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International Immunology Advance Access originally published online on May 15, 2006
International Immunology 2006 18(7):1043-1054; doi:10.1093/intimm/dxl038
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Defective cell cycle induction by IL-2 in naive T-cells antigen stimulated in the presence of refractory T-lymphocytes

Hiroto Inaba1 and Terrence L. Geiger2

1 Department of Hematology and Oncology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA
2 Department of Pathology, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA

Correspondence to: T. L. Geiger; E-mail: terrence.geiger{at}stjude.org


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T cells enter a transient refractory period after stimulation. Upon re-stimulation the refractory cells produce little IL-2 and show diminished proliferation. We previously demonstrated that refractory T cells can also, like anergic and CD4+CD25+ regulatory T cells, suppress in trans the proliferation of antigen-stimulated naive T cells. The suppressed T cells up-regulate high-affinity IL-2R but do not produce IL-2. This IL-2 deficit could potentially explain the proliferation failure, but does not appear to do so. Supplementation of refractory-naive co-cultures with exogenous IL-2 fails to alleviate both the proliferation suppression and IL-2 production defects. This does not result from a failure of IL-2 to stimulate its receptor. Proximal IL-2 signaling into suppressed T cells through STAT5 and Akt is intact. However, refractory cell-co-cultured T cells fail to up-regulate cyclins and c-myc and incompletely down-regulate p27kip1 in response to IL-2, and the downstream consequences of this signaling are therefore dissociated. IL-2 signaling is not fully disabled as IL-2 up-regulates the anti-apoptotic protein Bcl-xL to control levels. This up-regulation correlates with enhanced survival of refractory cell-co-cultured T cells placed in IL-2 when compared with cells cultured without IL-2. Thus, refractory T cells are able to suppress naive T-cell proliferative responses in part by blocking both IL-2 production and the mitogenic but not anti-apoptotic effects of IL-2. These results have implications for how activation-refractory T cells may influence nascent immune responses.

Keywords: apoptosis, cytokine, proliferation, signal transduction, suppression, T-cell


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The T-cell response to antigen is contextually modified by both autocrine and antigen-presenting cell (APC)-derived mediators, including cytokines and co-stimulatory molecules. T–T interactions may further influence the outcome of antigen stimulation. Activated CD8+ T cells are able to suppress naive responses by inducing apoptosis in a Fas-dependent manner (1–3). CD4+CD25+ regulatory T cells can suppress the proliferation of and IL-2 production by naive T cells stimulated with antigen or mitogen (4–8). Other populations of anergic T cells possess a similar capacity to suppress naive T-cell responses (9–11).

After CD4+ T cells are stimulated with antigen, they enter a refractory period during which they exhibit diminished proliferation and IL-2 production if re-stimulated (12–14). This period generally begins several days after stimulation and lasts for several additional days. The addition of IL-2 to cultures lengthens the refractory period. Recently, we showed that stimulation-refractory CD4+ T cells, like CD4+CD25+ regulatory T cells and anergic T cells, are able to suppress naive T-cell responses (14). Like these other suppressive T-cell populations, the refractory cells block both the proliferation of and IL-2 production by stimulated naive T cells. Suppression is not antigen specific or MHC restricted. The refractory and target T cells need not even recognize antigen on the same APC. Inhibition is, however, contact or proximity dependent, failing to occur when the refractory and naive T cells are independently stimulated across a semi-permeable barrier.

Normally, IL-2 production and proliferation pathways are fully enabled in T cells after only several hours of contact with antigen/APC (15, 16). After this time, removal of the antigen/APC does not prevent T-cell proliferation. When naive T cells are stimulated in the presence of refractory cells, they are partially activated, up-regulating CD25 and CD69. However, when these cells are isolated from the refractory cells and antigen/APC 24–72 h later, unlike control cells, they do not produce IL-2 or spontaneously proliferate when re-cultured in medium. The suppressed T cells are not anergic, however, and when purified away from the refractory cells are able to fully respond to antigen. Thus, refractory cells induce a transient blockade that leads to incomplete T-cell activation.

IL-2 is critical for TCR stimulation-induced proliferation in vitro (17, 18). T cells from animals unable to produce IL-2 show a diminished proliferative response (19). As naive T cells stimulated in the presence of refractory T cells fail to produce IL-2, a deficiency in IL-2 production and availability may constrain naive T-cell expansion. Indeed, one hypothesis for the mechanism of suppression by CD4+CD25+ regulatory T cells is that the regulatory cells consume the IL-2 present and/or prevent IL-2 production by naive T cells, thereby limiting their ability to respond to antigen (20, 21).

In this study, we characterize the role of IL-2 in antigen-refractory T-cell-mediated suppression. We find several defects in the naive T-cell stimulation program. The cells fail to produce IL-2 despite up-regulation of high-affinity IL-2R. They also fail to expand in response to IL-2 when it is present. Upstream IL-2 signaling is intact. IL-2, though, fails to induce downstream mediators required for cell cycling. This proliferation defect is dissociated from the anti-apoptotic effect of IL-2, which is preserved in the suppressed T cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
AND mice, transgenic (Tg) for a rearranged pigeon cytochrome c (PCC)-specific TCR, were bred >20 generations onto the B10.BR background. B10.BR and OT-1 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Media, reagents and antibodies
Cells were grown in Eagle's-Hank's amino acid (EHAA; BioSource) supplemented with 10% heat-inactivated Premium FCS (BioWhittaker), penicillin G (100 U ml–1), streptomycin (100 µg ml–1), 292 µg ml–1 L-glutamine (Invitrogen Life Technologies) and 50 µM 2-mercaptoethanol (FisherBiotech). PCC (KAERADLIAYLKQATAK) and ovalbumin (OVA) (SIINFEKL) peptides were synthesized and HPLC purified by the St Jude Children's Research Hospital Hartwell Center for Biotechnology. FITC-conjugated or biotinylated anti-CD25 (7D4) and allophycocyanin-conjugated anti-CD4 (L3T4), anti-CD8 (53-6.7) and anti-CD16/CD32 Fc block (2.4G2) were purchased from BD-PharMingen.

