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The Journal of Immunology, 2006, 177: 4451-4457.
Copyright © 2006 by The American Association of Immunologists, Inc.

Cell-Autonomous CCL5 Transcription by Memory CD8 T Cells Is Regulated by IL-41

Antoine Marçais, Charles-Antoine Coupet, Thierry Walzer2, Martine Tomkowiak, Raffaella Ghittoni and Jacqueline Marvel3

Institut National de la Santé de la Recherche Médicale, Unité 503, Lyon, France; Institut Fédératif de Recherche 128, BioSciences Lyon-Gerland, Lyon, France; and Université Lyon 1, Villeurbanne, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunological memory is associated with the display of improved effector functions. The maintenance by CD8 memory cells of high levels of untranslated CCL5 mRNA allows these cells to immediately secrete this chemokine upon Ag stimulation. Untranslated mRNA storage is a newly described process supporting the immediate display of an effector function by memory lymphocytes. We have tested the capacity of different cytokines to regulate the memorization of CCL5 by memory CD8 T cells. We found that IL-4 treatment of murine CD8 T cells impairs immediate CCL5 secretion capacity by inhibiting CCL5 mRNA transcription through a STAT6-dependent pathway. The inhibition by IL-4 is reversible, as memory CD8 T cells reconstitute their CCL5 mRNA stores and reacquire their immediate CCL5 secretion capacity when IL-4 is withdrawn. This recovery is cell autonomous because it proceeds in culture medium in the absence of exogenous growth factors, suggesting that CCL5 expression by memory CD8 T cells is a default process. Overall, these results indicate that the expression of CCL5 is an intrinsic property acquired by memory CD8 T cells that is regulated by environmental factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A characteristic of the adaptive immune system is its capacity, following the first encounter with an Ag, to modulate the effector functions that will be displayed upon recall. Indeed, following the differentiation of naive CD8 T cells into memory cells, several new functions acquired during the effector stage will be retained. Examples range from surface phenotype to effector capacities and homing properties (1). The maintenance by memory CD8 T cells of untranslated mRNA coding for the CCL5 chemokine is a newly identified characteristic of memory T cells that supports immediate CCL5 secretion capacity following TCR-dependent rechallenge (2, 3). CCL5 is a CC chemokine that binds three receptors: CCR1, CCR3, and CCR5 (4). Several cell types such as endothelial cells, epithelial cells, or monocytes express elevated levels of CCL5 mRNA and proteins within hours of exposure to proinflammatory stimuli, including TNF-{alpha}, IFN-{gamma}, viruses, or LPS (5, 6, 7, 8, 9). In contrast, in naive T lymphocytes the ccl5 gene is expressed at a late stage of cell activation (3, 10, 11), its mRNA appearing 3 to 4 days following priming. Recently, we and others have demonstrated that, unlike naive cells, resting memory CD8 T cells produce CCL5 protein immediately upon TCR triggering (2, 3, 12). This immediate CCL5 secretion correlates with the maintenance of high levels of stored CCL5 mRNA. Indeed, the CCL5 mRNA levels acquired by activated cells are maintained in CD8 T cells that survive the deletion phase and differentiate into memory cells (3). These elevated CCL5 mRNA levels have been observed in memory CD8 T cells generated in a variety of systems (2, 3, 12, 13). They are maintained through constitutive transcription of the ccl5 gene and increased stabilization of the mRNA (A. Marçais, M. Tomkowiak, T. Walzer, C. A. Coupet, A. Ravel-Chapuis, and J. Marvel, manuscript in preparation).

The immediate CCL5 production by memory CD8 T cells could significantly accelerate the onset of a secondary immune response, particularly outside lymphoid structures. Indeed, production of CCL5 at the site where memory CD8 T cells are activated could lead to the recruitment of immature dendritic, NK, or memory T cells that will accelerate the initiation of an efficient immune response. Moreover, CCL5 secretion could favor memory CD8 T cell stimulation, as it has recently been demonstrated that CCL5 stabilizes the immunological synapse (14).

The cytokine bias associated with Th1 or Th2 CD4 T cell differentiation and the accelerated production of cytokines by memory CD8 T cells are hallmarks of T lymphocyte differentiation toward memory. These processes, dependent on epigenetic modifications, are stable features that can be maintained during the life span of the cell (15, 16). However, recent data indicate that some memory functions can be modulated by the microenvironment (17, 18). The factors acting on memory cells that are leading to these modifications have not been identified. Cytokines could play a role in this process. Indeed, it is well established that cytokines modulate the differentiation pathway of naive cells in effector and memory cells (19, 20, 21). In contrast, the effect of cytokines on established memory CD8 T cell effector functions has been poorly investigated.

