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CD4⁺CD25⁺ T Cells Facilitate the Induction of T Cell Anergy

Joerg Ermann^*, Veronika Szanya^*, Gregory S. Ford^*, Violette Paragas^*, C. Garrison Fathman^1,* and Kristina Lejon

^* Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305; and UmanGenomics, Umestans Företagspark, Umea, Sweden

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
T cell anergy is characterized by the inability of the T cell to produce IL-2 and proliferate. It is reversible by the addition of exogenous IL-2. A similar state of unresponsiveness is observed when the proliferative response of murine CD4⁺CD25^- T cells is suppressed in vitro by coactivated CD4⁺CD25⁺ T cells. We have developed a suppression system that uses beads coated with anti-CD3 and anti-CD28 Abs as surrogate APCs to study the interaction of CD4⁺CD25⁺ and CD4⁺CD25^- T cells in vitro. CD4⁺CD25⁺ T cell-induced suppression, in this model, was not abrogated by blocking the B7-CTLA-4 pathway. When the CD4⁺CD25^- T cells were separated from the CD4⁺CD25⁺ suppressor cells after 24 h of coactivation by the Ab-coated beads, the CD4⁺CD25^- T cells were unable to proliferate or to produce IL-2 upon restimulation. The induction of this anergic phenotype in the CD4⁺CD25^- T cells correlated with the up-regulated expression of the gene related to anergy in lymphocytes (GRAIL), a novel anergy-related gene that acts as a negative regulator of IL-2 transcription. This system constitutes a novel mechanism of anergy induction in the presence of costimulation.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Tcell anergy is one of the mechanisms thought to act in the periphery to ensure tolerance to self. The term anergy was first used to describe T cell clones rendered unresponsive to subsequent restimulation by first activating them through the TCR (signal 1) without appropriate costimulation (signal 2) (1) or, more recently, using an altered peptide ligand for activation (2). The characteristic feature of this induced unresponsiveness was the inability of the anergic T cells to proliferate or produce IL-2 following subsequent optimal restimulation (3). Anergic T cells appeared to have a defect in signaling pathways upstream of transcription of the IL-2 gene (4, 5, 6). Additional studies demonstrated the presence of cis-dominant negative regulatory elements affecting IL-2 transcription (7, 8). The recent finding of increased general receptor of phosphoinositides-1 expression indicated that broader genetic changes may accompany the induction and maintenance of the anergic phenotype (9). Our laboratory identified a gene related to anergy in lymphocytes (GRAIL)² as a novel gene that is preferentially expressed in anergized T cells.³ Its translated protein product has high homology to the Drosophila protein Goliath (10) and other zinc ring finger-containing proteins. Overexpression of GRAIL in T cells dramatically reduced the transcription of IL-2.³

Anergy prevents the clonal expansion of T cells under stimulatory conditions. A similar block in proliferation was observed when activated murine CD4⁺CD25^- responder T cells were cocultured with activated CD4⁺CD25⁺ suppressor T cells in vitro. CD4⁺CD25⁺ suppressor T cells comprise 5–10% of CD4⁺ T cells in naive adult mice (11). Their special immunoregulatory properties were first described by Sakaguchi et al. (12), who showed that the adoptive transfer of CD4⁺CD25⁺ T cells could prevent the autoimmune diseases that develop in mice thymectomized on day 3 of life. Similar findings were later obtained in adoptive transfer models of gastritis (13), diabetes (14), and colitis (15). Suppression of CD4⁺CD25^- T cells in vitro by CD4⁺CD25⁺ T cells required activation of the CD4⁺CD25⁺ T cells as well as direct cell-cell contact. Interestingly, suppression could be abrogated by increasing costimulation via CD28 or by the addition of exogenous IL-2 (16).

The close relationship of costimulation, IL-2 production, and proliferation in the CD4⁺CD25⁺ models was highly reminiscent of anergy systems and prompted us to look more closely at the suppressed CD4⁺CD25^- T cells. Using a system that substitutes beads coated with anti-CD3 and anti-CD28 Abs for APC function, we demonstrated that, upon activation and coculture in vitro, CD4⁺CD25⁺ T cells rendered CD4⁺CD25^- T cells anergic. Once unresponsiveness was established, the presence of the CD4⁺CD25⁺ T cells was no longer required. After removal of the suppressors from coculture, the CD4⁺CD25^- T cells proliferated only when exogenous IL-2 was added during restimulation. Suppression by CD4⁺CD25⁺ T cells thus appears to be an alternative mechanism for the induction of anergy in Ag-reactive CD4⁺ T cells in the presence of costimulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

DBA/2J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were kept under specific pathogen-free conditions in the Department of Comparative Medicine at Stanford University School of Medicine (Stanford, CA).