Cell purification
Lymph nodes (axillary, inguinal, superficial cervical, mandibular and mesenteric) and spleen were harvested from 6- to 12-week old mice and single-cell suspensions prepared by forced passage through a cell strainer. Erythrocytes were lysed with Gey's solution, and T cells were purified by panning with goat anti-mouse Ig antibody (Jackson ImmunoResearch). CD4+CD25– or CD4+CD25+ cells were purified by staining with Fc block, allophycocyanin–anti-CD4 and FITC–anti-CD25 antibodies in PBS with 5% FCS for 20 min before flow cytometric sorting on a MoFlo high-speed sorter (DakoCytomation). Sorted cell purity ranged from 97 to 99%.

Cell culture
To produce activated refractory T-cells, freshly sorted CD4+CD25– AND T-cells were cultured with 3000-rad irradiated B10.BR splenocyte feeders, 5 µM PCC peptide and 100 U ml–1 recombinant human IL-2 (rhIL-2; NCI BRB Repository) in EHAA complete medium. After 2 days, viable cells were purified by Ficoll-Hypaque centrifugation (lymphocytes separation media; BioWhittaker) and then cultured in growth factor-enriched medium. Cells were stimulated every 10 days with feeders and antigen for up to three passages. AND TCR Tg CD4+CD25+ regulatory T cells were expanded for one generation as described prior to use (8).

[3H]-thymidine proliferation assays
A total of 5 x 104 CD4+CD25– flow cytometrically isolated T cells from 6- to 12-week old AND mice were cultured for 72 h at 37°C and 5% CO2 in EHAA complete medium in round-bottom 96-well plates (Corning-Costar, Cambridge, MA, USA) with 2.5 x 105 3000-rad irradiated B10.BR splenocytes, with or without 5 µM PCC, 5 x 104 activated AND T-cells and rhIL-2. Cultures were pulsed with [3H]-thymidine ([3H]TdR) (1 µCi per well) for 18–20 h, harvested onto filtermat and scintillation counted.

5,6-Carboxylfluorescein diacetate succinimidyl ester proliferation assays
Flow cytometrically purified CD4+CD25– AND T-cells were washed and re-suspended at 10 x 106 to 50 x 106 cells ml–1 in 5 µM 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR, USA)/PBS/5% FCS for 8 min at 37°C. The cells were then washed three times and mixed in a 6- or 96-well plate at a 1:1 ratio with unlabeled refractory AND T cells, CD4+CD25+ regulatory T cells or control freshly isolated naive CD4+CD25– AND T-cells. The cells were stimulated with a 3- to 5-fold excess of irradiated splenocyte feeders and 5 µM PCC peptide. The co-cultures were analyzed by flow cytometry at 24, 48 or 72 h. In some cases, the CFSE+ cells were resorted and analyzed after re-culturing them in different conditions. For CD8+ T-cell cultures, CFSE-labeled OT-1 T cells were stimulated as above, though with 5 µM OVA peptide/syngeneic APCs.

Flow cytometry
Cells were stained and analyzed on a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). Quantitative flow cytometry to determine viable cell counts was performed by enumerating all cells in a culture well. Cells were stained with propidium iodide (PI), and CFSE+ cells were gated for viability using forward/side scatter (FSC/SSC) and PI.

Cytokine secretion analysis
Cell-free supernatants (50 µl), harvested at the designated times, were tested for IL-2 content by Bio-Plex assay according to the manufacturer's instructions (Bio-Rad).

Reverse transcription–PCR for FoxP3
Total RNA from cells was extracted using Trizol (Invitrogen) as per the manufacturer's instructions. Extracted RNA was quantified by optical density, and identical quantities (1 µg) were reverse transcribed (Omniscript, Qiagen) and analyzed by simultaneous PCR (32 cycles) with FoxP3- and internal control (hypoxanthine phosphoribosyl transferase, HPRT)-specific primers. Primer sequences were: FoxP3: 5'-CAGCTGCCTACAGTGCCCCTAG-3' and 5'-CATTTGCCAGCAGTGGGTAG-3' and HPRT: 5'-GCTGGTGAAAAGGACCTCTCG-3' and 5'-CCACAGGACTAGAACACCTGC-3'.

Western analysis
CFSE-labeled responder cells were flow cytometrically sorted at the designated times after culture and, where indicated, activated with 100 U ml–1 rhIL-2 for 30 min. They were washed thrice with ice-cold PBS, and equal numbers of cells were lysed in ice-cold cell lysis buffer (Cell Signaling; 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate) with 1 mM Na3VO4, 1 mM phenylmethylsulphonylfluoride, 10 mM NaF and protease inhibitor mixture (Roche). Equivalent quantities of protein was separated by 12% SDS-PAGE, blotted and probed with anti-mouse p27kip1 (Santa Cruz Biotechnology); anti-cyclin D2, D3 or E (prepared from hybridomas, gift from M. Roussel); anti-mouse c-Myc (Santa Cruz Biotechnology), anti-mouse Bcl-xL (BD Bioscience), anti-phospho–Akt (Cell signaling) or anti-phospho–STAT5 (BD Bioscience), followed by anti-Akt (Cell signaling), anti-STAT5 or anti-actin (Santa Cruz Biotechnology). Blots were washed, incubated with HRP-conjugated secondary antibody and developed using chemiluminescence as per manufacturer's instructions (Amersham Biosciences).