In this study, we sought to identify signals responsible for the maintenance of high levels of CCL5 mRNA in memory CD8 T cells. To this aim, we have investigated the effect of several proinflammatory or anti-inflammatory cytokines on the immediate CCL5 secretion capacity of memory CD8 T cells. We found that IL-4, but not the other cytokines tested, down-regulates CCL5 mRNA levels in a STAT6-dependent manner, leading to the loss of the immediate CCL5 secretion capacity of these memory CD8 T cells. However, this inhibition is reversible, as CCL5 mRNA levels and immediate CCL5 secretion capacity are restored upon IL-4 withdrawal.


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

F5 TCR-transgenic mice were gifts from D. Kioussis (National Institute for Medical Research, London, U.K.) (22), and STAT6–/– mice were obtained from S. Akira (Osaka University, Osaka, Japan) (23) and crossed with F5 TCR-transgenic mice. Mice were bred in our animal facility, "Plateau de Biologie Expérimentale de la Souris" (Lyon, France). Thymectomies were performed on 5- to 6-wk-old mice. Three weeks after the thymectomy, F5 mice were injected twice at 24-h intervals with 50 nmol of the A/NT/60/68 influenza virus nucleoprotein peptide 366–374 (Ala-Ser-Asn-Glu-Asn-Met-Asp-Ala-Met; from D. Ficheux, Plateforme Institut Fédératif de Recherche 128, Institut de Biologie et Chimie des Protéines, Lyon, France) in PBS in the peritoneal cavity (15). Memory CD8 T cells were recovered 6 wk after peptide injection. Primed cells were recovered 7 days after immunization of nonthymectomized mice and were similar to memory cells in terms of CCL5 regulation (our unpublished data). They were preferentially used for ethical reasons, as the mice did not need to be thymectomized, and for practical reasons when large numbers of cells were necessary. However, every key result was confirmed using memory cells. In some experiments, F5 lymphocytes purified from spleen and lymph nodes (inguinal, mesenteric, brachial, and axillary) were CFSE stained and adoptively transferred in C57BL/10 recipient mice by intravenous injection. Transferred cells were recovered 6 days later from host spleens and lymph nodes (inguinal, mesenteric, brachial, and axillary) and sorted according to CD8 expression and CFSE staining. All protocols using live mice were performed in accordance with institutional guidelines. Experimental procedures were approved by the Comité Régional d’Ethique pour l’Expérimentation Animale (Lyon, France).

Cells and cell cultures

The medium used for all cultures was DMEM (Invitrogen Life Technologies) supplemented with 6% FBS (PAN Biotech), 50 µg/ml gentamicin, 2 mM L-glutamine (Invitrogen Life Technologies), 10 mM HEPES, and 50 µM 2-ME (Sigma-Aldrich). Serum-free medium was DMEM prepared as described above, except that FBS was not added.

CD8 T cells were purified from spleen and lymph nodes (inguinal, mesenteric, brachial, and axillary) using a negative selection strategy as previously described (15). For sorting, purified CD8 T cells were stained using anti-CD8 (YTS169.4-TC; BD Pharmingen) and anti-CD44 (IM-781-FITC; homemade) Abs. CD8 T cells were sorted into CD44low, CD44intermediate, or CD44high expression levels as previously described (24).

To measure the immediate cytokine secretion capacity of F5 CD8 T cells in response to TCR triggering, 1 or 2 x 105 cells were stimulated for 4 h in 200 µl of medium in U-bottom 96-well plates with nucleoprotein peptide 366–374 at a concentration of 10 nM. CD44high memory phenotype CD8 T cells from non-TCR transgenic mice were stimulated by anti-CD3 Abs coated to microtiter plates in the presence of 2 µg/ml anti-CD28 (clone 37.51; BD Pharmingen). To measure the effect of cytokines on CCL5 production, cells were cultured overnight in the presence or absence of cytokines at a concentration of 3–5 x 106 cells/ml. The next day, viable cells were separated from dead cells by centrifugation on a Ficoll gradient (Cedarlane, Tebu). Live cells were either stimulated in culture as indicated or 4–5 x 106 cells were transferred i.v. into a syngeneic host.

