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Keywords:

  • CD4+CD25+ T cells;
  • Cytotoxic T lymphocyte-associated antigen 4;
  • GITR;
  • ICER/CREM;
  • IL-2

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Here, we report that inducible cAMP early repressor/cAMP response element modulator (ICER/CREM) is induced early in CD25+CD4+ regulatory T cell (TR) assays mainly in activated Foxp3 effector T cells and this induction correlates with sharp decrease in number of IL-2-expressing T cells. Importantly, RNAi targeting of ICER/CREM in responder CD25CD4+ T cells antagonizes TR-mediated suppression. Moreover, forced expression of Foxp3 in naive CD25 T cells induces constitutive expression of ICER/CREM in T cells with a regulatory phenotype. Foxp3 facilitates expression of ICER/CREM both in Foxp3 transductants as well as CD25 responder T cells suggesting that induction of TR function in suppression assays may utilize contact-dependent interaction. Indeed, CTLA-4 blockade or use of B7-deficient CD25 responder T cells prevents ICER/CREM accumulation and leads to the rescue of IL-2 expression. Therefore, we propose that CTLA-4 binding to B7 ligands expressed on activated ligand-bearing Foxp3 effector T cells results in ICER/CREM-mediated transcriptional attenuation of IL-2. Collectively, these data suggest that Foxp3 expression in TR cells imposes suppression in contact-dependent fashion by induction of constitutive ICER/CREM expression in activated CD25+ Foxp3 T cell effectors thus preventing them from producing IL-2.

Abbreviations:
B7DKO:

B7-deficient mice

GITR:

glucocorticoid-induced TNF receptor

ICER/CREM:

inducible cAMP early repressor/cAMP response element modulator

nDC:

nature DC

PGE2:

prostaglandin E2TR: regulatory T cell

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Foxp3 encodes a forkhead/winged-helix transcription factor termed Scurfin, which is tightly associated with the development and function of regulatory T (TR) cells in mice 13. Foxp3 is expressed in naturally occurring CD25+CD4+ TR cells and retroviral transduction or transgenic expression of Foxp3 can convert CD25CD4+ naive T cells to those with a regulatory phenotype up-regulating CTLA-4 and GITR expression. Furthermore, augmented Foxp3 expression regulates transcription, either directly or indirectly, and thus reduces the proliferative capacity of activated T cells and their IL-2 expression 4. Moreover, even though it has been reported that DNA-binding sites of Foxp3 are adjacent to NFAT sites critical for TCR-mediated induction of IL-2 expression, it is not clear at present, how Foxp3 represses the transcription of IL-2 in Foxp3effector T cells 5.

Importantly, signaling through the second messenger cyclic adenosine monophosphate (cAMP) up-regulates CTLA-4 in resting CD4+ T lymphocytes 6. Furthermore, CTLA-4 increases T cell motility and overrides TCR induced stop signals required for stable conjugate formation between T cells and antigen presenting cells (APC) 7. Critical importance of cAMP/protein kinase A (PKA) pathway for regulation of immune tolerance 8 is further supported by inability to develop T and B lymphocytes in mice conditionally deficient in PKA-RIα subunit (Dr. Hua Gu, Columbia University, personal communication). Moreover, overexpression of a newly established marker of TR cells termed G protein-coupled receptor (GPR) 83, inducible by cAMP in naive CD4+CD25T cells, leads to the induction of regulatory Foxp3+ T cells in vivo 9, 10. In addition, cAMP-elevating agonists such as prostaglandin E2 (PGE2) induce Foxp3 gene expression in adaptive Foxp3+CD4+CD25+ regulatory T (TR) cells during peripheral conversion to the regulatory phenotype 11. This finding is further supported by the observations that T cells expressing cyclooxygenase-2 (COX-2) suppress effector Foxp3 T cells by a PGE2-dependent mechanism 12. Importantly, PGE2 elevates intracellular levels of cAMP, which is a key physiological inducer of inducible cAMP early repressor/cAMP response element modulator (ICER/CREM) 1315. ICER/CREM is also transiently induced by forskolin, which elevates intracellular levels of cAMP 13. However, transient, forskolin-mediated induction of ICER, which leads to transcriptional attenuation of IL-2 upon TCR activation 13, 14, does not endow T cells with regulatory properties and in fact it down-regulates Foxp3 (our unpublished observations). In contrast, constitutive ICER expression in T cells from mouse made transgenic with ICER showed upon TCR stimulation (i) a profound defect in IL-2 expression and (ii) diminished proliferation either upon TCR activation or in mixed lymphocyte reaction response (MLR) 16. It is tempting to speculate that ICER-mediated suppression is manifested in MLR by regulatory phenotype acquired by ICER-transgenic T cells. Therefore, we propose that Foxp3 expression in TR cells may impose suppression in contact-dependent fashion by induction of constitutive ICER/CREM expression in Foxp3 effector T cells preventing them from IL-2 production 5.