Quantitative PCR
Real-time reverse transcription (RT)–PCR was performed using iQ SYBR Green Supermix (Bio-Rad) and an iCycler thermocycler (Bio-Rad). cDNA was prepared as above. Primers specific for IL-2 (F 5'-CCTGAGCAGGATGGAGAATTACA, R 5'-TCCAGAACATGCCGCAGAG), Bcl-2 (F 5'-GTCGTGACTTCGCAGAGATGT, R 5'-TCACCCCATCCCTGAAGAGTT), Bcl-XL (F 5'-GCAGGTATTGGTGAGTCGGATT, R 5'-CATTGTTCCCGTAGAGATCCACA), Bax (F 5'-ATCATGGGCTGGACACTGGA, R 5'-GGGTCCCGAAGTAGGAGAGG) and mouse ubiquitin (F 5'-TGGCTATTAATTATTCGGTCTGCAT, R 5'-GCAAGTGGCTAGAGTGCAGAGTAA) were used under optimized cycling conditions that included denaturing at 95°C for 10 s and annealing at 60°C for 45 s for 40 cycles. Mouse ubiquitin primers were used to standardize RNA levels in all samples. RNA level is plotted relative to expression in unstimulated naive T-cells.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
IL-2 fails to alleviate refractory T-cell-mediated suppression
To produce stimulation-refractory T cells, we flow cytometrically sorted CD4+CD25– PCC-specific AND TCR Tg T cells, thereby excluding CD4+CD25+ regulatory T cells. These were expanded with irradiated syngeneic feeders, PCC peptide and rhIL-2. The IL-2, which has been shown to enhance stimulation refractoriness (12), was replenished every 2–3 days. The cells were re-stimulated after 10 days and studied 4 days after stimulation.

We previously showed that naive T cells stimulated in the presence of refractory T cells up-regulate high-affinity IL-2R and therefore should be responsive to IL-2 (14). To assess the role of IL-2 in the suppressive effects of the refractory T cells, we analyzed the circumvention of suppression by exogenous IL-2. We stimulated the refractory T-cells, freshly sorted CD4+CD25– AND T cells or a 1:1 mixture with irradiated APCs/PCC peptide in the presence or absence of added rhIL-2 and measured proliferation in a 72-h [3H]TdR incorporation assay (Fig. 1A). As we previously observed (14), antigen alone induced limited proliferation of the refractory cells. A concentration of 100 U ml–1 and, to a lesser extent, 10 U ml–1 rhIL-2 induced some increased proliferation. Response magnitude was significantly less than that of equal numbers of similarly stimulated naive CD4+CD25– T-cells. Interestingly, the response of co-cultures of naive and refractory cells was not increased compared with refractory cells alone. This suggested that the response we detected was due to expansion of the refractory T cells and that naive CD4+CD25– T-cell proliferation was suppressed even in the presence of exogenous IL-2.


Figure 1
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  		Fig. 1 Exogenous IL-2 fails to alleviate suppression by refractory T cells. (A) A total of 5 x 104 freshly sorted CD4+CD25– AND T cells, refractory T cells or a 1:1 mixture of AND and refractory cells were stimulated with irradiated APC/PCC and the designated rhIL-2 concentration for 72 h prior to pulsing with [3H]TdR. Mean CPM [3H] incorporation from triplicate samples is shown on the abscissa. (B) Sorted CD4+CD25– AND T cells were labeled with CFSE and then co-cultured in 96-well plates in the presence of APC/PCC and an equivalent number of unlabeled CD4+CD25– T cells (control) or refractory T cells (co-culture). At the designated times, triplicate wells of each condition were harvested, stained with PI and analyzed by flow cytometry. CFSE+ PI– FSC/SSC-gated cells are shown as an overlay in the histogram plots. One of three essentially identical flow cytometry profile replicates from a representative experiment is shown.

 
We confirmed this result by specifically visualizing the proliferation of antigen-stimulated CFSE-labeled CD4+CD25– T cells co-cultured with refractory T cells by loss of fluorescence (Fig. 1B). As a control, the CFSE-labeled cells were also co-cultured with identically isolated unlabeled naive T cells. Cell populations were assessed 24, 48 and 72 h after stimulation by flow cytometry. In contrast to control cultures, which strongly proliferated, naive T cells proliferated little in co-cultures with refractory T cells. This was true regardless of the addition of exogenous IL-2, confirming that proliferation suppression is IL-2 independent.

Effective T-cell stimulation induces IL-2 production by T-lymphocytes. Although exogenous IL-2 could not mitigate the proliferation defect induced by the refractory T cells, it was possible that it could alleviate the defect in IL-2 synthesis. To test for this, we stimulated cultures containing CD4+CD25– T cells, refractory T cells or both of these with antigen in the presence or absence of rhIL-2. We then specifically measured the production of non-cross-reactive murine IL-2 (mIL-2). After primary stimulation of control naive cells, mIL-2 was detectable in the culture supernatant by 24 h regardless of the presence of rhIL-2 (Fig. 2A). In contrast, little mIL-2 was detected at this time in cultures of refractory cells or in co-cultures of naive and refractory cells. At 48 h (not shown) or 72 h, no mIL-2 was detected in these populations (Fig. 2B).


Figure 2
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  		Fig. 2 Effect of IL-2 supplementation on IL-2 production. Specific production of mIL-2 by cells co-cultured in the presence or absence of rhIL-2 was monitored. Assays were set up as in Fig. 1 except at 24 (A) or 72 h (B) supernatants were harvested and mIL-2 was determined using an mIL-2-specific assay. (C) CFSE-labeled responder cells assayed as in (A and B) were flow cytometrically isolated after 24-h culture, and IL-2 mRNA levels were determined by quantitative RT–PCR. Fold increase is relative to naive unstimulated T-lymphocytes. Results from one of two (A and B) or three (C) essentially identical experiments are shown. Error bars show ±1 SD.

