Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability

Maintenance of CCL5 mRNA stores by post-effector andmemory CD8 T cells is dependent on transcription and iscoupled to increased mRNA stability

Antoine Mar�ais1,2,3, Martine Tomkowiak1,2,3, Thierry Walzer*1,2,3,Charles-Antoine Coupet1,2,3, Aymeric Ravel-Chapuis2,3,4 andJacqueline Marvel1,2,3

1 INSERM, U503, Lyon, France2 IFR128, BioSciences Lyon-Gerland, Lyon, France3 Universit� Lyon 1, Villeurbanne, France4 CNRS, UMR5161, Lyon, France

Immunological memory is associated with the display of improved effector functions bycells of the adaptive immune system. The storage of untranslated mRNA coding for theCCL5 chemokine by CD8 memory cells is a new process supporting the immediatedisplay of an effector function. Here, we show that, after induction during the primaryresponse, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We haveinvestigated the mechanisms involved in the long-term maintenance of CCL5 mRNAlevels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life isincreased in memory CD8 Tcells and that these cells constitutively transcribe ccl5 gene.By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role oftranscription in the maintenance of CCL5 mRNA stores. Finally, we show that thesestores are spontaneously reconstituted when the inhibitory signal is removed, indicatingthat the transcription of ccl5 is a default feature ofmemory CD8 Tcells imprinted in theirgenetic program.

Introduction

Chemokines are a family of proteins that play anessential role in the recruitment of leukocytes toinflammatory sites. CCL5 is a CC chemokine previouslytermed RANTES, which can bind three differentreceptors: CCR1, CCR3 and CCR5 [1]. Several celltypes express elevated levels of CCL5 mRNA andproteins within hours of exposure to pro-inflammatorystimuli including TNF-a, IFN-c, viruses or LPS [2–6]. In

naive T lymphocytes, the ccl5 gene is expressed at a latestage of T cell activation [7–9], its mRNA appearing 3 to4 days following priming. Recently, others and we havedemonstrated that, in contrast to naive cells, memoryCD8 T cells produce CCL5 protein immediately uponTCR triggering [9 11]. This immediate production issimilar to what has been described for cytokines such asIFN-c [12, 13] and is a hallmark of a memorizedfunction. The immediate CCL5 production by memoryCD8 T cells could significantly accelerate the onset of asecondary immune response.

The immediate CCL5 secretion correlates with themaintenance of high levels of untranslated CCL5 mRNA.Indeed, the CCL5 mRNA levels acquired by activated

Molecular immunology

Correspondence: Dr. Jacqueline Marvel, Equipe Immuno-Apoptose, INSERM U503, 21 avenue Tony Garnier, 69365 Lyoncedex 07, FranceFax: +33-437-282-341e-mail: [email protected]

Received 10/5/06Revised 18/7/06

Accepted 21/8/06

[DOI 10.1002/eji.200636259]

Key words:CCL5 � Memory

CD8 T cells � mRNAstabilization

� Transcription

Abbreviations: ChIP: chromatin immunoprecipitation �NP: nucleoprotein peptide � RPA: RNase protection assay

Eur. J. Immunol. 2006. 36: 2745–2754 Molecular immunology 2745

f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

* Present address: Centre d'Immunologie deMarseille-Luminy, Campus de Luminy, case 906,13288 Marseille cedex 09, France

cells are maintained in CD8 T cells that survive thedeletion phase and differentiate into memory cells[9–11, 14]. In the absence of TCR stimulation, memoryCD8 T cells do not secrete any CCL5 protein. However,following TCR triggering, they are able to produce it, in amatter of minutes, by translating the stored mRNA [9,10]. The accelerated production of CCL5 by memoryCD8 T cells is the first memory effector functiondescribed relying on the storage of untranslated mRNA.

Different processes that are characterized by a rapidor accelerated response also rely on the presence ofpools of untranslated mRNA. In differentiated NKT ormast cells, the immediate cytokine production capacityis associated with the presence of untranslated cytokinemRNA [15–17]. Similarly, in the nervous system, long-termpotentiation, amemorization process, is dependenton the storage of mRNA at the level of synapses [18].However, in those systems, themechanisms allowing themaintenance of the mRNA pool are still unknown.Indeed, post-transcriptional mechanisms leading to anincreased mRNA half-life or sustained transcriptioncould both be involved in this process.