The profound proliferative defect characterized by the lack of IL-2 production observed in T cells from transgenic mice expressing a dominant-negative form of CREB 17 is remarkably similar to the phenotype observed in T cells from ICER-transgenic mice 16. This suppressive phenotype, mimicked by physiologically relevant expression of ICER in human T cells acts at least in part by transcriptional attenuation of IL-2. ICER is a master regulator of the CREB/CREM family of transcription factors 18. Our investigations along with those of others concluded that the –160 to –180 region of the IL-2 promoter, also known as CD28-responsive element (CD28RE), serves as a target of ICER/CREM transcriptional repressors that contribute to the inhibition of IL-2 production during T cell anergy 14, 19. Furthermore, previously reported data indicate that suppression of ICER/CREM mRNA synthesis by antisense CREM plasmid results in decreased ICER/CREM binding to the IL-2 promoter and increased expression of IL-2 20. ICER and CREM are synthesized from two distinct promoters of the CREM gene 18. However, only ICER represents inherent subfamily of transcription repressors in T cells since it is transcribed from internal P2 promoter positioned downstream of CREM activation domain 13. Therefore, the absence of activation domain makes ICER dominant-negative master regulator playing a pivotal role in suppression of TCR-induced transcription by CD4+CD25+ TR cells 21 even though definitive experimental proof was not yet firmly established.

CTLA-4 is a member of the CD28-B7 immunoglobulin superfamily of immune regulatory molecules 22. Although CTLA-4 undoubtedly has a cell-autonomous role in controlling helper and effector T cell function, an additional cell-non-autonomous role in activity of TR cell population, which expresses high levels of CTLA-4, remains more controversial. TR-mediated CTLA-4 engagement of B7 expressed on APC has been suggested to result in the induction of indoleamine 2,3-dioxygenase (IDO), which in turn leads to immune suppression as a consequence of tryptophan depletion and production of proapoptotic metabolites 23. However, IDO does not seem to be an exclusive mechanism of suppression since direct TR-mediated suppression can be achieved even in the absence of APC at least in vitro 24. Additional evidence supports the possibility of a similar direct effect on T cells, which up-regulate both B7.1 and B7.2 following TCR activation 25, 26. Therefore, B7 expression on T cells is likely to be relevant since TR cells effectively limit B7 expression on DC 27. It was proposed that T cells, in particular naturally occurring Foxp3+CD25+ TR cells, could utilize an interaction based on CTLA-4 binding to B7 ligands expressed on activated T cells resulting in outside-in ‘reverse’ signaling into the ligand bearing Foxp3 responder T cells 28, 29. Such a notion is consistent with susceptibility of Foxp3 responder T cells to Foxp3+CD25+ TR cell suppressive activity mediated early by transcriptional attenuation of IL-2 30, 31.

ICER/CREM transcription factors play an important role in transcriptional attenuation of IL-2 expression in CD4+ T cells presumably by forming dominant inhibitory complex between NFAT and ICER/CREM (Supporting Information Figs. S1–S3) 13, 14, 32. To investigate further their function in CD25+CD4+ TR cell-mediated suppression we examined ICER/CREM expression in suppression assays. ICER/CREM accumulated significantly in the presence of natural TR cells inhibiting IL-2 transcription. However, in mock assays, in the absence of TR cells, ICER/CREM failed to accumulate and IL-2 was transcribed vigorously. Moreover, naive CD25CD4+ responder T cells retrovirally-transduced with Foxp3 induced the expression of ICER/CREM in CD25 T cells and replaced natural TR cells in their suppressive activity. Importantly, RNAi targeting of ICER/CREM in CD25CD4+ responder T cells antagonized TR-mediated suppression thus making these responders, similarly to B7-deficient responders, refractory to TR-cell function. These data suggest that ICER/CREM conveys Foxp3 function into Foxp3 effector T cells through contact-dependent transcriptional attenuation of IL-2 since abrogation of TR-mediated suppression by CTLA-4 blockade prevents its accumulation and can rescue IL-2 synthesis. Therefore, ICER/CREM appears to be an important component of Foxp3-mediated suppressive function mediated at least in part by reverse signaling triggered via CTLA-4/B7 interaction. We conclude that ICER/CREM is a dominant repressor of contact-dependent TR-mediated suppression that effects transcriptional attenuation of IL-2 production in CD25+ Foxp3 effector T cells.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Single-cell kinetic analysis of CD25+CD4+ T cell suppression

Upon stimulation with ConA (1 μg/mL) T cells positive for IL-2 secretion were measured in TR assays by single-cell IL-2 capture assay (Miltenyi Biotec, CA) (Fig. 1A). Negatively enriched CD4+ T cells subsequently sorted for CD25+ TR and CD25 responder T cells using CD25 mAb alone were incubated in TR assays for 5 h and collected in parallel with the cells from unstimulated TR assays (unstimulated or ConA-stimulated TR assays; 5 h, Fig. 1A). In addition, ConA-stimulated TR assays were incubated overnight (17 h; Fig. 1A). Single-cell capture assays showed approximately 100-fold increase in the number of IL-2-producing cells during the first 5 h (compared to unstimulated TR assays). However, number of IL-2 producing cells in ConA-stimulated TR assays precipitously declined overnight (approximately 5-fold after 17 h; Fig. 1A). Intracellular single-cell staining of ICER/CREM showed that this precipitous decline in the number of IL-2-expressing T cells tightly correlated with the induction of ICER/CREM expression in activated CD25+ T cell population (Fig. 1B). Cells from ConA-stimulated TR assays were collected either after 5 or 17 h of cocultivation and evaluated for ICER/CREM expression in single-cell analysis by intracellular staining of ICER/CREM (black) vs. isotype (grey) (Fig. 1B, ICER/CREM; 5 h, 17 h) or FITC-conjugated Foxp3 antibodies (Fig. 1B, Foxp3; 5 h, 17 h) using Foxp3 Staining Set (eBioscience). ICER/CREM expression gated on CD25+ cells was significantly increased during the first 17 h of coculture, while expression of Foxp3 was increased albeit modestly. To examine which population (Foxp3 or Foxp3+) is responsible for observed ICER/CREM induction after 17 h of incubation we double stained cells with ICER/CREM-FITC and Foxp3-PE conjugated antibodies and then gated on CD25+ Foxp3 T cells. Our analysis showed that CD25+ Foxp3 T cells expressed elevated levels of ICER/CREM in TR assays but not in mock assays (Fig. 1C, ICER/CREM; 5 h, 17 h). ICER/CREM expression in CD25+ Foxp3+ cells, although substantial, was changed only little (99% positive; not shown). These data suggest that elevated levels of ICER/CREM tightly coincide with reduction of IL-2 expression in activated CD25+ Foxp3 responder T cells. Furthermore, they show that CD25+ Foxp3+ TR cells do not suppress the initial activation of CD25responder T cells, but mediate suppressive effect in the target CD25+ Foxp3 effector T cells following production of IL-2. This may result in TR–mediated suppression supported by both the expansion of CD25+ Foxp3+ TR cells (from 10.9% to 14.1%) (Foxp3; Fig. 1B) and/or suppression exhibited through induction of ICER/CREM-mediated inhibition of IL-2 in CD25+ Foxp3 effectors (Fig. 1C).