 
It was possible that the deficit in supernatant mIL-2 resulted from increased consumption by the CD25+ T-lymphocytes present rather than from a production defect. To exclude this possibility, we CFSE labeled naive T cells prior to their stimulation, flow cytometrically sorted them after 24 h and measured IL-2 mRNA levels by quantitative PCR. An ~90-fold increase of IL-2 mRNA was observed in the naive T cells cultured with antigen when compared with cells pre-culture. In contrast, only a 2-fold increase was observed in the presence of refractory cells, and this was independent of the presence of exogenous IL-2 (Fig. 2C). Therefore, refractory T cells cause at least two defects in the naive T-cell population that are not correctable by IL-2, diminished IL-2 production and diminished proliferation.

Intact survival of refractory cell co-cultured T cells
Although naive T cells failed to proliferate when antigen stimulated in the presence of refractory T cells, their survival was intact. Enumeration of CFSE-labeled T cells demonstrated that naive T cells cultured in the absence of antigen progressively died over a 72-h time course (Fig. 3A). Naive T cells similarly died when refractory T cells were added in the absence of antigen, but this was slightly delayed, potentially reflecting the production of survival factors by the refractory cells. As expected, numbers of T cells increased with antigen stimulation in the absence of refractory T cells beginning ~48–72 h after stimulation (data not shown). In contrast, numbers of naive cells stimulated with antigen in the presence of refractory cells remained relatively unchanged over the 72-h culture period. Because these cells failed to proliferate in the presence of refractory cells (Fig. 1B), this enhanced survival compared with the antigen-deficient condition seems to wholly reflect a block in cell death. Thus, the co-cultured T cells receive effective survival though not proliferative signals after antigen stimulation in the presence of refractory T-lymphocytes.


Figure 3
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  		Fig. 3 Survival of naive T cells in co-cultures. Replicate cultures for each culture condition were established as in Fig. 1. At the indicated times post-culture, triplicate wells for each culture condition were harvested, stained with PI and quantitatively analyzed by flow cytometry. Mean numbers of responder cells (A) or refractory cells (B) were determined using Cellquest® software and are plotted. Data are representative of three similar experiments. Error bars show ±1 SD.

 
We similarly analyzed survival of the refractory T cells in the co-cultures. Numbers of these cells remained relatively stable (Fig. 3B) or increased mildly (data not shown) during the course of culture in different experiments, demonstrating that the refractory cells remain available throughout the culture as a source of suppressive activity.

Refractory T-lymphocytes are not classical regulatory T cells
The suppressive activity of the refractory T cells mimics that observed by us and others using CD4+CD25+ regulatory T cells. Suppression in both cases is contact or proximity dependent and occurs in an antigen- and MHC-independent manner (4, 8, 14). Recent reports have shown that CD4+CD25– cells may transform into FoxP3+CD25+ regulatory T cells in vitro, particularly when stimulated in the presence of exogenous transforming growth factor (TGF)-ß (22–24). This would not be anticipated here as our refractory cells were induced using standard stimulation regimens. Nevertheless, to exclude conversion of the refractory cells into regulatory T cells, we analyzed FoxP3 mRNA expression in activated T cells by RT–PCR. We did not detect induction of FoxP3 in these cells for up to 2 weeks after initial stimulation, demonstrating that the refractory T cells had not converted into classic regulatory cells (Fig. 4A).


Figure 4
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  		Fig. 4 Refractory T cells phenotypically and functionally differ from CD4+CD25+ regulatory T cells. (A) FoxP3 expression by activated T cells is shown. CD4+CD25– AND T cells were stimulated with APC/PCC/rhIL-2 and after 10 days re-stimulated. At the designated times, viable cells were purified and 1 µg RNA used for RT–PCR with FoxP3- (upper band) and control HPRT (lower band)-specific primers. As controls, RT–PCR with RNA from 1 x 106 CD4+CD25+ T cells or 70 ng of plasmid containing FoxP3 was simultaneously performed. (B) Refractory T-cell-mediated suppression of CD4+ versus CD8+ T-cell cultures. In the upper rows, naive CFSE-labeled AND T cells were stimulated with APC/PCC in the absence or presence of equal numbers of refractory AND T cells or pre-stimulated CD4+CD25+ AND regulatory T cells. CFSE+ CD4+ PI– FSC/SSC-gated histograms are plotted. In the lower rows, CFSE-labeled CD8+ OT-1 T cells were stimulated with APC/OVA in the absence or presence of equal numbers of refractory AND T cells or activated CD4+CD25+ AND T cells. The presence (shown) or absence of B10.BR APC/PCC did not influence OT-1 T-cell suppression. CD8+ PI– FSC/SSC-gated cell histograms are plotted. Data are representative of three experiments.

 
CD4+CD25+ regulatory T cells, in addition to non-specifically suppressing CD4+ T-cell responses in vitro, have been reported to potently suppress CD8+ T-cell responses (25). We therefore tested whether refractory AND T cells could suppress responses by naive class I MHC-restricted, OVA-specific, OT-1 TCR Tg T-cells (Fig. 4B). Suppression by refractory T cells was directly compared with that mediated by AND TCR Tg CD4+CD25+ regulatory T cells. As expected, the refractory and regulatory T cells strongly inhibited CD4+ T-cell proliferation. The regulatory T cells were similarly effective in inhibiting CD8+ T-cell responses. In contrast, the refractory cells had only a limited influence on the rate of CD8+ T-cell cycling (Fig. 4B). These results show that in contrast to regulatory T cells, the target specificity of the refractory cells differs with T-cell type, with potent activity against CD4+ but not CD8+ T cells.