Reagents

Actinomycin D (Sigma-Aldrich) was used at a concentration of 10 µg/ml. Cytokines were used at the following concentrations: murine (m)4 IL-2, 200 U/ml (homemade); mIL-4, 20 ng/ml (PeproTech); mIL-7, 10 ng/ml (R&D Systems); human IL-15, 20 ng/ml (R&D Systems); mIL-10, 50 ng/ml (PeproTech); mIL-13, 50 ng/ml (PeproTech); human TGF-beta1, 10 ng/ml (PeproTech); mIFN-{alpha}A: 1500 U/ml (Tebu); mIFN-beta, 1500 U/ml (Tebu); and mIFN-{gamma}, 50 ng/ml (R&D Systems).

Multiprobe RNase protection assay

CCL5 mRNA level was measured by RNase protection assay (RPA) using the RiboQuant kit (BD Pharmingen) following the supplier’s instructions. The quantity of protected RNA was determined using a PhosphorImager and the ImageQuant software (both from Molecular Dynamics). Relative CCL5 mRNA level was normalized using the internal control, L32 mRNA.

CCL5 and IFN-{gamma} ELISA

ELISA (R&D Systems) was used to measure the CCL5 and IFN-{gamma} contents in culture supernatants according to the supplier’s instructions.

Extraction and retrotranscription of nascent mRNA transcripts

To measure gene transcription level, 2–5 x 107 CD8 T cells were purified and treated as indicated, and nuclear chromatin was isolated as described (25, 26). All steps were performed at 4°C with RNase-free reagents. Briefly, T cells were lysed for 5 min in 0.5% Nonidet P-40, 140 mM NaCl, 1.5 mM MgCl2, 50 mM Tris (pH 8.0), 1 mM DTT, and 1000 U/ml RNase inhibitor. Nuclei were pelleted at 300 x g for 5 min and lysed for 10 min in 1% Nonidet P-40, 300 mM NaCl, 7.5 mM MgCl2, 1 M urea, 0.5 mM EDTA, 20 mM HEPES (pH 7.6), 1 mM DTT, and 1000 U/ml RNase inhibitor. Chromatin was isolated by centrifugation at 15,000 x g for 10 min. Nascent mRNA transcripts bound to chromatin were purified using the NucleoSpin RNA II kit (Macherey-Nagel) with DNase I treatment. Purified RNAs were resuspended in 40 µl of water. Reverse transcription was performed using 9.3 µl of RNA solution using SuperScript II (Invitrogen Life Technologies) and random primers according to the manufacturer’s instructions. The cDNAs obtained were diluted three times, and 5 µl thereof was subjected to real-time PCR. The relative level of the CCL5 mRNA (target sequence) was normalized to that of the ubiquitin mRNA (reference sequence). To monitor genomic contamination, a control was prepared for each sample following the same procedure except for the reverse transcription step. In all experiments performed, no CCL5 sequence could be detected in these control samples.

Quantification by real-time PCR

Real-time PCR analysis was conducted using Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen Life Technologies) on an ABI Prism 7700 (PerkinElmer). The mix contained 10 µl of Platinum mix, 0.8 µl of primers (10 µM each), 0.6 µl of ROX reference dye, 3.6 µl of H2O, and a template in 5 µl of water. For PCR amplification, samples were incubated at 50°C for 2 min followed by 10 min at 95°C and then 40 cycles of incubation at 95°C for 10 s and 60°C for 1 min. The dissociation curve was analyzed for each sample. Relative level of the target sequence against the reference sequence was calculated using the {Delta}{Delta} cycle threshold method with calculated real efficiencies (27). The sequence of primers used are available upon request.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-4 treatment inhibits CCL5 secretion by memory CD8 T cells