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Figure 1. Single-cell kinetic analysis of CD25+CD4+ T cell suppression. (A) CD25+CD4+ TR cells cultured with negatively selected CD25CD4+ T cells (1:5) ratio in TR assays were either unstimulated or stimulated with ConA (1 μg/mL). Cells were collected after 5 and 17 h of co-cultivation and evaluated for IL-2 secretion by the cytokine capture assay. FACS plots are gated on live CD4+ DAPI cells. (B) ICER/CREM or Foxp3 expression was evaluated by intracellular ICER/CREM or Foxp3 single-cell staining using FITC-labeled ICER/CREM (CS4) (black) vs. isotype (grey) or FITC-conjugated Foxp3 antibodies using Foxp3 Staining Set (eBioscience, CA). T cells evaluated for ICER/CREM expression are gated on CD25+ T cells. (C) In order to distinguish between ICER/CREM expression in Foxp3 and Foxp3+ T cells, CD25+CD4+ TR cells cultured with CD25CD4+ T cells (1:5) ratio in TR assays (black line) or without TR cells in mock assays (grey line) were collected to be evaluated by double intracellular staining using FITC-labeled ICER/CREM and PE-conjugated Foxp3 antibodies and gated first on CD25+ T cells and then on Foxp3 or Foxp3+ population (not shown).

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RNAi targeting of ICER/CREM in CD25 responder T cells antagonizes TR-mediated suppression

Next, we examined the expression of ICER/CREM mRNA in TR suppression assays and compared this with accumulation of ICER/CREM in mock assays, where CD25+CD4+TR cells had been replaced by non-suppressive CD25CD4+ responder T cells. Real-time quantitative PCR analyses revealed that after 48 h ICER/CREM mRNA levels were approximately 30-fold higher in the TR suppression assays than in the mock assays, and increased to approximately 40-fold difference after 72 h (Fig. 2A). These data indicate that ICER/CREM is significantly accumulated in TR suppression assays and that only a low level of expression could be seen in mock assays replacing TR cells with CD25 responder cells, which show little or no regulatory activity 1. Importantly, ICER/CREM expression in TR suppression assays as well as mock assays correlated inversely with synthesis of IL-2 mRNA (Fig. 2A and B), suggesting that ICER/CREM is associated with TR-mediated transcriptional attenuation of IL-2 expression. To examine directly the role of ICER/CREM in TR mediated suppression, enriched CD4+ T cells were sorted for CD25+ or CD25 T cells. CD4+CD25 responder T cells were electroporated with ICER/CREM RNAi and incubated in TR suppression assays. Approximately 3-fold increase in proliferation was observed in TR assays incubated with CD25 responders electroporated with ICER/CREM RNAi versus CD25 responders electroporated with non-target RNAi (Fig. 2C). Specificity and efficiency of ICER/CREM RNAi was estimated against forskolin-mediated induction of ICER/CREM in CD4+ T cells (Fig. 2D). Collectively, data reported in Fig. 1 and 2 suggest that ICER/CREM conveys suppressive function of CD25+ TR cells acting through transcriptional attenuation of IL-2 in CD25+ Foxp3 responder T cells.

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Figure 2. RNAi targeting of ICER/CREM in CD25 responder T cells antagonizes TR-mediated suppression. Quantification ICER/CREM (A) or IL-2 mRNA levels (B) in suppression assays with (TR assay) or without TR cells (mock assay). CD25+ TR cells and CD25CD4+ responder T cells were mixed at 1:1 ratio along with irradiated APC and stimulated 48 or 72 h with 1 μg/mL ConA. cDNA samples were subjected to real-time quantitative PCR. (C) ICER/CREM RNAi antagonizes TR-mediated suppression. Negatively selected CD25CD4+ T cells were electroporated according to Amaxa (Amaxa, Gaithersburg, MD) with either control plasmid pMaxGFP or ICER/CREM RNAi (Santa Cruz, CA). Nonspecific effects of RNAi were monitored by RISC-free non-target RNAi (Dharmacon, Chicago, IL). CD25responder cells in TR assays proliferated approximately threefold as compared to those electroporated with non-target RNAi. Proliferation was determined by measuring incorporation of [3H]thymidine during the last 6 h of 72-h culture. (D) ICER/CREM mRNA in CD4+ T cells is specifically down-regulated by ICER/CREM RNAi. Negatively selected CD4+ T cells (Dynal, Oslo, Norway) were electroporated with ICER/CREM RNAi or non/target RNAi control and incubated overnight as above. Next day cells were treated with forskolin (100 μM final) and RNA was isolated 5 h later. cDNA samples were subjected to real-time quantitative PCR and evaluated for ICER/CREM mRNA levels.