Target T cells have intact IL-2R signaling
Ligation of the IL-2R stimulates the Jak-STAT and phosphatidylinositol 3-kinase (PI3-K) signal transduction pathways (18, 26–28). These pathways are essential for the mitogenic and anti-apoptotic effects of IL-2. Jak kinase tyrosine phosphorylates STAT5, thereby promoting its dimerization and nuclear translocation. PI3-K activates Akt, which then phosphorylates downstream molecules related to cell survival, cell cycle and protein synthesis. It would have been anticipated considering the up-regulation of the high-affinity IL-2R in refractory cell co-cultured cells that the addition of exogenous IL-2 would activate these pathways prompting cell cycling. One possible explanation for the failure of this to occur is that the IL-2R was disengaged from its signaling pathways. To test for this, we analyzed STAT5 and Akt phosphorylation in response to added IL-2. CFSE-labeled naive CD4+CD25– cells were cultured with PCC/APC and either unlabeled refractory T cells or control naive T cells. The CFSE+ T cells were flow cytometrically isolated ~40 h later, at which time they had up-regulated high-affinity IL-2R (14). They were then cultured for an additional 30 min with or without IL-2 prior to western blot analysis.

Basal phosphorylation of STAT5 was detected in cells isolated from control cultures, presumably resulting from the IL-2 accumulating in the medium during culture (Fig. 5A). In contrast, cells derived from co-cultures with refractory T cells lacked this basal phosphorylation. This would be expected due to the limited amount of IL-2 generated in the co-cultures. After pulsing with IL-2, effective phosphorylation of STAT5 was observed in both cells co-cultured with refractory cells and control T cells. Therefore, IL-2R signaling is intact in cells after co-culture with refractory T cells, permitting STAT5 phosphorylation.


Figure 5
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  		Fig. 5 Western blot analysis of upstream IL-2R signaling. CFSE-labeled CD4+CD25– T cells were stimulated in the presence of refractory T cells or control CD4+CD25– T cells for ~40 h and then flow cytometrically purified. The cells were kept in medium or pulsed with 100 U ml–1 rhIL-2 for 30 min and then lysed. Control freshly isolated, unstimulated CD4+CD25– T cells were simultaneously analyzed. Equivalent quantities of lysates were analyzed by SDS-PAGE and western blotted for either phospho-STAT5 (p-STAT5) or phospho-Akt (p-Akt). Blots were sequentially stripped and re-probed for total STAT5 or Akt and actin as content controls. Results are representative of four independent experiments.

 
We performed similar analyses of Akt phosphorylation. CFSE+ T cells isolated from co-cultures with refractory T cells showed a low level of basal phosphorylation that was comparable to that present in freshly isolated naive (day 0) CD4+CD25– T cells (Fig. 5B). Akt phosphorylation was modestly increased in the cells isolated from the control culture, likely due to the accumulation of IL-2 in these cultures. After IL-2 stimulation, there was a mild increase in phosphorylated Akt levels in the control cells. CFSE+ T cells isolated from co-cultures with refractory cells and stimulated with IL-2 also phosphorylated Akt and, interestingly, the extent of phosphorylation of Akt was increased compared with control cultures. Possibly this resulted from the development of tachyphylaxis along this pathway in the control cultures due to chronic IL-2 exposure, limiting further Akt phosphorylation. This result shows that IL-2 is able to induce Akt phosphorylation and that the major upstream signal transduction pathways for the IL-2R are functional in cells stimulated in the presence of refractory T cells.

IL-2-independent block of cell cycle protein induction by refractory T cells
STAT5 is essential for IL-2-induced proliferation, and the PI3-K pathway can further potentiate this (26, 28). Through these mediators, IL-2 induces mitogenic genes, including cyclins which bind to and up-regulate the activity of cyclin-dependent kinases (cdk) (29, 30). In addition, signals from the IL-2R down-regulate expression of cdk inhibitors, particularly p27kip1 (31). The c-myc proto-oncogene, also up-regulated by IL-2, is critical to cell cycle induction, acting in part through the transcriptional up-regulation of cyclin genes (32, 33). As we had established that upstream IL-2 signaling was intact in the co-cultured cells, we were next interested in determining whether these downstream cell cycle proteins were appropriately regulated. We CFSE labeled CD4+CD25– T cells and antigen stimulated them in the presence of unlabeled refractory cells or control naive cells with or without exogenous IL-2. After 40 h, the CFSE-labeled cells were flow cytometrically isolated and analyzed for cell cycle proteins by western blotting.

As expected, cyclins D2, D3 and E and c-myc were up-regulated in control-cultured cells compared with freshly isolated (day 0) naive T cells (Fig. 6A and B). Expression of these proteins was, however, substantially reduced in cells co-cultured with refractory T cells. Importantly, the diminished expression of these cell cycle proteins was not altered by the addition of exogenous IL-2 during culture. This demonstrates that despite the presence of both a functional IL-2R and IL-2, even at high doses, downstream IL-2 signaling is defective.


Figure 6
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  		Fig. 6 Western blot analysis of cell cycle proteins. CFSE-labeled CD4+CD25– AND T cells were stimulated with antigen in the presence of refractory T cells or control CD4+CD25– T cells for ~40 h in the presence or absence of 100 U ml–1 rhIL-2 and then flow cytometrically purified. The cells were immediately lysed, and equivalent quantities of protein separated by SDS-PAGE and analyzed by western blot with antibody probes for the indicated proteins. Alternatively, lysate was obtained and blotted from freshly isolated CD4+CD25– AND T cells or cells stimulated for 4 days in the absence of refractory T cells. Actin was analyzed as a control for protein content. Blots are representative of four to five independent experiments.