To study the role of cytokines in the maintenance of CCL5 mRNA levels by memory CD8 T cells, we studied the effects of a panel of proinflammatory or anti-inflammatory cytokines on the immediate CCL5 secretion capacity of memory CD8 T cells. Naive or memory F5 CD8 T cells were sorted and cultured in the presence of cytokines, and 20 h later their capacity to produce CCL5 protein following TCR engagement was measured. As shown in Fig. 1A, when memory cells are maintained overnight in culture with or without cytokines, the immediate CCL5 secretion capacity is maintained, if not increased, as compared with directly ex vivo isolated cells. However, in contrast to the other cytokines tested, IL-4 induces a strong decrease in the immediate CCL5 secretion capacity in response to TCR stimulation. IL-13, which is closely related to IL-4 (28), did not induce a decrease in the immediate CCL5 secretion capacity or an increase in cell viability as compared with control cells grown in medium only, confirming the lack of IL-13-specific receptors at the surface of CD8 T cells (29). None of the cytokines was able to induce naive cells to produce CCL5. Importantly, the effect of IL-4 on the immediate CCL5 production capacity did not result from a general impairment of memory cell functions, as the TCR induced immediate IFN-{gamma} secretion capacity was not decreased by IL-4 treatment. Indeed, the production of IFN-{gamma} by memory cells following TCR stimulation was maintained or significantly increased (Fig. 1B). The effect of IL-4 on the capacity of memory cells to produce CCL5 and IFN-{gamma} is also observed when non-TCR-transgenic C57BL/10 CD8 T cells with a memory phenotype are used (Fig. 1C). Finally, the effect of IL-4 is not likely due to an increased mortality, because similar cell viabilities were observed when cells were maintained with IL-4, IL-2, IL-7, or IL-15 (Fig. 1A). Moreover, we compared in parallel the effect of IL-4 on memory CD8 T cell viability and on their immediate CCL5 secretion capacity and showed that as little as 0.1 ng/ml IL-4 is sufficient to partially inhibit the immediate CCL5 secretion capacity while maintaining cell viability (Fig. 1D). The maximal inhibition is obtained when IL-4 concentrations are superior or equal to 10 ng/ml. These results indicate that IL-4 inhibits the immediate CCL5 secretion capacity of memory CD8 T cells at concentrations that induce other cellular responses such as inhibition of apoptosis. Thus, IL-4, but none of the other cytokines tested, is able to suppress the immediate CCL5 secretion capacity of memory CD8 T cells.


Figure 1
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  		FIGURE 1. IL-4 suppresses the immediate CCL5 secretion capacity of memory CD8 T cells. Sorted naive or memory F5 CD8 T cells were incubated without cytokine or with IL-2, IL-4, IL-7, IL-15, IL-10, IL-13, TGF-beta, IFN-{alpha}, IFN-beta, or IFN-{gamma} for 20 h. The immediate CCL5 secretion capacity of cells in response to TCR engagement was measured before treatment (ex vivo) and following incubation with cytokine. A, The quantity of CCL5 produced in a 4-h assay was quantified by ELISA. The percentage of viable cells at the end of the cytokine treatment is given. The mean of three independent experiments is shown (±SD). B and C, The quantity of CCL5 or IFN-{gamma} produced by memory CD8 T cells in response to TCR triggering was measured in a 4-h assay before (ex vivo) or after a 20-h IL-4 treatment (IL-4). Protein secretion is expressed as the percentage of protein secreted by CD8 T cells directly stimulated ex vivo. Memory CD8 T cells purified from immunized F5 mice (B) or memory phenotype CD8+CD44high T cells sorted from 6- to 9-mo-old C57BL/10 mice (C) were used. The mean of four independent experiments is shown (±SD). D, F5 memory CD8 T cells were purified and incubated with different concentrations of IL-4 for 20 h. At the end of the IL-4 treatment, the percentage of apoptotic cells and the quantity of CCL5 produced in response to TCR triggering in a 4-h assay are given. CCL5 secretion is expressed as percentage of the secretion of cells maintained without IL-4. The mean (±SD) of two independent experiments is shown. Statistically significant differences, compared with ex vivo cells, are marked (Student’s t test: n.s., nonsignificant; **, p < 0.01; ****, p < 10–4).