The Abs

Purified anti-CD3 (145-2C11), anti-CD28 (37.51), anti-CTLA-4 (UC10-4F10-11), anti-CD80 (16-10A1), anti-CD86 (PO3), anti-FcR (2.4G2), and FITC-conjugated anti-CD4 (GK1.5) were purchased from BD PharMingen (San Diego, CA). Biotinylated anti-CD25 (PC61) and PE-conjugated streptavidin were purchased from Caltag Laboratories (Burlingame, CA).

Media

Cell preparation for FACS analysis and sorting was performed in Dulbecco’s PBS (Life Technologies, Gaithersburg, MD) plus 5% heat-inactivated FBS (HyClone Laboratories, Logan, UT). For proliferation assays, lymphocytes were cultured in RPMI-C, i.e., RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 1% nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin plus 100 µg/ml streptomycin, 2 mM L-glutamine (all obtained from Life Technologies), and 50 µM 2-ME (Sigma, St. Louis, MO).

Coating of latex beads

The 5-µm latex beads (Interfacial Dynamics, Portland, OR) were coated with 2.5 µg/ml anti-CD3 (145-2C11; BD PharMingen) and varying amounts of anti-CD28 (37.51; BD PharMingen) in PBS for 90 min at 37°C. The coated beads were then washed in RPMI-C, resuspended in RPMI-C, and stored at 4°C until use.

Preparation of CD4⁺CD25⁺ cells

Female mice, 6–8 wk old, were sacrificed, spleen and lymph nodes (inguinal, axillary, brachial, submandibular, mesenteric, pancreatic, and paraaortic) were harvested, and single-cell suspensions were prepared. CD4⁺ cells were then enriched using anti-CD4 magnetic MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. After staining with FITC-conjugated anti-CD4 and biotinylated anti-CD25 followed by PE-conjugated streptavidin, cells were sorted into CD4⁺CD25^- and CD4⁺CD25⁺ populations.

Cell sorting

Cells were sorted on a FACStar cell sorter (BD Biosciences, Mountain View, CA) in the Shared FACS Facility, Center for Molecular and Genetic Medicine at Stanford University.

In vitro proliferation assay

A total of 12,500 sorted cells/well was incubated with equal numbers of Ab-coated beads in RPMI-C in a 96-well U-bottom plate (BD Biosciences). Beads were coated with 2.5 µg/ml anti-CD3 and 1.25 µg/ml anti-CD28, unless stated otherwise. Cells were pulsed with 1 µCi [³H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) per well for the last 15 h of the 72-h culture period. Cells were then harvested onto filter membranes using a Wallac harvester (PerkinElmer Life Sciences, Gaithersburg, MD), and the amount of incorporated [³H]thymidine was measured with a Wallac Betaplate counter (PerkinElmer Life Sciences). For coincubation experiments, CD4⁺CD25^- and CD4⁺CD25⁺ cells were mixed at a 1:1 ratio and stimulated with equal numbers of Ab-coated beads. Where indicated, recombinant murine IL-2 (R&D Systems, Minneapolis, MN) was added at 50 U/ml.

Labeling of cells

For experiments requiring the separation of CD4⁺CD25^- and CD4⁺CD25⁺ cells after coincubation, the CD4⁺CD25^- cells were labeled with CFSE (Molecular Probes, Eugene, OR). Sorted cells were incubated in PBS with 5 µM CFSE for 10 min at 37°C, washed, and resuspended in RPMI-C.

Analysis of resorted cell populations

Coincubated CD4⁺CD25⁺ and CFSE-labeled CD4⁺CD25^- cells (50,000 cells each type with equal numbers of beads per well of a 96-well U-bottom plate) were harvested, washed, resuspended in FACS buffer with propidium iodide (0.1 µg/ml, Sigma), and resorted based on their CFSE signal. CD4⁺CD25^- cells stimulated alone were treated identically and served as control. The resorted cells were frozen for subsequent PCR analysis or counted and resuspended in RPMI-C for functional studies (IL-2 secretion, proliferation). In some experiments, the resorted populations were rested for 2 days at 25,000 cells/well together with 200,000 T cell-depleted (using anti-CD4 plus anti-CD8 magnetic MicroBeads) DBA/2 splenocytes irradiated with 3000 rad. After the rest period, the cells were washed and restimulated with soluble anti-CD3 (0.5 µg/ml), murine IL-2 (10 U/ml), or medium. Proliferation was measured after 72 h as described.