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Foxp3 induces ICER/CREM in a similar fashionto natural TR cells

Next, we determined whether forced expression of Foxp3 in naive CD25 T cells could facilitate accumulation of ICER/CREM in suppression assays. Bicistronic retroviral vectors expressing Foxp3 and green fluorescent protein (GFP) (Foxp3/MIGR1) or GFP alone (MIGR1) were generated as described previously 1 (Fig. 3A). One week after infection, GFP positive T cells were sorted and a small aliquot of purified cells was analyzed for ICER/CREM mRNA expression by real-time quantitative PCR. GFP-sorted Foxp3/MIGR1 infected GFP+ cells accumulated approximately twice as much ICER/CREM mRNA as compared to GFP+ MIGR1-infected cells (transduction in Fig. 3B). In contrast, positively selected freshly purified TR cells stained with FITC-anti-CD4 and PE-anti-CD25 and purified by MoFlo cell sorter showed elevated levels of ICER/CREM mRNA but only moderate differences between CD25+CD4+ TR and responder CD25 T cells (fresh cell in Fig. 3B, suggesting that anti-CD4 mAb may trigger induction of ICER as previously observed in human T cells (16 and unpublished data). Despite this initial increase in ICER/CREM mRNA levels, CD25CD4+ responder cells do not acquire a regulatory phenotype in suppression assays after 3-day coculture (fresh cell in Fig. 3C). They also do not accumulate ICER/CREM in suppression assays in a fashion comparable to virally transduced Foxp3+ cells (transduction in Fig. 3C). Suppression assays with Foxp3/MIGR1 infected GFP+ cells expressing Foxp3 accumulated significant, albeit slightly lower amounts of ICER/CREM, in comparison with natural CD25+CD4+ TR cells (coculture in Fig. 3C). Both Foxp3-negative populations (GFP+ MIGR1-infected cells as well as non-regulatory CD25CD4+ T cells) accumulated in mock assays a low but comparable amount of ICER/CREM mRNA.

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Figure 3. Naive CD25 T cells retrovirally transduced with Foxp3 can induce accumulation of ICER/CREM in suppression assays and replace natural TR cells. (A) Foxp3/MIGR1 simultaneously expresses two cDNA, Foxp3 and GFP, with the use of an internal ribosomal entry site (IRES). (B) Expression of ICER/CREM in a T cell population of GFP+ MIGR1 and Foxp3/MIGR1 transductants, analyzed in parallel with freshly sorted natural CD25+ TR cells and CD25CD4+ responder T cells using FITC-anti-CD4 and PE-anti-CD25 mAb. cDNA samples were subjected to real-time quantitative PCR. (C) Foxp3-expressing cells, whether natural TR cells (fresh cells) or Foxp3 transductants (transduction), effectively accumulate ICER/CREM mRNA in 3-day TR assays (coculture) as compared with empty vector transductants (GFP-sorted MIGR cells) or mock T cells (non-regulatory CD25CD4+ responders) mixed at a 1:1 ratio. (D) After 3-day coculture (Foxp3/MIGR1 in panel C) re-sorting of Foxp3/MIGR1 infected GFP+ cells and their comparison with GFP naive CD25 responder T cells showed that ICER/CREM mRNA is equally well accumulated also in GFPCD25 responders. (E) To demonstrate that GFP+ Foxp3 expressors retain their suppressive activity even after the second GFP sort, GFP sorted Foxp3/MIGR1 infected GFP+ cells were put into standard suppression assay at indicated ratios with CD25CD4+ responder T cells second time and evaluated for proliferation in 3-day coculture.

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Next we asked which cells actually accumulate ICER/CREM in suppression assays with Foxp3/MIGR infected GFP+ cells. We thus re-sorted GFP positive (GFP+) and GFP negative (GFP) cells after 3-day (72 h) of coculture and evaluated ICER/CREM mRNA levels in both populations (Fig. 3D). Interestingly, GFP responder T cells expressed ICER/CREM equally to GFP+ Foxp3 transductants despite the fact that even after the second GFP sort the suppressive activity of Foxp3/MIGR1 infected GFP+ cells was unabated in control suppression assays performed in parallel (Fig. 3E). These data indicate first, that in suppression assays Foxp3 plays a critical role in ICER/CREM accumulation in TR cells and secondly, that Foxp3 expression in TR cells mediates ICER/CREM induction also in the target population of naive CD25 responder T cells.