 
Elevated levels of the cdk inhibitor p27kip1 plays a significant role in restraining cell cycling in T cells (34, 35). p27kip1 levels were dramatically decreased in activated control T cells when compared with freshly isolated naive T cells. In co-cultured cells this decline in p27kip1 was not observed. The addition of exogenous IL-2 partially reversed this effect, with variable reduction in p27kip1 expression observed in most experiments, though never to the extent seen in the control cells (Fig. 6B). These results confirm that despite up-regulation of CD25, T cells stimulated in the presence of refractory cells fail to initiate a mitotic program. Proteins required for cell cycle progression are ineffectively regulated, even in the presence of exogenous IL-2.

IL-2 promotes survival of refractory cell co-cultured responder T cells
In addition to its role in promoting mitosis, IL-2 signaling influences cell death pathways. IL-2 may enhance susceptibility to activation-induced cell death (36). However, in vitro, the dominant effect of IL-2 is typically anti-apoptotic. Although regulation of anti- and pro-apoptotic proteins with T-cell stimulation is complex and involves multiple proteins, up-regulation of the Bcl family member Bcl-xL by IL-2 is believed to be significant for its anti-apoptotic effects (32, 37). Bcl-xL is induced in naive T cells with activation and reverts to baseline levels as T cells return to rest. Indeed, 40 h after stimulation, flow cytometrically isolated control cultured cells showed significant up-regulation of Bcl-xL, independent of the addition of exogenous IL-2 (Fig. 7). This up-regulation was consistently reduced in the refractory cell co-cultured T cells, potentially resulting from stimulation in the IL-2-deficient environment of this co-culture. Indeed, when exogenous IL-2 was added to the co-cultured cells, the level of Bcl-xL increased to that observed in the control cultured cells. This result implies that the presence of refractory T cells induces a selective defect in the regulation of cell cycle proteins. However, IL-2-induced up-regulation of the anti-apoptotic protein Bcl-xL is intact.


Figure 7
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  		Fig. 7 Western blot analysis of Bcl-xL. Analysis was performed as in Fig. 6 on flow cytometrically purified responder cells using a Bcl-xL-specific antibody to probe the western blot. Blot is representative of four independent experiments.

 
The increase in Bcl-xL in co-cultured cells stimulated in the presence of IL-2 suggested that, although not cycling, cells co-cultured with exogenous IL-2 should show increased survival when compared with those co-cultured without IL-2. In Fig. 3, we observed that naive T cells stimulated in the presence of the refractory T cells remained viable throughout 72 h of culture whereas those that were not stimulated died. In these conditions survival factors may be derived from many sources, including factors produced by the refractory T cells, APC or resulting from antigenic stimulation. To specifically isolate the effect of IL-2 on cell survival, we flow cytometrically purified CFSE-labeled responder cells 24–48 h after stimulation in the presence of unlabeled refractory cells or control CD4+CD25– cells, and re-cultured these with or without exogenous IL-2 (Fig. 8 and data not shown). In the control-cultured population, which produced its own IL-2 (Fig. 2), addition of exogenous IL-2 mildly enhanced viability and proliferation (Fig. 8A). When the co-cultured cells were re-cultured in the absence of IL-2, they failed to divide and largely died over the course of 72 h (Fig. 8A and B). In contrast, there was significantly enhanced survival among co-cultured cells that were re-cultured in the presence of exogenous IL-2. Sixty-one percent of cells viable 24 h after re-culture in the presence of 100 U ml–1 rhIL-2 were still alive at 72 h whereas only 8% of cells re-cultured in the absence of IL-2 remained viable. The majority of these cells failed to divide at 72 h. Fewer than 2% of viable co-cultured cells had divided one or more times in the absence of rhIL-2, 18% divided after the addition of 10 U ml–1 rhIL-2 and 27% divided after the addition of 100 U ml–1 rhIL-2. The enhanced cell viability in the presence of IL-2 did not result from this limited expansion. Disregarding the divided cells, there was still 7.7 times more surviving undivided cells at 72 h in the presence of 100 U ml–1 rhIL-2 than in its absence (Fig. 8C). This demonstrates that removal of the suppressive refractory T cells and provision of mitogenic cytokine is insufficient to break the proliferative block in most of the target T cells and that in the absence of IL-2 these cells then die. In contrast, IL-2 promotes the survival of these cells even when they fail to proliferate. Importantly, this defect in cytokine-induced proliferation is selective. The cells are not anergic as we previously demonstrated that re-stimulation of co-cultured responder cells with APC/PCC is able to induce their proliferation (14).


Figure 8
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  		Fig. 8 Anti-apoptotic effect of IL-2 on co-cultured T cells. (A) CFSE-labeled CD4+CD25– T cells were stimulated with APC/PCC in the presence of equal numbers of refractory T cells (co-culture) or unlabeled CD4+CD25– cells (control). After ~24 h, the CFSE+ cells were re-isolated by flow cytometric sorting for CFSE and equal numbers of cells (~5 x 104) cultured in wells of a 96-well plate with or without 10 or 100 U ml–1 rhIL-2. These were analyzed at the indicated times after re-culture by flow cytometry with PI/FSC/SSC gating for viable cells. (B) Quantitative flow cytometry was performed on cells treated as in (A). Mean numbers of viable cells detected in wells 24, 48 and 72 h after re-culture from three replicates of each data point are plotted. (C) Quantitative flow cytometry plots were gated for individual cell cycle peaks based on CFSE staining intensity. Absolute numbers of viable cells were enumerated within each peak and are plotted. (D) Analysis was performed as in (A–C) except flow cytometrically isolated cells were re-cultured in the presence or absence of irradiated syngeneic APC (3 x 105 per well) or 80% cell supernatant isolated 24 h after antigenic stimulation of refractory T cells. (E) Quantitative PCR was performed for Bcl-2, Bcl-xL and Bax on control or co-cultured responder cells that were flow cytometrically isolated after 24 h and re-cultured with or without exogenous rhIL-2 for an additional 24 h. Expression relative to naive unstimulated T cells is shown. (F) Western blot analysis for Bcl-xL. Control or co-cultured responder cells were isolated after 24 h and then re-cultured for 36 h in the presence or absence of rhIL-2. Samples analyzed include freshly purified naive T cells, responder cells at the time of isolation and responder cells after re-culture. Data are representative of three independent experiments, except for (E) in which data are from one of two similar experiments. Error bars show ±1 SD.