IL-2 ELISA

Samples and serial dilutions of recombinant murine IL-2 were incubated overnight at room temperature on Maxisorb ELISA plates (Nalge Nunc International, Rochester, NY) coated with 1 µg/ml anti-mouse IL-2 (1JES6-1A12; BD PharMingen). This was followed by incubation with 0.5 µg/ml biotinylated anti-mouse IL-2 (1JES6-5H4; BD PharMingen) for 3 h at 4°C and extravidin/peroxidase (Sigma, St. Louis, MO) for 30 min at room temperature. The plate was washed extensively between all steps with PBS plus 0.1% Tween 20 (Fisher Scientific, Pittsburgh, PA). Finally, tetramethylbenzidine liquid substrate system (Sigma) was added for 20 min, the reaction was stopped with 1 N HCl (Mallinckrodt, St. Louis, MO), and extinction at 450 nm was read on a Victor 1420 ELISA reader (PerkinElmer Wallac, Gaithersburg, MD). Analysis was performed using Microsoft Excel (Microsoft, Redmond, WA). The sensitivity of the assay was 4 mU/ml.

Real-time quantitative PCR

The mRNA was extracted from frozen cell pellets with a QIAGEN RNeasy mini kit (QIAGEN, Valencia, CA), DNase treated (DNA-free, Ambion, Austin, TX), and reverse transcribed with MultiScribe reverse transcriptase in the presence of random hexamers (PerkinElmer Applied Biosystems, Foster City, CA). Real-time quantitative PCR (17) for GRAIL and ribosomal RNA for normalization was performed using the ABI Prism 7700 Sequence Detection System that contains a Gene-Amp PCR System 9600 (Perkin-Elmer Applied Biosystems). Primers and probes were synthesized by PerkinElmer Applied Biosystems. All samples were analyzed in triplicates. A cDNA pool from DBA/2 CD4⁺ lymphocytes served as reference standard. The arbitrary units used to express results are multiples of this standard.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Ab-coated beads can replace APC function in suppression assays in vitro

CD4⁺CD25⁺ T cells have been demonstrated to inhibit the proliferation of CD4⁺CD25^- T cells in vitro. This suppressor activity required activation of both the CD4⁺CD25⁺ and the CD4⁺CD25^- T cells as well as direct cell contact between these two cell types and APCs (16, 18). To address the role of the APCs in the interaction between CD4⁺CD25⁺ and CD4⁺CD25^- T cells, we stimulated FACS-sorted cell populations with latex beads coated with anti-CD3 and anti-CD28 Abs. These beads could replace APCs without changing the characteristics of the system (Fig. 1 and Refs. 16 and 18). CD4⁺CD25^- T cells, but not CD4⁺CD25⁺ T cells, proliferated when stimulated with Ab-coated beads alone. Coincubation of both populations led to inhibition of the CD4⁺CD25^- T cell response. Exogenous IL-2 rescued the proliferation of the CD4⁺CD25⁺ T cells stimulated alone as well as of cocultured CD4⁺CD25^- and CD4⁺CD25⁺ T cells (Fig. 1A). The amount of costimulation provided to the T cells was easily controlled by changing the concentration of anti-CD28 during the coating procedure. Stronger costimulation enhanced the proliferative response of CD4⁺CD25^- T cells and abrogated suppression in the coincubation assay (Fig. 1B), as previously described (16).

View larger version (28K): [in this window] [in a new window]   FIGURE 1. CD4+CD25+ T cells can suppress the proliferation of CD4+CD25- T cells in vitro with latex beads coated with anti-CD3 and anti-CD28 Abs used as surrogate APCs. A, A total of 12,500 FACS-sorted CD4+CD25- and/or CD4+CD25+ T cells per well was incubated for 72 h with equal numbers of beads (coated with 2.5 µg/ml anti-CD3 and 1.25 µg/ml anti-CD28). Murine rIL-2 (50 U/ml) was added as indicated. Proliferation was measured as incorporated radioactivity after a 15-h pulse with [3H]thymidine at the end of the 72-h culture period. B, Beads were coated with a constant amount of anti-CD3 (2.5 µg/ml) and titrated amounts of anti-CD28 (0.62, 1.25, 2.5, and 5 µg/ml). The proliferation assay was performed as in A. Results are representative of at least three experiments performed.