 
IL-4 down-regulates the level of CCL5 mRNA by inhibiting ccl5 gene transcription

The immediate CCL5 secretion capacity of memory CD8 T cells relies on the presence of a pool of untranslated CCL5 mRNAs. We have previously shown that the maintenance of this pool of CCL5 mRNAs by CD8 T cells results from the constitutive transcription of the gene associated with an increased stability of the mRNA (A. Marçais, M. Tomkowiak, T. Walzer, C. A. Coupet, A. Ravel-Chapuis, and J. Marvel, manuscript in preparation). The IL-4-mediated inhibition could thus act on the maintenance of the CCL5 mRNA pool by interfering with its production or stabilization. Alternatively, IL-4 could act by inhibiting the translation or secretion of CCL5 in response to TCR engagement. We first tested the effect of IL-4 treatment on CCL5 mRNA levels. F5 memory CD8 T cells were purified, and CCL5 mRNA levels were measured by RPA before or after IL-4 treatment. As shown in Fig. 2A, a 20-h IL-4 treatment induces a dramatic decrease in the CCL5 mRNA content of memory cells. This effect of IL-4 was not immediate, as a time course analysis showed a slow decrease in CCL5 mRNA levels during the treatment (Fig. 2B). As expected, in the absence of cytokines the CCL5 mRNA content of CD8 T cells was maintained (Fig. 2B). The 10-h estimated half-life of CCL5 mRNA in the presence of IL-4 was similar to the one measured using actinomycin D to inhibit transcription (data not shown). Moreover, the ratio between the CCL5 mRNA levels remaining after 10 h of treatment with IL-4 plus actinomycin D and treatment with actinomycin D alone is very close to 1 (0.97 ± 0.05), suggesting that IL-4 principally acts on mRNA generation rather than stabilization. We next measured the effect of IL-4 on the transcription of ccl5. The level of ccl5 transcription in memory CD8 T cells was measured by quantifying the nascent CCL5 mRNA that was associated with nuclear chromatin as described in Materials and Methods. The results in Fig. 2C show that the level of transcription observed in memory cells is strongly inhibited by incubation with IL-4. In contrast, the transcription of the antiapoptotic gene bcl2 was induced by IL-4. This finding is in agreement with a previous report showing that IL-4 induces Bcl-2 transcript accumulation in CD8 T cells (30).


Figure 2
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  		FIGURE 2. IL-4 down-regulates the level of CCL5 mRNA by inhibiting ccl5 transcription. A, The level of CCL5 mRNA was measured by RPA in memory F5 CD8 T cells before or after incubation in medium with IL-4 for 20 h. CCL5 mRNA level normalized by L32 mRNA is shown. One representative experiment is shown, and the relative CCL5 mRNA level representing the mean (±SD) of three independent experiments is given. B, CCL5 mRNA level in primed F5 CD8 T cells was measured by RPA at different times following IL-4 addition; control cells were incubated in culture medium only. The CCL5 mRNA level measured by RPA and normalized by L32 is shown for one representative experiment. C, The ccl5 and bcl-2 transcription levels were measured by quantifying nascent mRNA transcripts as described in the methods before and after a 4-h IL-4 treatment of memory F5 CD8 T cells. The mean of three independent experiments (±SD) is shown. D, Memory F5 CD8 T cells were stimulated by peptide (Pep) in the presence (+) or absence of IL-4 for 4 h. The quantity of CCL5 produced was measured by ELISA. Protein production is expressed as the percentage of protein secreted by CD8 T cells stimulated without IL-4. The mean of three independent experiments (±SD) is shown. Statistically significant differences are marked (Student’s t test: n.s., non significant; **, p < 0.01; ***, p < 10–3).

 
Finally, the capacity of IL-4 to interfere with CCL5 mRNA translation or secretion was measured by comparing the quantity of CCL5 produced by freshly isolated memory CD8 T cells in a 4-h assay in response to TCR engagement in the presence or absence of IL-4. The results shown in Fig. 2D indicated that IL-4 does not act on the translation or secretion of CCL5, as similar quantities of cytokine were produced in its presence or absence.

Overall, these results indicate that IL-4 inhibits the immediate CCL5 secretion capacity of memory CD8 T cells by inhibiting its constitutive transcription, and they stress that the transcription of ccl5 is a major factor controlling the immediate CCL5 secretion capacity of memory CD8 T cells.