 
It was curious that the CFSE+ T cells when isolated from the refractory T cells after stimulation and re-cultured in cytokine-deficient medium die (Fig. 8). This contrasts with the survival of these cells when they remain in the presence of antigen–APC and refractory T cells (Fig. 3). One potential explanation for this is that a factor produced by the refractory T cells or by APCs during co-culture promotes the viability of the naive T-lymphocytes. To test for this, we set up analyses as in Fig. 8A and B. We re-cultured the isolated CFSE-labeled T cells without antigen, and in the presence or absence of APCs or supernatant derived from refractory cell cultures (Fig. 8D). The addition of APCs only minimally enhanced cell viability whereas supernatant from refractory T cells modestly, though reproducibly, promoted cell viability. Thus, preservation of viability in the naive cells stimulated in the presence of refractory T cells likely results from a combination of survival signals, including soluble mediators derived from the refractory T cells themselves as well as signals derived from ongoing antigenic stimulation. IL-2, if available, can also provide important survival signals to T cells stimulated in the presence of refractory T-lymphocytes, and is more effective at providing this than APCs or refractory cell supernatant alone.

We would predict that the increased survival of the T cells re-isolated after co-culture and placed in IL-2 should correlate with an increased expression of anti-apoptotic factors. To test for this, we analyzed the level of mRNA encoding the anti-apoptotic proteins Bcl-2 and Bcl-xL and the pro-apoptotic protein Bax by quantitative RT–PCR (Fig. 8E). When IL-2 was not added to the re-culture medium, the co-cultured cells showed diminished levels of each of the anti-apoptotic factors in comparison with control cells. Addition of IL-2 to the medium enhanced the level of the anti-apoptotic factors, with an ~4-fold increase in the levels of Bcl-2 and Bcl-xL mRNA in the co-cultured cells. Bax mRNA levels were also increased with the addition of exogenous IL-2. However, the magnitude of this increase was less, ~1.5-fold. The IL-2-mediated increase in mRNA for the anti-apoptotic Bcl-xL protein observed in co-cultured cells also correlated with an increase in Bcl-xL protein as shown by western analysis (Fig. 8F). Thus, exogenous IL-2 can augment the expression of anti-apoptotic proteins in isolated and re-cultured cells, potentially thereby promoting their survival.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Activated T cells, although not anergic, are transiently refractory to re-stimulation with antigen. We have shown that stimulation-refractory CD4+ T cells can also suppress the antigen-induced proliferation of naive CD4+ T cells and that this suppression shares many properties previously described with CD4+CD25+ regulatory T-cell-mediated suppression. Suppression is antigen/MHC independent and requires cell contact or proximity (14). However, the refractory cells, derived from a CD4+CD25– population, do not express FoxP3 and do not efficiently suppress CD8+ T-cell cycling, and are therefore distinct from classic regulatory T cells.

Using an antigen-specific system, we show that naive CD4+ T cells stimulated in the presence of refractory CD4+ T cells are incompletely activated, up-regulating CD25 though failing to produce IL-2 or proliferate, even after supplementation with exogenous IL-2. Proximal IL-2 signaling is intact, and T-cells antigen stimulated in the presence of refractory cells phosphorylate STAT5 and Akt in response to IL-2. However, the normal downstream consequences of this stimulation are severed. IL-2 fails to induce cyclins or c-myc, or fully down-regulate p27kip1. In contrast, IL-2 can up-regulate the anti-apoptotic protein Bcl-xL. This up-regulation is associated with enhanced survival when the co-cultured cells are placed in the presence of IL-2 (Fig. 8). Thus, the mitogenic and anti-apoptotic effects of IL-2 are dissociated in T cells stimulated in the presence of the refractory suppressive cells. Further, because exogenous IL-2 cannot overcome the suppression mediated by the refractory cells, the suppression appears dominant to the mitogenic effects of IL-2. This suggests that the block in cell cycling induced by refractory T cells occurs downstream of the intersection between the TCR/co-stimulation and IL-2 pathways.

Further analyses will be required to biochemically isolate the site of this defect and are ongoing. One clue may be in the dissociation between IL-2 synthesis, which is lost, and CD25 expression, which is preserved, in the co-cultured cells. Early CD25 expression depends on transcription factor binding to NF-{kappa}B, AP-1 and Elf-1/HMG-I(Y)-binding sites (38). This expression may be enhanced by STAT5 or STAT4 signaling, and can result from stimulation with IL-2 or other cytokines, including IL-1, IL-7, IL-12, IL-15 and tumor necrosis factor (39–41). The refractory cells do not appear to inhibit the signaling pathways required for CD25 expression. By exclusion, other signaling pathways, such as that mediated by c-Rel, which is more important for cellular proliferation and IL-2 production than CD25 expression (38), may have a role in refractory T-cell-mediated suppression. Indeed, similar to our results, c-Rel-knockout mice showed specific loss of IL-2 production without loss of CD25 or CD69 expression (42). c-Rel production, which appears necessary to initiate IL-2 synthesis, is dependent on sustained ERK signaling, and the ability of APC to maintain signal into T cells may therefore be important in relation to refractory T-cell-mediated suppression (43).