Blocking signaling through CTLA-4 does not abrogate suppression

Freshly isolated CD4⁺CD25⁺ T cells have high intracellular levels of CTLA-4 (14, 21). Takahashi et al. (21) abrogated suppression of the CD4⁺CD25^- response by CD4⁺CD25⁺ T cells in vitro with Abs against CTLA-4. The same result was obtained with CD4⁺CD25^- responders from CTLA-4^-/- mice, suggesting that the anti-CTLA-4 Ab affected the CD4⁺CD25⁺ suppressors. These authors proposed that CD4⁺CD25⁺ T cells might suppress by competing with CD4⁺CD25^- T cells for B7 ligands on the surface of the APCs (as CTLA-4 has a higher affinity for CD80/CD86 than CD28) and/or that the balance between signals received through CD28 and CTLA-4 might influence the suppressive capacity of the CD4⁺CD25⁺ T cells. However, our experiments using Ab-coated beads for stimulation show that an APC-derived signal via CTLA-4 is not required for the suppressive activity of the CD4⁺CD25⁺ T cells. Interestingly, CD4⁺CD25⁺ T cells express higher levels of B7 molecules than CD4⁺CD25^- T cells (data not shown). To exclude the possibility that CTLA-4 signals were provided by neighboring T cells, we blocked all potential B7-CTLA-4 interactions by adding anti-CTLA-4 (clone 4F10) at high doses with additional anti-FcR Ab or a combination of Abs against CD80 and CD86 to the culture. In our system, anti-B7 Abs do not interfere with T cell activation. As shown by data presented in Fig. 2, neither anti-CTLA-4 nor the mixture of anti-CD80 and anti-CD86 Abs abrogated suppression. This argues against a role for CTLA-4 in the suppressive mechanism in our system.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. CTLA-4 blockade does not abrogate the suppression of CD4+CD25- by CD4+CD25+ T cells in vitro. CD4+CD25- T cells and/or CD4+CD25+ T cells were stimulated with Ab-coated beads in the presence or absence of Abs blocking the interaction between B7 molecules and CTLA-4. Anti-CTLA-4 (UC10-4F10-11) was added at 100 µg/ml, and anti-CD80 (16-10A1), anti-CD86 (PO3), and anti-FcR (2.4G2) were used at 10 µg/ml. Proliferation was measured as incorporated radioactivity after a 15-h pulse with [3H]thymidine at the end of the 72-h culture period. Results are representative of five experiments performed. The Abs were shown to be effective in a primary T cell activation assay (data not shown).

A block in T cell proliferation in vitro due to insufficient IL-2 production that can be overcome by the addition of exogenous IL-2 is the hallmark of T cell anergy. The suppressed CD4⁺CD25^- T cells demonstrated all of these characteristics. However, anergy is an acquired endogenous phenotype of T cells resulting from insufficient activation and has not been demonstrably dependent on the presence or actions of other cells. To ask whether the "suppressed" CD4⁺CD25^- T cells had become anergic, we FACS sorted CD4⁺CD25^- T cells after 24 h of coincubation with CD4⁺CD25⁺ T cells and analyzed their functional response to restimulation. The CD4⁺CD25^- T cells were labeled with CFSE to allow differentiation of the two populations by FACS, as virtually all cells expressed CD25⁺ by that time (data not shown). In contrast to control CD4⁺CD25^- T cells that had been stimulated alone, CD4⁺CD25^- T cells initially coincubated with CD4⁺CD25⁺ T cells showed defective IL-2 production upon restimulation and proliferated only when exogenous IL-2 was added (Fig. 3A). In a subsequent set of experiments, we rested the cells for 2 days before restimulation. Data presented in Fig. 3B show that under these experimental conditions, the previously coincubated CD4⁺CD25^- T cells also showed an anergic phenotype. They did not respond to restimulation through the TCR but proliferated well to exogenous IL-2.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. CD4+CD25+ T cells facilitate the induction of anergy in the suppressed CD4+CD25- cells. CFSE-labeled CD4+CD25- and CD4+CD25+ T cells were coactivated with Ab-coated beads. Cells were harvested after 24 h and FACS sorted based on their CFSE signal. CD4+CD25- T cells stimulated alone served as control. A, Resorted cells were counted, restimulated with fresh beads, and proliferation was measured at the end of an additional 48 h of restimulation with [3H]thymidine added for the last 15 h. B, Resorted CD4+CD25- T cells were rested on irradiated splenocytes for 2 days and were then washed and restimulated with medium, IL-2, or soluble anti-CD3. These experiments were done three times with similar results.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Defective IL-2 production in anergized CD4+CD25- T cells correlates with up-regulated expression of of GRAIL. A, CD4+CD25- T cells suppresssed through coincubation with CD4+CD25+ T cells (as described in Fig. 3) and control CD4+CD25- T cells were restimulated with beads. IL-2 was measured by ELISA in the supernatant after 24 h. B, For analysis of gene expression, RNA was isolated from resorted cells, and real-time quantitative PCR was performed for GRAIL. Results expressed as arbitrary units (see Materials and Methods) are representative of three experiments performed.