IL-4-mediated inhibition of CCL5 expression is dependent on STAT6

STAT6 is the main transcription factor activated by IL-4 (23, 31, 32), Indeed, when IL-4 binds to its receptor it induces the activation of JAK 1 and 3 that, in turn, phosphorylate cytoplasmic STAT6, which then undergoes dimerization and is translocated to the nucleus where it can either activate (23) or repress gene expression (33). We thus decided to study its involvement in the regulation of CCL5 expression by IL-4. Memory CD8 T cells were generated in F5 or STAT6–/– x F5 mice, and the capacity of IL-4 to inhibit the immediate CCL5 secretion capacity was measured in both cell types. We first measured the capacity of memory CD8 T cells to produce CCL5 or IFN-{gamma} protein following TCR engagement. It was similar in both types of memory CD8 T cells. As shown in Fig. 1, in F5 memory CD8 T cells CCL5 and IFN-{gamma} production were strongly modulated by IL-4 pretreatment. In contrast, in STAT6–/– x F5 memory CD8 T cells the production of both cytokines was not affected by IL-4 pretreatment (Fig. 3A). Similar results were obtained using memory phenotype CD8 T cells from non-TCR transgenic wild-type (WT) and STAT6–/– mice (Fig. 3B). Consistent with the results shown in Fig. 2A, no IL-4-induced decrease in the CCL5 mRNA content was observed in the STAT6–/– x F5 memory CD8 T cells (Fig. 3C). In contrast, the prosurvival activity of IL-4 was not affected by the absence of STAT6 (Fig. 3D). Thus, these results showed that the inhibition of CCL5 expression by IL-4 in memory CD8 T cells is dependent on STAT6.


Figure 3
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  		FIGURE 3. Inhibition of immediate CCL5 secretion capacity by IL-4 is STAT6 dependent. A and B, Memory CD8 T cells sorted from F5 or F5 x STAT6–/– mice (A) or memory phenotype CD8+CD44high T cells sorted from 6- to 9-mo-old WT or STAT6–/– mice (B) were treated with IL-4 for 20 h. Immediate CCL5 and IFN-{gamma} secretion capacity were measured before (filled histograms) or after IL-4 treatment (open histograms). Protein secretion is expressed as the percentage of protein secreted by CD8 T cells directly stimulated ex vivo. C, CCL5 mRNA content of F5 or F5x STAT6–/– memory F5 CD8 T cells was measured by RPA before or after IL-4 treatment. CCL5 mRNA content normalized by L32 mRNA is shown. One representative experiment and the mean of three independent experiments (±SD) are shown. D, The percentage of viable cells at the end of the IL-4 treatment is given for F5 or F5 x STAT6–/– memory CD8 T cells. The mean of three independent experiments is shown (±SD). Statistically significant differences are marked (Student’s t test: n.s., non significant; *, p < 0.05; **, p < 0.01; ***, p < 10–3).

 
IL-4-mediated CCL5 mRNA decrease is reversible

We next tested whether the inhibition of the immediate CCL5 secretion capacity by IL-4 was reversible; i.e., could CD8 T cells reconstitute their CCL5 mRNA content when IL-4 was removed? We first measured the CCL5 mRNA pool reconstitution by CD8 T cells following in vivo parking (Fig. 4A). Memory F5 CD8 T cells were sorted from spleen and lymph nodes and labeled with CFSE before culture with IL-4 for 20 h. Cells were then transferred in syngeneic mice, and the CFSE-positive CD8 T cells were recovered 6 days later from spleen and lymph nodes. CFSE labeling of CD8 T cells did not detect cell division during the experiment (data not shown). The mRNA content and the capacity of memory cells to produce CCL5 were measured at different stages of the experiment as depicted on Fig. 4A. Naive CD8 T cells were used as control. As expected, compared with directly isolated ex vivo cells, the mRNA levels of memory CD8 T cells and their immediate CCL5 production capacity in response to TCR triggering were strongly reduced after a 20-h culture in the presence of IL-4. However, after parking for 6 days in vivo, CFSE-positive CD8 T cells had recovered high levels of CCL5 mRNA and their immediate CCL5 secretion capacity (Fig. 4, B–D). In contrast, the IFN-{gamma} secretion-capacity of memory CD8 T cells was similar at all steps (Fig. 4E). The increase in CCL5 mRNA levels was not due to the selection of a small subset of cells. Indeed, a similar percentage of transferred cells was recovered from hosts whether naive or memory cells were transferred (data not shown). Moreover, each subset displayed comparable surface phenotype in terms of CD8 and CD44 expression as it did before the parking. Additionally, culture conditions did not impair the capacity of CD8 T cells to settle in the host, as the same number of cells was recovered from host receiving freshly isolated cells or cells cultured for 20 h (data not shown). Finally, CCL5 re-expression is not due to T cell activation following transfer, as control naive cells did not express CCL5 mRNA after the same experimental procedure (Fig. 4, B–D). The reacquisition of CCL5 by memory cells is a relatively rapid phenomenon, as cells parked in vivo for 20 h have already recovered their immediate CCL5 secretion capacity (data not shown).