We have measured the relative contributions ofmRNA stabilization versus transcription to the main-tenance of high levels of CCL5 mRNA in memoryCD8 T cells. We show that there is an increase in thestability of CCL5 mRNA. However, the maintenance ofthe CCL5 mRNA pool is strongly dependent on thetranscription of the gene. Indeed, by directly measuringccl5 gene transcription level, we show that ccl5 isconstitutively transcribed by memory cells. The main-tenance of transcription is correlated with ccl5 promoteracetylation. We also show that the immediate CCL5-production-capacity is lost following IL-4 treatment ofmemory cells and that this results from an inhibition ofthe ccl5 transcription. However, as soon as IL-4 iswithdrawn, transcription of ccl5 resumes, indicatingthat transcription of ccl5 has become a dominant featureof memory CD8 T cells imprinted in their geneticprogram.

Result

CCL5 mRNA levels are maintained in primedCD8 T cells in the absence of specific signals

Memory CD8 T cells maintain the levels of CCL5 mRNAacquired by effector cells following the primarystimulation (Fig. 1A and B). This allows them toimmediately produce the protein in response to peptidestimulation (Fig. 1C and [9]). We first characterized themaintenance of CCL5 mRNA in the absence ofexogenous signals in post-effector (cells recovered7 days after immunization) or memory (cells recovered

40 days after immunization) CD8 T cells compared tofibroblasts. CCL5 expression by NIH 3T3 fibroblasts wasinduced with TNF-a and IFN-c. The capacity to maintainCCL5 mRNA levels in F5 CD8 T cells in the absence ofTCR signaling or in NIH 3T3 fibroblasts after withdrawalof the cytokines was measured. At different time points,cells were harvested and CCL5 mRNA was quantified byan RNase protection assay (RPA). We found that in NIH3T3 fibroblasts, CCL5 mRNA levels decreased steadilyover an 8-h period (Fig. 2A). In contrast, in post-effectoror memory CD8 T cells the CCL5 mRNA level does notdecrease over the same period (Fig. 2B and C). Thedecrease observed in fibroblasts was not due to dilutionassociated with proliferation, as the number of livefibroblasts in culture remained constant over the time ofthe experiment (data not shown). These results indicate

Figure 1. In CD8 T cells, CCL5 mRNA levels are maintained lateafter priming. F5 mice were immunized and at the indicatedtimes, mice were sacrificed and single-cell suspension wereprepared from spleen and lymph nodes. (A) The total numberof CD8 per spleen determined by CD8 staining and lymphocytecount is shown (dotted line). CCL5 mRNA content of purifiedCD8 T cells determined by RNase protection assay (RPA) is alsoshown (continuous line). The mean of four independentexperiments (� SD) is shown. Statistically significant differ-ences were found using a Student's t-test, when CCL5 mRNAcontent on day 2 was compared to mRNA content on day 7(p <0.05) and day 40 (p <0.0001). (B) A representative gel fromone experiment is shown. (C) The immediate CCL5-secretion-capacity of CD8 T cells purified 2, 3, 7 or 40 days followingpriming was measured by incubating cells 4 h in vitro with10 nMpeptide. The CCL5 spontaneous secretionwasmeasuredby incubating cells 4 h in medium alone. The mean (� SD) ofthree independent experiments is shown.

Antoine Mar�ais et al. Eur. J. Immunol. 2006. 36: 2745–27542746


that the maintenance of high CCL5 mRNA levels is aspecific feature of the cell type but not of the CCL5mRNA.

CCL5 mRNA half-life is enhanced in memoryCD8 T lymphocytes

This maintenance could result either from an increasedstability of the CCL5 mRNA or from the constitutivetranscription of the ccl5 gene by memory cells. Todetermine the mechanisms involved in the maintenanceof CCL5 mRNA levels by primed CD8 T cells we firstmeasured the half-life of CCL5 mRNA in post-effectorCD8 T cells by assaying mRNA levels by RPA after 5- or10-h incubation in the presence of the transcriptioninhibitor actinomycin D. The actinomycin D dose weused totally inhibits transcription in CD8 T cells (datanot shown). As controls, the level of two mRNAs codingone for the chemokine receptor CCR7 and the other for ahousekeeping gene, the ribosomal protein L32, werealso measured. As shown in Fig. 3A and B, the levels ofCCR7 and L32 mRNA decreased rapidly upon actino-mycin D treatment with an estimated half-life of less