We conclude that CD4⁺CD25^- T cells suppressed in vitro by CD4⁺CD25⁺ T cells are rendered anergic. They remain viable but do not produce the IL-2 needed for proliferation. This constitutes a novel mechanism for the induction of anergy as it occurs in CD4⁺CD25^- T cells through their interaction with activated CD4⁺CD25⁺ T cells and in the presence of costimulation. Increased expression of GRAIL seems to be part of complex changes within the suppressed CD4⁺CD25^- T cells that lead to defective IL-2 production upon restimulation.

How the presence of the CD4⁺CD25⁺ T cells causes these dramatic results is unknown. Functionally, CD4⁺CD25^- T cells coincubated with CD4⁺CD25⁺ T cells appear to need higher levels of costimulation for IL-2 production and proliferation. If these increased costimulatory requirements are not met, the expansion of the CD4⁺CD25^- T cells is suppressed and the cells acquire an anergic phenotype. In other words, the putative "suppressive" signal from the CD4⁺CD25⁺ T cells appears to raise the threshold of costimulation required for productive activation of the CD4⁺CD25^- T cells. Thus, CD4⁺CD25⁺ T cells facilitate the induction of T cell anergy in the presence of costimulation.

It could be inferred that, in our system, CTLA-4 signaling is not required for the induction of T cell anergy. This is a controversial issue (22, 23). However, the apparent exposure of naive T cells to B7 signals under tolerizing conditions in vivo (23) and the loss of suppression/anergy with higher degrees of costimulation through CD28 in our system suggest that the need for CTLA-4 signaling in anergy induction might be relative rather than absolute. In this scenario, CTLA-4 signals derived from APCs and the unknown negative signal(s) provided by CD4⁺CD25⁺ T cells would be complementary in opposing positive costimulatory signals.

The relevance of our in vitro findings for the in vivo situation remains to be shown. It is interesting to speculate about the potential role of CD4⁺CD25⁺ T cells and the requirements for suppression or control of responses to self in noninflammatory conditions (low expression of costimulatory products), vs immune responses to foreign proteins in inflammatory conditions (up-regulation of costimulatory products). The fact that CD4⁺CD25^- T cells from normal mice can induce autoimmune diseases when adoptively transferred into T cell-deficient hosts does not contradict the model proposed here. The apparent lack of anergy in autoreactive cells in these models could be due to transfer of not yet anergized recent thymic emigrants and/or to limited duration of the anergic phenotype in vivo, requiring the repeated interaction with CD4⁺CD25+ T cells.

In any event, it has been demonstrated that CD4⁺CD25⁺ T cells act to suppress CD4⁺CD25^- T cells by inducing anergy in the suppressed population in the presence of costimulation in vitro, creating unresponsiveness to restimulation and expression of the anergy-related gene GRAIL.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. C. Garrison Fathman, Stanford University School of Medicine, Center for Clinical Sciences Research Building, Room 2225, 300 Pasteur Drive, Stanford, CA 94305-5166. E-mail address: cfathman{at}stanford.edu

² Abbreviation used in this paper: GRAIL, gene related to anergy in lymphocytes.

³ G. Ford, D. Bloom, V. Paragas, N. Anandasapathy, H. Skrenta, L. Soares, J. Ermann, B. Moore, R. Cron, D. Lewis, and C. Fathman. GRAIL: an novel gene expressed in anergic T cells that inhibits NFAT/AP-1 activation and IL-2 transcription. Submitted for publication.

Received for publication May 18, 2001. Accepted for publication August 14, 2001.

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