Abrogation of TR-mediated suppression by CTLA-4 blockade prevents ICER/CREM accumulation

It was previously demonstrated that blockade with Fab-anti-CTLA-4 (UC-10) mAb abrogated TR-mediated suppression (Fig. 4B) 33. We wished to know whether costimulation blockade through CTLA-4, which is a major B7 receptor transducing inhibitory signal to T cells, would also affect ICER/CREM accumulation in TR assays. ICER/CREM expression was significantly reduced in the presence of Fab-anti-CTLA-4 (UC-10) mAb in a dose-dependent fashion to an extent, which was comparable to the reduction observed in mock assay without CD25+CD4+ TR cells (Fig. 4A). Importantly, engagement of GITR using anti-GITR mAb (DTA-1) caused CD25 responder T cells to escape suppression by CD25+CD4+ TR cells as well 34 (Fig. 4B) and abrogated accumulation of ICER/CREM in TR assays to levels comparable to Fab-anti-CTLA-4 (UC-10) mAb treatment (Fig. 4A). Moreover, blockade with Fab-anti-CTLA-4 (UC-10) mAb leads in TR assays to recovery of IL-2 production in a dose-dependent fashion (Fig. 4C). Similarly, triggering via DTA-1, a GITR mAb, released suppression of IL-2 although to lower extent, perhaps due to unopposed activation and expansion of CD25+CD4+ TR cells. Collectively, these data indicate that ICER/CREM does not accumulate in TR assays under conditions, which caused CD25 responder T cells to escape suppression by CD25+CD4+ TR cells and this lack of accumulation coincides with rescue of IL-2 production.

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Figure 4. ICER/CREM fails to accumulate in TR suppression assays under CTLA-4 blockade or GITR triggering allowing CD25 responder T cells to escape suppression and rescue IL-2 production. (A) ICER/CREM mRNA accumulation is prevented in suppression assays by the presence of Fab-anti-CTLA-4 mAb or DTA-1 mAb recognizing GITR. CD25+CD4+ TR cells and CD25CD4+ responder T cells were mixed in 1:1 ratio and stimulated with anti-CD3 mAb (0.5 μg/mL) and APC in the presence of indicated amounts of Fab-anti-CTLA-4 mAb or DTA-1 mAb recognizing GITR. cDNA samples were subjected to real-time quantitative PCR. Both Fab-anti-CTLA-4 mAb at 100 μg/mL or 20 μg/mL or DTA-1 mAb recognizing GITR at 50 μg/mL or 10 μg/mL abrogated suppression in proliferation assay (B). (C) Concentration of IL-2 in the culture supernatants was determined by ELISA.

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LPS-activated bone marrow-derived nature (m) DC in TR assays represent an important example of complete abrogation of TR- mediated suppression, which allows CD25 responder T cells to escape suppression otherwise provided by standard APC (irradiated T cell-depleted splenocytes, mostly B cells) 35, 36. We wished to know whether abrogation of TR-mediated suppression by LPS activated mDC (Fig. 5A) prevents ICER/CREM accumulation in TR assays. We also wished to know whether lack of ICER/CREM expression during mDC-mediated abrogation of TR suppression (Fig. 5B) is accompanied by a lack of IL-2 inhibition (Fig. 5C). Our studies clearly showed that abrogation of TR mediated suppression in the presence of limited number of mDC (1–5 × 103) completely prevented ICER/CREM accumulation and dramatically enhanced IL-2 expression.

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Figure 5. Abrogation of TR-mediated suppression in the presence of LPS-activated mDC prevents ICER/CREM accumulation and IL-2 inhibition. (A) Replacement of irradiated T cell-depleted splenocytes (standard APC) by LPS-activated bone marrow-derived mDC leads to complete abrogation of suppression reflected by enhanced proliferation in TR assays (TR/mDC; numbers indicate amount of mDC) perturbing CD25+CD4+ TR cell suppressive capacity (TR/B) imposed in the presence of irradiated T cell-depleted APC (mostly B cells). CD25+CD4+ TR cells and CD25CD4+ responder T cells were mixed in a 1:1 ratio with anti-CD3 mAb (0.5 μg/mL) or without stimulation (No Stim) along with the indicated number of irradiated mDC or B cells. Proliferation was determined by measuring incorporation of [3H]thymidine during the last 6 h of 72-h culture. (B) LPS-activated mDC prevented ICER/CREM mRNA accumulation in 72-h culture (TR/mDC vs. TR/B). (C) Diminished IL-2 inhibition is reflected by enhancement of IL-2 mRNA levels in TR suppression assays in accordance with increasing numbers of mDC (TR/mDC vs. Mock/B). cDNA samples were subjected to real-time quantitative PCR analyses as described above.

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Absence of B7 from responder T cells accounts for limited ICER/CREM accumulation