than 3 h for CCR7 and 5 h for L32. In contrast, the CCL5mRNA half–life was much longer being superior to 10 h.To determine if the increased half-life of CCL5 was anintrinsic property of CD8 T cells or if this property wasacquired by post-effector cells that differentiate intomemory cells, we also monitored the half-life of CCL5mRNA in freshly activated CD8 T cells compared tomemory CD8 Tcells. Activated CD8 Tcells at the peak ofthe primary response, 3 days after priming (Fig. 1A), ormemory CD8 Tcells were purified and CCL5mRNA half-life was measured using actinomycin D. The half-life ofCCL5 mRNA was strongly increased in memory cellscompared to activated cells (Fig. 3C). This was not dueto a general stabilization of mRNA in memory cells assimilar half-lives were found in freshly activated andmemory cells for the control mRNA CCR7 and L32(Fig. 3C). These results indicate that following activa-tion CD8 Tcells that differentiate in memory CD8 Tcellsbecome able to stabilize the CCL5 mRNA. This increasedstability could contribute to the establishment of theCCL5 mRNA stores by memory cells. However, althoughthe CCL5 mRNA is more stable in memory CD8 T cellscompared to the other mRNA species that we havemonitored or to what was found in freshly activatedcells, its half-life does not support the hypothesis thatmRNA produced during the primary response aremaintained for up to 40 days (Fig. 1) after priming.Hence, these results suggest that the maintenance ofCCL5 mRNA levels in memory CD8 T cells is alsodependent on an active transcription mechanism.

Maintenance of ccl5 transcription accounts for themaintenance of high mRNA levels

The level of ccl5 transcription at different times afterimmunization was measured by quantifying nascenttranscripts using the technique described byWuarin andSchibler [19]. Mice were immunized and freshlyactivated, post-effector and memory CD8 T cells werepurified 3, 7 and 40 days later. Naive CD8 T cells wereused as control. In parallel, to assess the opening of thelocus, the levels of histone H3 acetylation associatedwith the ccl5 promoter were measured by chromatinimmunoprecipitation (ChIP). As shown in Fig. 4A thetranscription level reaches a maximum on day 3 afterimmunization. By day 7 the transcription level drops, toa level that is, however, three- to fourfold above the onefound in naive cells. This transcription level is main-tained in the memory phase. The increased transcriptionof ccl5 coincides with an increased association ofacetylated-histone H3 with the promoter. The presenceof acetylated-histone H3 on the ccl5 promoter wasmaintained in the memory phase paralleling what wasobserved for the transcription level (Fig. 4B). Incontrast, as shown in Fig. 4B, acetylation level of H3

Figure 2. CCL5 mRNA levels are specifically maintained inCD8T cells. 3T3 fibroblast cells (A), pre-treated overnightwith acombination of IFN-c/TNF-a, or purified post-effector (B) ormemory (C) F5 CD8 T cells were maintained in medium alone.At the indicated time, CCL5 mRNA levels were quantified byRPA. Relative values are given with the CCL5 mRNA content ofeach cell type on time 0 being set to 1. Themean (� SD) of threeindependent experiments is shown. Statistically significantdifferences compared to naive cells are marked (Student'st-test, n.s., non-significant *p <0.05).



on the il-4 promoter remained very low at all time-pointsassessed. Altogether, these results indicate that themaintenance of high CCL5 mRNA levels by memoryCD8 Tcells is correlated with the persistent transcriptionof the gene combined with high transcript stability.

Constitutive transcription of ccl5 is essential forthe maintenance of the CCL5 mRNA pool

Incubation of memory CD8 T cells with IL-4 leads to astrong decrease in their CCL5 mRNA content (Fig. 5A).We thus decided to use IL-4 as a tool to furthercharacterize the parameters controlling CCL5 mRNAlevels maintenance by memory CD8 T cells. We firstmeasured the effect of IL-4 on CCL5 transcription andmRNA stability. The CCL5 mRNA stability was deter-mined as previously described by treating the cells withactinomycin D in the presence or absence of IL-4. Asexpected, CCL5 mRNA levels steadily decreased whencells were treated with actinomycin D. The addition ofIL-4 did not accelerate the process, suggesting that IL-4does not act by destabilizing CCL5 mRNA (Fig. 5B). Incontrast, the transcription of ccl5 was strongly inhibitedby a 4-h incubation in the presence of IL-4 (Fig. 5C); thiswas concomitant with a decrease in the cytoplasmic

CCL5 mRNA levels (Fig. 5D). This inhibition was notassociated with a decrease in the level of acetylated-histone H3 associated with the CCL5 promoter (data notshown). These results indicate that inhibiting ccl5transcription without affecting mRNA stability issufficient to induce a loss of CCL5 mRNA stores inmemory CD8 T cells.

