjo. 1994. IL-2 can support growth of CD8⁺ T cells but not CD4⁺ T cells of human IL-2 receptor ß-chain transgenic mice. J. Immunol. 153:5373.[Abstract]
22. Karnitz, L. M., R. T. Abraham. 1995. Cytokine receptor signaling mechanisms. Curr. Opin. Immunol. 7:320.[Medline]
23. Schindler, C., J. E. J. Darnell. 1995. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621.[Medline]
24. Lin, J. X., T. S. Migone, M. Tsang, M. Friedmann, J. A. Weatherbee, L. Zhou, A. Yamauchi, E. T. Bloom, J. Mietz, S. John. 1995. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 2:331.[Medline]
25. Frasca, L., P. Carmichael, R. Lechler, G. Lombardi. 1997. Anergic T cells effect linked suppression. Eur. J. Immunol. 27:3191.[Medline]
26. Berg, L. J., A. M. Pullen, D. S. Fazekas, D. Mathis, C. Benoist, M. M. Davis. 1989. Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand. Cell 58:1035.[Medline]
27. Hedlund, G., M. Dohlsten, T. Herrmann, G. Buell, P. A. Lando, S. Segren, J. Schrimsher, H. R. MacDonald, H. O. Sjogren, T. Kalland. 1991. A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells. J. Immunol. 147:4082.[Abstract]
28. Mott, H. R., B. S. Baines, R. M. Hall, R. M. Cooke, P. C. Driscoll, M. P. Weir, I. D. Campbell. 1995. The solution structure of the F42A mutant of human interleukin 2. J. Mol. Biol. 247:979.[Medline]
29. De Jong, J. L., N. L. Farner, M. B. Widmer, J. G. Giri, P. M. Sondel. 1996. Interaction of IL-15 with the shared IL-2 receptor ß and c subunits: the IL-15/ß/ c receptor-ligand complex is less stable than the IL-2/ß/ c receptor-ligand complex. J. Immunol. 156:1339.[Abstract]
30. Sogaard, M., L. Ohlsson, K. Kristensson, A. Rosendahl, A. Sjöberg, G. Forsberg, T. Kalland, M. Dohlsten. 1999. Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors. Int. J. Oncol. 15:873.[Medline]
31. Dohlsten, M., M. Bjorklund, A. Sundstedt, G. Hedlund, D. Samson, T. Kalland. 1993. Immunopharmacology of the superantigen staphylococcal enterotoxin A in T-cell receptor Vß3 transgenic mice. Immunology 79:520.[Medline]
32. Giri, J. G., M. Ahdieh, J. Eisenman, K. Shanebeck, K. Grabstein, S. Kumaki, A. Namen, L. S. Park, D. Cosman, D. Anderson. 1994. Utilization of the ß and chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
33. Nakajima, H., X. W. Liu, A. Wynshaw-Boris, L. A. Rosenthal, K. Imada, D. S. Finbloom, L. Hennighausen, W. J. Leonard. 1997. An indirect effect of Stat5a in IL-2-induced proliferation: a critical role for Stat5a in IL-2-mediated IL-2 receptor chain induction. Immunity 7:691.[Medline]
34. Willerford, D. M., J. Chen, J. A. Ferry, L. Davidson, A. Ma, F. W. Alt. 1995. Interleukin-2 receptor chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3:521.[Medline]
35. Johnston, J. A., C. M. Bacon, D. S. Finbloom, R. C. Rees, D. Kaplan, K. Shibuya, J. R. Ortaldo, S. Gupta, Y. Q. Chen, J. D. Giri. 1995. Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15. Proc. Natl. Acad. Sci. USA 92:8705.[Abstract/Free Full Text]
36. Boussiotis, V. A., D. L. Barber, T. Nakarai, G. J. Freeman, J. G. Gribben, G. M. Bernstein, A. D. D’Andrea, J. Ritz, L. M. Nadler. 1994. Prevention of T cell anergy by signaling through the _c chain of the IL-2 receptor. Science 266:1039.[Abstract/Free Full Text]
37. Miyazaki, T., A. Kawahara, H. Fujii, Y. Nakagawa, Y. Minami, Z. J. Liu, I. Oishi, O. Silvennoinen, B. A. Witthuhn, J. N. Ihle. 1994. Functional activation of Jak1 and Jak3 by selective association with IL- 2 receptor subunits. Science 266:1045.