As both CD28 and CTLA-4 molecules are implicated in the function of TR cells, we investigated the ability of their two natural ligands B7.1 (CD80) and B7.2 (CD86) to influence the TR- suppressive capacity 37. The relative expression levels of B7.1 and B7.2 on DC is modulated by TR cells during progression from an immature to a mature state, and this correlates with the ability of TR cells to suppress responses 27, 38. We asked whether upon TCR stimulation specific mAb against B7 molecules expressed on Foxp3 CD25 responder T cells could induce ICER/CREM in the absence of APC. Indeed, ICER/CREM levels were increased early in ConA-stimulated CD25CD4+ responders following a 3-h treatment with anti-B7.1 (16–10A1) while ConA and anti-B7.2 mAb (PO3) triggered only minor accumulation of ICER/CREM (Supporting Information Fig. S4A). This engagement had a profound inhibitory effect on IL-2 production (Supporting Information Fig. S4B). Importantly, B7.1 and B7.2 surface expression was clearly detectable on both CD25CD4+ responder T cells as well as in CD25+CD4+ TR cells (data not shown) 26, 39. Next, we asked whether B7 expression on activated T cells is necessary to effect TR-suppressive activity using cells from B7-deficient mice (B7DKO). B7-DKO CD25CD4+ responder T cells showed increased proliferation and IL-2 production in TR assays in comparison with wild-type responder T cells even in the absence of TR cells (Supporting Information Fig. S4C). Thus, unexpectedly, despite the fact that CD28/B7 costimulation provided by wild-type APC in suppression assays was intact, B7-DKO responder T cells seemed unable to engage CTLA-4/B7 interaction, which at least in part provided for T cell inhibition. Interestingly, only TR suppression assays with wild-type responder T cells but not with B7DKO responders accumulated significant amounts of ICER/CREM after 72 h of incubation (Supporting Information Fig. S4D). Furthermore, B7-DKO responders produced more IL-2 in TR assays both on the mRNA level as well in the form of secreted protein than wild-type responder T cells (Supporting Information Figs. S4E and F). Collectively, our data implicated B7 expressed on activated CD25+ Foxp3 effector T cells in ICER/CREM induction leading to transcriptional attenuation of IL-2 in Foxp3 effectors based on CTLA-4 binding provided by CD25+CD4+ TR cells.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

In sum, these studies identify novel role of ICER/CREM in the suppressive interaction between naturally arising Foxp3+ CD25+ TR cells and target population of Foxp3 effector CD4+ T cells producing IL-2. These conclusions are supported by forced expression of Foxp3 in naive CD25CD4+ T cells, which demonstrate that Foxp3 expression alone is sufficient for induction of constitutive ICER/CREM expression in Foxp3population of responder T cells. Furthermore, transductants expressing Foxp3 can induce ICER/CREM in naive CD25CD4+ T cells and thus replace natural TR cells in suppression assays. Importantly, RNAi targeting of ICER/CREM in naive CD25CD4+ T cells makes these cells refractory to TR-mediated suppression (Fig. 1, 2 and 3 and reviewed in 5). Furthermore, our data suggest that TR-mediated constitutive ICER/CREM expression is induced in contact dependent fashion and attenuates IL-2 production (Fig. 4). Thus, CTLA-4/B7 blockade and/or use of B7-deficient CD25 target T cells (Supporting Information Fig. S4) clearly implicate CTLA-4/B7 interaction in the contact-dependent ICER-mediated inhibition of IL-2 presumably via general inhibitory mechanisms of transcription such as ICER-mediated uncoupling of CBP/p300 5 and/or formation of inhibitory complex between NFAT and ICER (Supporting Information Figs. S1-S3 and reviewed in 32, 40). Specifically, we propose that contact-dependent CTLA-4/B7 interaction provided by Foxp3+ CD25+ TR cells induces ICER/CREM in Foxp3CD4+ T cells and thus directly attenuates their IL-2 transcription (Fig. 6). Indeed, abrogation of TR suppression by LPS-activated DC expressing high levels of B7 prevents ICER/CREM accumulation and allows profound IL-2 production (Fig. 5). Notably, this mechanism is strikingly different from the recently proposed model 41, suggesting that Foxp3-mediated control of TR function operates in TCR-activated T cells through direct cooperation of Foxp3 with NFAT. Such interaction does not seem to be applicable for TR-mediated suppression within population of Foxp3CD4+ T cells, which in mice represent vast majority of IL-2-producing T cells in vivo42.

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Figure 6. Putative model of reverse signaling: CTLA-4-induced amplification of ICER-mediated ‘infectious’ inhibition of IL-2 expression in CD25 responder T cells as a mechanism of TR suppression. Upon TCR activation, CTLA-4 is deployed to the surface of TR cells and a high affinity CTLA-4/B7 interaction leads to the immediate early induction of ICER in TCR-activated Foxp3 responder T cells. Initial IL-2 expression is subsequently attenuated by ICER after 2–4 h of delay necessary for ICER synthesis in Foxp3 responder T cells. Next, activated Foxp3 responders amplify the original TR suppressive signal generated by reverse signaling on the next neighboring TCR-activated Foxp3 responders via CTLA-4/B7 interaction. Thus, the first suppressed Foxp3 responder is mimicking TR cells in its ability to induce ICER in the next neighboring activated Foxp3 responder via CTLA-4/B7 interaction in an ‘infectious’ fashion leading thus to processive ICER-mediated transcriptional attenuation of IL-2 expression. In this model, TR cells modulate activity of APC and/or autoreactive T cells through high affinity CTLA-4/B7 and class II-TCR interactions.

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IL-2 is a well-known T cell growth factor, which is crucial to maintaining peripheral tolerance by supporting the survival and function of CD25+CD4+TR cells 43. Recent studies examining the role of IL-2 in the peripheral formation of TR cells address a seemingly opposing function of IL-2 in vivo44. In the view of the comparisons of NFAT and AP-1 in TCR-mediated transcription of the IL-2 promoter in proliferating T cells 32, 40, we have previously shown that ICER and NFATc2 can also form inhibitory NFAT/ICER complexes on several NFAT/AP-1 DNA-binding sites in vitro14 (see inhibitory model of NFAT/ICER ternary complex provided by courtesy of Dr. Lin Chen, University of Southern California; Supporting Information Figs. S1–S3). We propose that these inhibitory NFAT/ICER complexes could confer TR-mediated suppression into Foxp3CD4+ T cells thus antagonizing highly coordinated expression of NFAT-driven IL-2 promoter.