Reversible inhibition of ccl5 transcription by IL-4

To determine if CD8 T cells are able to reacquire CCL5mRNA stores after IL-4 treatment, we followed the CCL5mRNA levels in post-effector cells in vitro after IL-4withdrawal. Post-effector F5 CD8 T cells were cultured20 h in the presence of IL-4. The cells were then washedand re-plated in medium alone or medium plus IL-4.Twenty hours later, cells were harvested, and their CCL5mRNA content as well as their immediate CCL5secretion capacity in response to TCR triggering wereassessed. As shown in Fig. 6A and B, in cells re-plated inmedium containing IL-4, CCL5 mRNA levels andimmediate production capacity were maintained in-hibited. In contrast, the steady state mRNA content andthe capacity to rapidly produce CCL5 protein in responseto TCR triggering were recovered when cells were re-

Figure 3. CCL5 mRNA half-life isenhanced following memory differ-entiation. (A, B) Post-effector F5CD8 T cells from spleen and lymphnodes were purified and maintainedin medium in the presence of acti-nomycin D. At the indicated time,absolute mRNA levels of CCL5, CCR7and L32 were quantified by RPA. Themean of three independent experi-ments (� SD) is shown. (C) F5CD8 T cells from spleen and lymphnodes were purified 3 days (acti-vated) or 40 days (memory) afterimmunization and treated as above.The mean of three independentexperiments (� SD) is shown.



plated 20 h in medium alone. The rapid recovery wasfound in post-effector cells as well as in memory cells(Fig. 6C). Thus, maintenance of high CCL5 mRNA andimmediate secretion capacity is dependent on themaintenance of constitutive transcription. Overall, theseresults indicate that the transcription of CCL5 by post-effector as well as by memory cells is a default feature ofthese cells.

Discussion

In this study, we demonstrate that the long-termmaintenance of the CCL5 mRNA levels by memoryCD8 T cells is dependent on transcription and isassociated with increased transcript stability (summar-ized in Fig. 7).

Indeed, by directly measuring ccl5 transcription, weshow that following primary stimulation ccl5 transcrip-tion is initiated and is then maintained at a low level inpost-effector and memory cells. This is correlated withan increased association of acetylated-histone-H3 with

the ccl5 promoter. Thus, in contrast to many other pro-inflammatory cytokines genes that are only transientlyexpressed following activation [20], ccl5 transcription ismaintained. The stability of this memorization processcould be demonstrated using IL-4. Indeed, we show thatin memory CD8 T cells IL-4 inhibits the transcription ofthe ccl5 gene. This leads to a slow decrease in their CCL5mRNA content and impairs their immediate CCL5production capacity following TCR engagement. How-ever, as soon as cells are removed from IL-4, transcrip-tion resumes and their CCL5 mRNA stores as well astheir immediate CCL5 production capacity are rapidlyrecovered. This rapid recovery was not due to anongoing effector response in post-effector cells, asmemory cells recovered from mice at least 40 daysafter priming showed a similar pattern of loss andrecovery when subjected to the same experiment. Thisalso shows that ccl5 expression pattern is establishedearly after primary stimulation of CD8 T cells. Thechronic ccl5 transcription is thus necessary for themaintenance of high CCL5 mRNA levels.

Although, the chronic transcription of CCL5 isessential for the maintenance of the immediate CCL5secretion capacity, mRNA stabilization also plays a role

Figure 4. ccl5 transcription is maintained in memory cells andcorrelates with an increased acetylated-histone H3 level.Spleen and lymph nodes CD8 T cells were recovered from F5mice, naive or different times after priming. (A) The ccl5transcription level was measured as described in Materials andmethods. The signal obtained with the control reaction (- RT)rules out the possibility of genomic DNA contamination. Themeanof four independent experiments (� SD) is shown. (B) Thepresence of acetylated histone H3 was determined by ChIP onthe ccl5 and il-4 genes promoters regions and normalized tocd8a gene promoter. The mean of three independent experi-ments (� SD) is shown. In (A) and (B), values are given relativeto the one found in naive cells. Statistically significantdifferences compared to naive cells are marked (Student'st-test, *p <0.05, **p <0.01).