It is also possible that the refractory T cells do not act on an intermediate signaling stage but rather act directly on downstream genes. For example, TGF-ß signaling inhibits IL-2-dependent T-cell cycling in a Smad-dependent manner through direct effects on cyclins and other cell cycle genes (44). Although our findings parallel those with TGF-ß treatment, if a direct transcriptional effect is occurring here, it is unlikely that Smad activity is responsible. Anti-TGF-ß treatment of co-cultures was not able to overcome the anti-proliferative effects of the refractory T cells (C. Duthoit and T. Geiger, unpublished results).

An uncoupling of IL-2 from the mitotic machinery has also been observed in several other circumstances. This suggests that the dissociation of mitogenic and anti-apoptotic IL-2 signaling pathways may form a common approach to limit T-cell expansion. In one report, activation of T cells in the presence of ryanodine receptor (RyR) inhibitors led to the up-regulation of CD25 though it suppressed proliferation, even in the presence of exogenous IL-2 (45). RyR is responsible for IP3-independent, nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent calcium mobilization from intracellular stores (46), and this similarity with our system would lend support to the hypothesis that the proliferative defect we observe here may result from defective calcium signaling.

Alternatively, rapamycin has also been observed to block IL-2-driven T-cell cycling without affecting IL-2-mediated survival (47). Rapamycin acts by inhibiting mTOR signaling which is activated through Akt by serine phosphorylation (48). Because we observed normal Akt phosphorylation in our suppressed cells in response to IL-2, we would expect that any block along this pathway is downstream to mTOR itself. This is further suggested by preliminary data demonstrating phosphorylation of the ribosomal protein S6, a target downstream of mTOR, after stimulation of naive T cells in the presence of refractory cells and IL-2 (H. Inaba and T. Geiger, unpublished results).

In other studies, a subset of T cells were identified that failed to initiate cell cycling after stimulation. As in our studies, these cells were found to have up-regulated CD25, but failed to proliferate to IL-2 despite showing enhanced survival, a phenomenon termed as division arrest anergy or IL-2-independent anergy. These cells also up-regulated Bcl-xL in response to IL-2 (49, 50).

Finally, we have previously shown that naive T cells antigenically stimulated in the presence of pre-activated antigen-specific TCR Tg CD4+CD25+ regulatory T cells display a proliferation defect similar to that shown here (8). There too CD25 is highly up-regulated on the naive T cells despite minimal IL-2 production, and proliferation suppression is independent of IL-2. The functional similarity of some of the suppressive properties of pre-activated regulatory T cells and refractory T cells may further suggest a common mechanism for this suppression. This remains speculative however, as effector molecules responsible for suppression have yet to be identified in either case.

The similarities between refractory and regulatory cell activity in vitro also does not imply any functional link between refractory T cells and the immunomodulatory activity of regulatory T cells observed in vivo. In contrast to their in vitro effects, in most model systems, regulatory T-cell function is cytokine dependent in vivo, with IL-10 and TGF-ß secretion by these cells of particular importance (51–53). These cytokines, in general, would not be abundantly produced by refractory cells. Further, it would seem unlikely considering the transient nature of the refractory state, that refractory T cells would have a sustained down-modulatory effect on immune responses as regulatory T cells do.

Despite this, the in vitro activity of refractory T cells may have in vivo correlates. If suppression by refractory T cells occurs in vivo, it may restrict the recruitment of naive T cells into an active immune response, thereby influencing its magnitude and potentially the development of the responding T-cell repertoire. Further, refractory cells would be expected to limit the recruitment of naive cells late in the immune response, after a significant primary response has been generated and refractory cells accumulate. They may therefore preferentially restrict responses to subdominant or cryptic epitopes as well as auto-antigens that are secondarily released from locally inflamed tissues. Therefore, it is possible that refractory cells, which may be abundant in an active immune response, may guide the evolution of that response. It is of interest that cross-competition between T cells has been observed in some in vivo models, and it is conceivable that suppression by activated T cells provides one potential mechanism for this (54).

In summary, we demonstrate that activation-refractory cells are able to suppress the proliferation of target naive cells independently of IL-2 priming or IL-2 competition. Antigenic signaling in the presence of refractory cells leads to an altered cellular activation program, characterized by up-regulation of high-affinity IL-2R, but a failure to produce IL-2. Survival and cell proliferation signals mediated by the IL-2 signaling pathway are further dissociated. The ability of activation-refractory T cells to suppress naive T-cell responses may impact the quality and magnitude of developing T-cell responses.


   Acknowledgements
 
The authors thank Richard Cross and Jennifer Smith for assistance with flow cytometry and cell sorting, Susan Rowe for assistance with IL-2 cytokine assays and Martine Roussel for assistance with western analyses. This work was supported by National Institutes of Health grants R21 AI49872 and R01 AI056153 (to T.L.G.) and by the American Lebanese Syrian Associated Charities/St Jude Children's Research Hospital (to T.L.G. and H.I.).


   Abbreviations
 
APC, antigen-presenting cell
cdk, cyclin-dependent kinase
CFSE, 5,6-carboxylfluorescein diacetate succinimidyl ester
FSC, forward scatter
HPRT, hypoxanthine phosphoribosyl transferase
[3H]TdR, [3H]-thymidine
mIL-2, murine interleukin 2
OVA, ovalbumin
PCC, pigeon cytochrome c
PI, propidium iodide
PI3-K, phosphatidylinositol 3-kinase
rhIL-2, recombinant human IL-2
RT, reverse transcription
RyR, ryanodine receptor
SSC, side scatter
Tg, transgenic
TGF, transforming growth factor

   Notes
 
Transmitting editor: R. A. Flavell

Received 14 October 2005, accepted 13 April 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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