Figure 4
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  		FIGURE 4. IL-4-mediated CCL5 mRNA decrease is reversible. CD8 T cells from naive or memory F5 mice were sorted. After CFSE labeling they were cultured for 20 h with IL-4 before i.v. transfer into C57BL/10 recipient mice. After 6 days, recipient mice were sacrificed and CD8+CFSE+ cells were sorted. At different steps as depicted in A, the CCL5 mRNA levels and the immediate CCL5 and IFN-{gamma} secretion capacity of naive (open histogram) or memory (filled histogram) CD8 T cells were measured. The CCL5 mRNA levels measured by RPA for one representative experiment are shown in B. The mean of three independent experiments (±SD) is shown in C. The levels of CCL5 (D) and IFN-{gamma} proteins (E) in the culture supernatant after a 4-h TCR stimulation are given. Statistically significant differences are marked between points indicated by brackets (Student’s t test: n.s., non significant; *, p < 0.05; **, p < 0.01).

 
The recovery of CCL5 mRNA is cell autonomous

To determine whether exogenous signals present in vivo are necessary for CD8 T cells to restore their immediate CCL5 secretion capacity, we followed it in vitro. Primed F5 CD8 T cells were cultured 20 h in the presence of IL-4. The cells were then washed and replated in complete culture medium, serum-free medium, or medium plus IL-4. Five hours later cells were harvested, and their immediate CCL5 secretion capacity in response to TCR triggering was assessed. As shown in Fig. 5A, the capacity to rapidly produce CCL5 protein in response to TCR triggering is recovered as soon as IL-4 is withdrawn, even when cells were plated in a culture medium that does not contain serum-derived factors. In contrast, when cells were replated in culture medium containing IL-4, immediate CCL5 production capacity was still inhibited. In parallel, we measured the ccl5 transcription levels. As shown in Fig. 5B, ccl5 transcription resumed as soon as IL-4 was withdrawn, paralleling what was observed for immediate CCL5 secretion capacity. These results indicate that the reacquisition of immediate CCL5 secretion capacity by memory CD8 T cells is cell autonomous and is due to a restart of ccl5 transcription.


Figure 5
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  		FIGURE 5. The reconstitution of CCL5 mRNA stores is a cell-autonomous process. A, Primed CD8 T cells were purified and cultured for 20 h with IL-4. Cells were then washed, and live cells were replated for 5 h in medium ({square}), medium plus IL-4 ({diamondsuit}), or serum-free medium ({triangleup}). Immediate CCL5 secretion capacity was measured at each time point and expressed as percentage of ex vivo secretion (not shown). The mean of three independent experiments (±SD) is shown. B, Primed CD8 T cells were purified and cultured for 20 h with IL-4. Cells were then washed, and live cells were replated for 4 h in medium alone. The ccl5 transcription level was measured at each step as described in Materials and Methods. The mean of three independent experiments is shown (±SD). Statistically significant differences are marked between points indicated by brackets (Student’s t test: *, p < 0.05; ***, p < 10–3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we show that immediate CCL5 secretion capacity of memory CD8 T cells can be shut down by IL-4. Indeed, following IL-4 treatment of memory CD8 T cells, ccl5 constitutive transcription is stopped, and this leads to depletion of the CCL5 mRNA stores, abolishing the immediate CCL5 secretion capacity of memory CD8 T cells that is induced by TCR engagement. IL-4 acted on the ccl5 transcription at concentrations that induce other physiological responses of the cell. Indeed, cell survival and inhibition of ccl5 transcription were induced by similar concentrations of IL-4. The ability of IL-4 to down-regulate immediate CCL5 secretion capacity is not a general property of anti-inflammatory cytokines, as TGF-beta or IL-10 had no effects on the immediate CCL5 secretion capacity of memory CD8 T cells. The fact that IL-13 had no effect in contrast to IL-4 is likely due to the lack of IL-13R{alpha} expression by CD8 T cells (29). Moreover, the effect of IL-4 is specific, because other cytokines that signal through the {gamma}c common receptor chain, such as IL-2, IL-7, or IL-15, did not inhibit this function of memory CD8 T cells. This ability of IL-4 to inhibit CCL5 expression is not restricted to memory CD8 T cells, as it was previously shown that IL-4 is able to inhibit CCL5 expression by endothelial cells (7).