Figure 5. Arrest of ccl5 transcription decreases CCL5 mRNAstores. Spleen and lymph nodes CD8 T cells were recoveredfrommemory F5mice. (A) CCL5mRNA levelsweremeasured byRPA before (ex vivo) or after 20-h incubation in the presence ofIL-4 (IL-4). (B) Purifiedmemory CD8 T cellswere incubatedwithactinomycin D in the presence or absence of IL-4 and CCL5mRNA levels were measured at different time points. Arepresentative experiment out of three is shown. (C, D) Purifiedmemory CD8 T cells were incubated 4 h in the presence of IL-4.ccl5 transcription level (C) and cytoplasmic CCL5 mRNA store(D) were measured before or after treatment using real-timeRT-PCRas described inMaterials andmethods. Themeanof threeindependent experiments (� SD) is shown. Statisticallysignificant differences are marked between points indicatedby brackets (Student's t-test, *p <0.05, ***p <0.001).



in this process. In CD8 T cells, we show that with a half-life of 10 h the CCL5mRNA is comparatively more stablethan other mRNA contained in the same cells. This longhalf-life was not due to a bystander stabilization effectmediated by actinomycin D [2], as when using IL-4 as aninhibitor of ccl5 transcription we found a similar half-life(data not shown). This is in contrast to a number of othercytokine or chemokine mRNA that are naturallyunstable with a half-life inferior to 1 h [21].One ofthe most common stability determinant are the AU-richelements (ARE) and they are found in the 30UTRofmanyunstable messengers coding for cytokines [22]. We did

not find any ARE sequences [23, 24] in the 30UTR ofCCL5. Conversely, the presence of stabilization se-quences in the 30UTR of CCL5mRNA in human epithelialcells has been described [2]. These sequences areinvolved in the stabilization of the CCL5 mRNA that isobserved following infection by RSV. In this system,similar levels of ccl5 transcription are induced by live orUV-inactivated RSV. However, CCL5 mRNA only accu-mulates after exposure to live RSV; this is due to anincrease mRNA half-life from less than 1 to 7 h. Weobserve a similar phenomenon in CD8 Tcells, where thehalf-life of CCL5 is increased in memory compared toactivated cells. This allows memory cells to maintain thesame mRNA levels as activated cells with a significantlylower transcription level (Fig. 7). The stabilizationmechanism and the macromolecules involved in thisprocess are still unknown, as are those responsible forthe silencing of CCL5 mRNA in CD8 T cells. However,these two processes could be linked. Indeed, thesilencing of stored mRNA involved in the establishmentof long-termpotentiation in the nervous system relies onmolecular complexes that also control their stability[25–27].

In memory CD8 T cells, the maintenance of highCCL5 mRNA stores is cell autonomous as it proceeds invitro in the absence of exogenous signaling. Thisproperty is not a general feature of all mRNA containedin memory CD8 T cells. Other mRNA, such as the oneencoding CCR7, needs a signal present in vivo to bemaintained and thus rapidly decrease when the cells areplaced in vitro (data not shown). In addition, cell-

Figure 6. Post-effector and memory CD8 T cells are able toreconstitute CCL5 mRNA stores following IL-4 withdrawal.Post-effector CD8 T cells were purified from spleen and lymphnodes and cultured for 20 h with IL-4. Cells were then washedand live cells were re-plated in medium alone or medium plusIL-4 for an additional 20-h incubation period. CCL5 mRNAcontent (A) as well as the immediate CCL5-secretion-capacity(B) were measured before treatment (ex vivo), after 20 h IL-4(IL-4), after 20 h IL-4 followed by 20 hmedium (IL-4?Medium)or after 20 h IL-4 followed by 20 h IL-4 (IL-4 ? IL-4). (B) CCL5secretion was measured without (-) or with (+) antigenicstimulation. A representative gel and the mean of threeindependent experiments are shown (� SD). (C) The capacityof memory CD8 T cells to recover their immediate CCL5-secretion-capacity following IL-4 treatment was compared tothe one of post-effector CD8 T cells. Memory CD8 T cells werepurified from mice at least 40 days after priming and werecultured with IL-4 as described above. The recovery of theimmediate CCL5-secretion-capacity following TCR stimulationwas measured after 5-h culture in medium alone or mediumplus IL-4. Themean of three independent experiments (� SD) isshown. Statistically significant differences are markedbetween points indicated by brackets (Student's t-test,***p <0.001, ****p <10–4).