Additional support for this mechanism is provided by tight link between cAMP-mediated induction of CTLA-4 and ICER in CD4+ T cells 6, 11, 13. This is consistent with our data indicating that CTLA-4 blockade can disrupt accumulation of ICER/CREM, thus releasing contact-dependent TR-mediated suppression. Therefore, we propose that both autonomous as well as reverse signaling may be used by CTLA-4 for ‘processive’ ICER-mediated transcriptional attenuation of IL-2 expression in effector Foxp3 CD4+ T cells (Fig. 6) 5. Moreover, previous reports indicated that CTLA-4/B7 engagement could inhibit extracellular signal-regulated kinases (ERK1 and ERK2) 45 thus protecting ICER from ERK-mediated phosphorylation and subsequent degradation by ubiquitination 46. Therefore, contact-dependent ‘infectious’ tolerance acting through ICER/CREM-mediated inhibition of IL-2 synthesis could explain (i) how a relatively small population of TR cells effectively suppresses a much larger population of Foxp3 responders, and (ii) how inducible CTLA-4 and B7 expression contributes to the spread of this infectious suppression conveyed by induction of ICER/CREM in antigen-nonspecific fashion (Fig. 6). At the same time, TR cells are thought to exert their regulatory potential by acting back on immature DC interfering with their maturation and consequent B7 up-regulation 27. These observations are consistent with our data indicating that LPS-activated bone-derived mDC up-regulating B7 showed lack of ICER/CREM accumulation accompanied by abrogation of TR-mediated suppression (Fig. 5). Thereby, we speculate that synergy between reverse and autonomous CTLA-4 signaling between TR cells and APC may lead to Foxp3-mediated protection of the inhibitory function of ICER/CREM in TR cells along with induction of ICER/CREM function leading to transcriptional attenuation of IL-2 in the suppressed population of Foxp3 effector T cells. We believe that dissecting this regulatory loop dictating induction of constitutive ICER/CREM expression is crucial for further understanding of the maintenance of suppressed phenotype triggered by TR cells in Foxp3 effector T cells (Fig. 6). However, it seems clear that signaling through other inhibitory receptor-ligand interactions such as Fc receptors may confer inhibitory signals reflecting striking mouse strain-specific differences between BALB/c and B6 mice (see Fig. 3E versus Supporting Information Fig. S4C) 47.

Clearly, contact-dependent suppression is conveyed by multiple costimulatory molecules (e.g. GITR) providing additional specificity to the CTLA-4/B7 interaction (Fig. 4) 48. For example, the glucocorticoid-induced leucine zipper (GILZ) gene is an important, yet unstudied, IL-2-inhibiting transcriptional repressor showing high homology with ICER through its leucine-zipper region 15, 49. Remarkably, GILZ is co-expressed in T cells with GITR 50. This relationship appears reminiscent of cAMP-mediated induction of ICER and CTLA-4 6, 13. Remarkably, in TR assays with B7-deficient CD25responders residual suppression is still detectable (Supporting Information Fig. S4), even though ICER/CREM fails to accumulate, perhaps due to the presence of additional transcriptional repressors such as GILZ. It remains to be seen whether GILZ, tightly linked to IL-10-mediated inhibition 51, 52, is also associated with TR-mediated suppression.

Overall, our data suggest that induction of constitutive ICER/CREM expression in Foxp3 effector T cells plays an important although non-exclusive role in contact-dependent TR-mediated suppression, which is conveyed at least in part through CTLA-4/B7 interaction. Such interaction could impose TR-mediated suppression via transcriptional repression facilitated by ICER-mediated uncoupling of CBP/p300 and/or formation of an inhibitory complex between NFAT and ICER. We believe that further explorations will shed more light on TR cell function, which may operate through relatively small number of ‘master’ and ‘effector ‘ regulators in order to induce tolerance in much larger population of Foxp3 effector T cells in contact-dependent infectious fashion.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Mice

BALB/c mice were purchased from CLEA Japan (Tokyo, Japan) or Jackson Laboratories (USA). With exception B7DKO mice of CB57BL/6 background and corresponding WT controls all mice have been BALB/c. Mice were used at 6 to 12 weeks of age and maintained under specific pathogen-free conditions in accordance with institutional guidelines for animal welfare.

Antibodies, reagents, and medium

Purified mAb were purchased from PharMingen (San Diego, CA). Mouse IL-2 was a gift from Shionogi (Japan). T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM 2ME, 10 mM HEPES, and 1 mM sodium pyruvate (all purchased from Sigma, St. Louis, MO). The packaging cell line, Plat-E, was grown in DMEM medium (Sigma) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 10 mM HEPES.

Preparation of T cell subpopulations and antigen-presenting cells

Purity of the CD25+ or CD25CD4+ population using standard protocols was typically >98% and ∼99%, respectively 35. In some experiments, CD25+ or CD25CD4+ cells were first negatively selected for CD4+ T cells (Dynal, Oslo, Norway) then stained by allophycocyanin-conjugated CD25 mAb (PharMingen, San Diego, CA) and then sorted (FACSAria, Becton Dickinson, CA). LPS-activated bone marrow-derived mDC were prepared as previously described 35.