Treatment of memory CD8 T cells by IL-4 resulted in the inhibition of ccl5 transcription. In contrast, the immediate IFN-{gamma} secretion capacity, as well as the survival of memory CD8 T cells, was increased by IL-4 treatment. This indicates that IL-4 does not act by inducing a general nonresponsive state of memory CD8 T cells that would inhibit their effector functions. The inhibition of ccl5 transcription induced by IL-4 was dependent on STAT6. IL-4 was, however, still active, as the IL-4-induced survival of CD8 T cells proceeded in the absence of STAT6. This finding is in agreement with previous studies (34). STAT6 can inhibit gene expression either by acting on the histone acetylation level of target gene promoter (35) or by interfering with the binding or the activity of transcription factors such as NF-{kappa}B (36, 37). For the ccl5 promoter, preliminary results suggest that the acetylation level of histones associated with the promoter is not decreased by IL-4 treatment. Thus, STAT6 could act by interfering with the transcription factors that are involved in the constitutive transcription of ccl5. NF-{kappa}B is involved in the expression of ccl5 (38) and could be the target of STAT6 in CD8 T cells. Additional experiments will be necessary to clarify this point.

Additionally, we show that the inhibition of ccl5 transcription by IL-4 is reversible. Indeed, as soon as the cells are placed in an environment devoid of IL-4, ccl5 transcription resumes, and the CCL5 mRNA stores as well as the immediate CCL5 secretion capacity are rapidly recovered. These results indicate that ccl5 expression by memory cells has become a stable feature, suggesting that the ccl5 gene is now epigenetically tagged for expression. This recovery occurs not only in vivo where cells could respond to exogenous signals but also in vitro in a medium that is devoid of FBS-derived growth factor or cytokines. In this situation, the only cell contact or cytokine signal would be derived from CD8 T cells. This suggests that ccl5 transcription in memory CD8 T cells is a cell-autonomous process that can take place in conditions under which signals from other cell types are absent.

IL-4 is principally known for its regulatory role in the Th1/Th2 differentiation pathway. However, the regulation of CD8 differentiation and effector functions by IL-4 has also been described. Indeed, IL-4 has been shown to enhance developing CD8 T cell cytolytic functions (39, 40) and to play a role in the differentiation and maintenance of memory CD8 T cells (21, 41, 42). Here we show that IL-4 is also able to act on established effector functions in differentiated memory cells. This type of regulation could be an important feature of immune response regulatory circuits. In a Th1-biased context such as the one associated with anti-viral response, CCL5 would not be down-regulated, and early CCL5 production could contribute to the accelerated memory response. In contrast, the down-regulation of the immediate CCL5 secretion capacity in the context of a Th2, IL-4-enriched environment could be an important regulatory mechanism in inflammatory pathologies. Indeed, infiltration of CD8 T containing high levels of CCL5 is associated with a number of inflammatory diseases (43, 44), and CCL5 invalidation in CD8 T cells leads to decreased inflammatory response (45, 46), highlighting the contribution of CCL5 production by CD8 T cells to these inflammatory pathologies. Thus, production of high levels of IL-4 by NKT cells or Th2 cells could down-regulate the CCL5 production of CD8 T cells and lead to a decreased inflammation.

In conclusion, depending on the context of the immunological response, IL-4 could act on memory CD8 T cells to fine tune their accelerated effector functions.


   Acknowledgments
 
We thank C. Arpin and Y. Leverrier for critical reading of this manuscript. We thank S. Akira and S. Uematsu for STAT6-deficient mice. We thank the staffs of the Plateau de Biologie Experimentale de la Souris and the Plateforme de Cytométrie en Flux for their technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche contre le Cancer, Ligue Régionale de Lutte Contre le Cancer, Région Rhône-Alpes (Contract 00816045), Université Claude Bernard Lyon 1, and Cancéropole Nationale. Back

2 Current address: Centre d’Immunologie de Marseille-Luminy, Campus de Luminy, Case 906, Marseille, F-13288 France. Back

3 Address correspondence and reprint requests to Dr. Jacqueline Marvel, Institut National et la Santé et de la Recherche Medicalé, Unité 503, 21 avenue Tony Garnier, Lyon, France. E-mail address: marvel{at}cervi-lyon.inserm.fr Back

4 Abbreviations used in this paper: m, murine; RPA, RNase protection assay; WT, wild type. Back

Received for publication June 1, 2006. Accepted for publication July 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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