Figure 7. Mechanisms involved in the maintenance of CCL5mRNA stores. ccl5 transcription is maintained at low levels inmemory CD8 T cells and the half-life of CCL5 is increased inmemory CD8 T cells compared to effector cells. IL-4 is able toinhibit the transcription of the ccl5 gene. This leads to a slowdecrease in their CCL5 mRNA content and impairs theirimmediate CCL5-production-capacity following TCR engage-ment. This inhibition is reversible as the transcription of ccl5resumes as soon as IL-4 is removed.



autonomous maintenance is not a specific feature ofCCL5 mRNA, as CCL5 mRNA levels decrease infibroblasts after removal of the inducing signal.

The storage of untranslated mRNA to support a rapidproduction of cytokine following TCR engagement has anumber of advantages in terms of speed of the responsebut also in terms of quality control. Indeed, it allows therapid production of newly synthesized proteins. Thiscould be important for CCL5, as chemokines arerelatively unstable proteins in vitro. Moreover, storage

of mRNA allows for a potential amplification step, aseach mRNA molecule will potentially give rise to severalproteins [28]. Finally, the combination of a lowtranscription level with an increased mRNA half-lifeallows the constant renewal of mRNA thus ensuring thepreservation of a functional pool of molecules. Inconclusion, our results show that the cell-autonomousbasal transcription of the ccl5 gene acquired byCD8 T cells following their differentiation in memorycells is necessary for the maintenance of the mRNA poolthat will be used for their immediate production of theprotein.

Materials and methods

Mice and immunizations

C57BL/10 F5 and RAG–/– F5 TCR-transgenic mice were giftsfrom D. Kioussis (London, UK) [29] and were bred in ouranimal facility, PBES “Plateau de Biologie Exp�rimentale de laSouris”. Thymectomies were performed on 5–6-week-oldmice.Three weeks after thymectomy, F5 or RAG–/– F5 mice wereinjected twice at 24-h interval with 50 nmol of the A/NT/60/68 influenza virus nucleoprotein peptide (NP-366–374: Ala-Ser-Asn-Glu-Asn-Met-Asp-Ala-Met, plateforme IFR128, IBCP,Dr. D. Ficheux) in PBS in the peritoneal cavity [30]. CD8 Tcellswere recovered 3 days (activated cells), 7 days (post-effectorcells) or 40 days (memory cells) after peptide injection.Memory cells recovered on day 40 have been previouslycharacterized for a number of surface markers or effectorfunctions [31]. These cells were compared to naive cells,activated cells or post-effector cells. Results in Fig. 8A showthat memory cells express higher levels of CD44, CD127 andCD122 than naive cells. In contrast to activated effector cells,memory cells did not contain granzyme B or express CD43.They were, however, able to rapidly produce IFN-c followingpeptide restimulation. Post-effector cells were similar tomemory cells in terms of average CCL5 mRNA content(Fig. 1A), protein secretion and regulation by macromoleculessynthesis inhibitors (Fig. 8C). Therefore, post-effector cellswere preferentially used for ethical reasons, as mice did notneed to be thymectomized to produce these cells and forpractical reasons when large numbers of cells were necessary.However, key results were confirmed using memory cells. Allprotocols using live mice were performed in accordance withour institutional guidelines. Experimental procedures wereapproved by the CREEA “Comit� R�gional d'Ethique pourl'Exp�rimentation Animale”.

Cells and cultures

The medium used for all cultures was DMEM (Gibco BRL, LifeTechnology, Cergy Pontoise, France) supplemented with 6%FBS (PAN biotech), 50 lg/mL gentamicin, 2 mM L-glutamine(Gibco BRL), 10 mM Hepes, and 50 lM b-mercaptoethanol(Sigma chemicals, L'Isle d'Abeau, France).