Retroviral constructs and transduction

Retroviral construct encoding the full-length mouse Foxp3 (amino acids 1–429) cloned into a MIGR1 vector was prepared as previously reported 1. Transfection of the packaging cell line, Plat-E, and transduction of primary T cells was performed as described 53.

Proliferation assays and cytokine measurement

Purified T cells (2.5 × 104/well, U-bottom 96-well plate) together with 5 × 104 APC were cultured for 72 h in the presence of 0.5 μg/mL anti-CD3 mAb or 1 μg/mL Con A. [3H]Thymidine (1 μCi/well; Du Pont/NEN) was added during the last 6 h of culture and incorporation was measured as proliferation. Results are expressed as the mean of triplicate cultures, and the SD was usually within 15% of the mean for each experiment. To measure IL-2 protein production, supernatants were harvested at 36 h and concentrations assessed by means of an ELISA (BioSource International, Camarillo, CA).

Single-cell capture assays of IL-2

The cytokine capture assay was performed essentially as described 31. In vitro-activated cells were labeled with the bifunctional Ab "catch" reagent (CD45/IL-2; Miltenyi Biotec, CA) for 5 min on ice and warmed to 37°C for 45 min to allow for IL-2 secretion. Trans-capture of the cytokine was avoided by reducing the cell number used (6 × 105 T cells per time point) and by performing the secretion step in a large volume of medium (3 × 104 cell/mL). Cell surface-bound IL-2 was detected using a second fluorochrome-conjugated IL-2 mAb and analyzed by FACS. T cells were gated for viability by exclusion of DAPI and expression of CD4.

Quantitative RT-PCR

Total RNA was extracted from sorted cells and first strand cDNA was synthesized according to standard protocols. The mRNA levels were quantified by real-time PCR with the ABI/PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). ICER/CREM-specific primers and an internal fluorescent TaqMan probe were designed as follows. ICER/CREM primers: 5′-CTG-CCT-CAC-CAG-GAA-G-3′ and 5′-CAG-CTC-CAG-CTT-GAG-AGT-TG-3′: ICER/CREM probe: 5′-FAM-ACA-GTC-CCC-AGC-AAC-TAG-CAG-AA-TAMRA-3′. HPRT primers: 5′-TGA-AGA-GCT-ACT-GTA-ATG-ATC-AGT-CAA-C-3′ and 5′-AGC-AAG-CTT-GCA-ACC-TTA-ACC-A-3′: HPRT probe: 5′-VIC-TGC-TTT-CCC-TGG-TTA-AGC-AGT-ACA-GCC-C-3′. IL-2 primers: 5′-CCT-GAG-CAG-GAT-GGA-GAA-TTA-CA-3′and 5′-TCC-AGA-ACA-TGC-CGC-AGA-G-3′: IL-2 probe 5′FAM-ACT-CCC-CAG-GAT-GCT-CAC-CTT-CAA-ATT-T-TAMRA-3′. Normalized mRNA values were obtained by dividing by the associated HPRT values. The relative quantity of ICER/CREM or IL-2 mRNA in each sample was normalized to the same sample's quantity of HPRT.

Intracellular ICER/CREM and Foxp3 single-cell staining

Single-cell intracellular staining was performed using Foxp3 Staining Set (eBioscience, CA) following manufacturer's instructions. Briefly, cells from TR assays stained for surface molecules such as CD4 and CD25 were fixed in freshly prepared eBioscience Fix/Perm Buffer and incubated at 4°C for 1 h in dark. After blocking step, Foxp3 antibody or ICER/CREM antibody (CS4 or CREM-1 (X-12) Santa Cruz) were added and anti-rabbit IgG conjugated to FITC was used to visualize CS4 or CREM-1 binding on FACS.

Electroporation of RNAi

Electroporation was performed according to instructions provided by Mouse T cell Nucleofector Kit (Amaxa Biosystems, Gaithersburg, MD). Briefly, freshly sorted CD25 CD4+ T cells (106) were pelleted and resuspended in reconstituted nucleofector solution with 1 μg of plasmid (maxGFP) or 2.5 μg of CREM siRNA (sc-37701; Santa Cruz Biotechnology, CA) and electroporated using Amaxa (program X-01). Nonspecific effects of siRNA were monitored by parallel electroporation using 2.5 μg of non-targeting siRNA (RISC free siRNA, Dharmacon, Lafayette, CO). Immediately after electroporation, cells were transferred into preincubated media in 24-well plate and incubated overnight. In parallel, cells electroporated with maxGFP plasmid were checked for electroporation efficiency under fluorescent microscope.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

We thank Drs. Hiroyuki Yoshitomi, Kyoko Nakamura, Keiji Hirota, Anfisa Stanevsky, John S. Manavalan, and Robert Winchester for helpful comments and suggestions and Dr. Naoshi Sugimoto for advice on gene transduction of Foxp3 to naive mouse lymphocytes. Furthermore, we acknowledge courtesy of Dr. Lin Chen at University of Southern California who kindly provided molecular models of inhibitory complex between NFAT and ICER. The authors have no conflicting financial interests. This work was supported by grants-in-aid from the Ministry of Education, Sports and Culture, the Ministry of Human Welfare of Japan to Shimon Sakaguchi and grants from National Institutes of Health, NIAMS (US) to Betty Diamond.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2007/36510_s.pdf or from the author.

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