Figure 8. Phenotypic and functional comparison of naive,activated, post-effector and memory CD8 T cells. Single- cellsuspensionswereprepared fromspleenobtained fromnaive F5mice or of peptide primed F5 mice. Cells were recovered 3(activated), 7 (post-effector) or 40 days (memory) after peptideimmunization. (A, B) The cell suspension was stained for CD8and the indicated markers and analyzed by flow cytometry.The mean of four to five mice from one representativeexperiment is shown (� SD). (C) Immediate CCL5-secretion-capacity was measured for naive, post-effector and memoryCD8T cells purified fromspleen and lymphnodes of F5mice. Invitro peptide restimulation in the presence or absence ofactinomycin D (10 lg/mL) or CHX (10 lg/mL) was done asdescribed in Materials and methods. The mean of sevenindependent experiments (� SD) is shown. Statisticallysignificant differences are marked between points indicatedby brackets (Student's t-test, *p<0.05, ***p<0.001, ****p<0.0001).



The 3T3 fibroblasts were cultured in DMEM supplementedwith 10% FBS. The expression of CCL5 was induced bygrowing cells for 24 h in the presence of IFN-c and TNF-a atconcentrations of 50 and 20 ng/mL, respectively.

CD8 T cells were purified from spleen and lymph nodes(mesenteric, inguinal, brachial and axillary) using a negativeselection strategy as previously described [30]. CD8+CD44int

T cells were sorted using FACS Vantage (Becton Dickinson)from purified CD8 Tcells using the anti-CD8 (YTS169.4-TC, BDPharMingen, Le Pont de Claix, France) and anti-CD44 (IM-781-FITC, home made) antibodies.

To measure the immediate cytokine secretion capacity of F5CD8 T cells in response to TCR triggering, 2 � 105 viable cellswere stimulated for 4 h in 200 lL in U-bottom 96-well plateswith NP366–374 peptide at a concentration of 10 nM. To studythe effect of IL-4 on the CCL5 memorization, cells werecultured overnight in the presence of IL-4 (R&D systems, Lille,France, 20 ng/mL) at a concentration of 3 to 5� 106 cells/mL.The next day, viable cells were separated from dead cells bycentrifugation on a Ficoll gradient (Cedarlane, Tebu, ParisFrance) and left in culture as indicated.

Cell staining

Spleen cells from F5 mice were stained with anti-CD8 incombination with anti-CD44, anti-CD122, anti-CD127 or anti-CD43 at 4�C for 20 min in FACS stain buffer. For intracellularcytokine staining; 1 � 106 spleen cells were incubated in 96-well plates with 0.67 ll/mL of GolgiStop (BD PharMingen) inpresence or absence of 10 nM NP68 peptide for 6 h. At the endof the incubation period, surface staining with anti-CD8 wasperformed for 20 min at 4�C. After one wash in FACS stainbuffer, cells were permeabilized for 20 min in Cytofix-Cytoperm solution (BD PharMingen) and washed withPermWash buffer (BD PharMingen). Anti-IFN-c antibodywas added to permeabilized cells for 30 min at 4�C. Afterone more wash in Perm/Wash buffer, cells were resuspendedin FACS stain buffer for flow cytometric analysis. Cells wereanalyzed on a FACSCalibur flowcytometer (BD Biosciences,Mountain view, CA). All antibodies were purchased from BDPharMingen. For granzyme B, after isolation and CD8 surfacelabeling, cell population was permeabilized and stained forgranzyme B expression.

Multiprobe RPA

Chemokine or cytokine mRNA levels were measured by RPAusing the Riboquant kit (BD PharMingen) following thesupplier's instructions. The quantity of protected RNA wasdetermined using a PhosphorImager and the ImageQuantsoftware (both from Molecular Dynamics, Sunnyvale, CA).Relative mRNA level was normalized using the internal controlL32 mRNA.

CCL5 ELISA assay

ELISA assay (R&D Systems) was used to measure the CCL5content in culture supernatant following manufacturer'sinstructions.

Chromatin immunoprecipitation

ChIP was done using the Upstate ChIP assay kit (Euromedex,Souffelweyersheim, France) following manufacturer's instruc-tions. Briefly, 4–5 � 106 purified CD8 T cells were fixed withformaldehyde (10 min, 37�C, 1% final), lysed in 0.5 mL lysisbuffer and sonicated on ice by ten pulses of 10 s. A volume of200 lL extract was used for each IP. To reduce nonspecificbinding to protein A, extract diluted ten times in dilution bufferwere incubated with 80 lL of protein A-agarose 30 min at 4�Cwith gentle rocking, before removal of the beads. Anti-acetyl-H3 polyclonal antibody (5 lg per IP, 06–599, Upstate) wasthen added to this diluted extract and incubation was carriedout overnight at 4�C with gentle rocking. Protein A-agarose(70 lL) was added to the chromatin antibody mix for 1 h at4�C with gentle rocking. The protein A-agarose and theunbound chromatin were separated by centrifugation(300 � g, 2 min, 4�C) and the complex protein A-boundchromatin was washed sequentially with high salt buffer, lowsalt buffer, LiCl buffer, and twice TE-buffer. Bound chromatinwas eluted twice with 250 lL of elution buffer (0.1 MNa2HCO3, 1% SDS) 15 min at room temperature. The elutedchromatin was incubated at 65�C with 20 lL of 5 M NaCl toreverse the crosslink. DNA was recovered by phenol/chloro-form extraction and ethanol precipitation and re-suspended in20 lL water; 0.5 lL was used per real-time PCR reaction.

Extraction and reverse transcription of nascent mRNAtranscripts

CD8 Tcells (2–5�107) were purified, treated as indicated, andnuclear chromatin was isolated as described [19, 32]. All stepswere performed at 4�C with RNase-free reagents. Briefly,Tcells were lysed (5 min in 0.5% NP40, 140 mMNaCl, 1.5 mMMgCl2, 50 mM Tris pH 8.0, 1 mM DTT, 1000 U/mL RNaseinhibitor). Nuclei were pelleted (300 � g, 5 min), the super-natant was kept, and cytoplasmic mRNA extracted. Nucleiwere then lysed (10 min in 1% NP40, 300 mM NaCl, 7.5 mMMgCl2, 1 M urea, 0.5 mM EDTA, 20 mM HEPES pH 7.6, 1 mMDTT, 1000 U/mL RNase inhibitor). Chromatin was isolated bycentrifugation, 15 000 � g for 10 min. Nascents mRNAtranscripts bound to chromatin were purified using theNucleoSpin RNA II kit (Macherey-Nagel, Hoerdt, France) withDNase I treatment. Purified RNA were re-suspended in 40 lLwater. Reverse transcription was performed using 9.3 lL ofRNA solution using SuperScript II (Invitrogen, Cergy-Pontoise,France) and random primers according to the manufacturer'sinstructions. For quantification of cytoplasmic CCL5 mRNAlevels, 1 lg of cytoplasmic RNA followed the same procedure.The obtained cDNA was diluted three times and 5 lL wassubjected to real-time PCR. The relative level of CCL5 mRNA(target sequence) was normalized to HPRT mRNA (referencesequence). The signal obtained with naive cells was used tonormalize the results obtained with the other populations. Fortranscription level measurements, primer detecting theunspliced or the spliced CCL5 mRNA gave similar results. Tomonitor genomic contamination, a control was prepared foreach sample following the same procedure except the reversetranscription step. In all experiments performed, no CCL5sequence could be detected in these control samples.



Quantification by real-time PCR

Real-time PCR analysis was carried out using Platinum SYBRGreen qPCR SuperMix UDG kit (Invitrogen) on an ABI Prism7700 (Perkin Elmer). Mix was as follows: 6 lL Platinum Mix,0.48 lL primer (10 lM), 0.52 lL Rox reference dye andtemplate in 5 lL. For PCR amplification, samples wereincubated at 50�C for 2 min, followed by 10 min at 95�C,followed by 40 cycles (95�C 10 s, 60�C 1 min). The dissocia-tion curve was measured for each sample. Relative level of thetarget sequence against the reference sequence was calculatedusing the DDCt method with calculated real efficiencies [33].The ccl5 promoter primers were designed to span the NFkB/Sp1/RFLAT-1 and the IRF3 sites in the ccl5 promoter [34]; forcd8a, primers are in the CII-3 DHS site [35]. The sequences ofprimers used are available upon request.

Acknowledgements: We thank JL. Darlix, P. Bouvet andL. Schaeffer for helpful discussions. C. Arpin, Y.Leverrier and J. Maryansky for critical reading of themanuscript. This work was supported by institutionalgrants from Institut National de la Sant� Et de laRecherche M�dicale, Association pour la Recherchecontre le Cancer, Ligue R�gionale de Lutte Contre leCancer, R�gion Rh�ne-Alpes (Contract 00816045), Uni-versit� Claude Bernard Lyon 1 and Canc�ropole natio-nale. We thank the staff of the Plateau de BiologieExperimentale de la Souris and of the Plateforme deCytom�trie en Flux for their technical assistance. Theauthors have no conflicting financial interests.

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Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability