Memory T Cells − CD27 + CD45RA + Reversible Senescence in Human

Home , APRIL (protein)

Reversible Senescence in Human CD4+ CD45RA+CD27− Memory T Cells Diletta Di Mitri, Rita I. Azevedo, Sian M. Henson, Valentina Libri, Natalie E. Riddell, Richard Macaulay, David Kipling, This information is current as Maria V. D. Soares, Luca Battistini and Arne N. Akbar of September 25, 2021. J Immunol 2011; 187:2093-2100; Prepublished online 25 July 2011; doi: 10.4049/jimmunol.1100978

http://www.jimmunol.org/content/187/5/2093 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2011/07/25/jimmunol.110097 Material 8.DC1 http://www.jimmunol.org/ References This article cites 70 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/187/5/2093.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Reversible Senescence in Human CD4+CD45RA+CD272 Memory T Cells

Diletta Di Mitri,*,† Rita I. Azevedo,*,‡ Sian M. Henson,* Valentina Libri,* Natalie E. Riddell,* Richard Macaulay,* David Kipling,x Maria V. D. Soares,{ Luca Battistini,† and Arne N. Akbar*

Persistent viral infections and inflammatory syndromes induce the accumulation of T cells with characteristics of terminal differentiation or senescence. However, the mechanism that regulates the end-stage differentiation of these cells is unclear. Human CD4+ effector memory (EM) T cells (CD272CD45RA2) and also EM T cells that re-express CD45RA (CD272CD45RA+;EMRA) have many characteristics of end-stage differentiation. These include the expression of surface KLRG1 and CD57, reduced replicative capacity, decreased survival, and high expression of nuclear gH2AX after TCR activation. A paradoxical observation was that although CD4+ EMRA T cells exhibit defective telomerase activity after activation, they have significantly longer Downloaded from telomeres than central memory (CM)-like (CD27+CD45RA2) and EM (CD272CD45RA2)CD4+ T cells. This suggested that telomerase activity was actively inhibited in this population. Because proinflammatory cytokines such as TNF-a inhibited telomerase activity in T cells via a p38 MAPK pathway, we investigated the involvement of p38 signaling in CD4+ EMRA T cells. We found that the expression of both total and phosphorylated p38 was highest in the EM and EMRA compared with that of other CD4+ T cell subsets. Furthermore, the inhibition of p38 signaling, especially in CD4+ EMRA T cells, significantly enhanced their telomerase activity and survival after TCR activation. Thus, activation of the p38 MAPK pathway is directly involved in http://www.jimmunol.org/ certain senescence characteristics of highly differentiated CD4+ T cells. In particular, CD4+ EMRA T cells have features of telomere-independent senescence that are regulated by active cell signaling pathways that are reversible. The Journal of Immunology, 2011, 187: 2093–2100.

uman T cell memory is mainly maintained throughout can exhibit potent effector functions (5, 7, 9, 10). Highly differ- life by episodes of proliferation induced by antigenic entiated populations of EM and EMRA-like CD4+ and CD8+ challenge and to a much lesser extent by continued gen- T cells have been shown to accumulate in older human (7, 11–13)

H by guest on September 25, 2021 eration of new cells as the thymus involutes early in life (1). The patients with persistent viral infections (14–18) and those with repeated lifelong stimulation of T cells induces them to differ- inflammatory syndromes such as rheumatoid arthritis (19–21). In entiate both functionally and phenotypically (1, 2). Since the addition, EMRA T cells are the dominant memory population that original use of CD45RA and CD45RO Abs to identify unprimed persists after some forms of vaccination (22). However, the exact and primed/memory subsets of T cells, respectively (3), it has be- nature of these T cells is not clear. come clear that some primed/memory T cells in both CD8 (4, 5) The maintenance memory T cells by proliferation of pre-existing and CD4 (6, 7) compartments can re-express the CD45RA mol- pools of cells may be limited because there is a finite limit to the ecule. These cells were subsequently shown to be largely CCR72 extent of proliferation that a cell population can experience before CD272CD282 and therefore have a highly differentiated effector growth arrest occurs (23). This constraint is set by the erosion of memory (EM)-like phenotype (EMRA) (4, 8). Although both EM repeating hexameric sequences of DNA at the ends of chromo- and EMRA T cells have proliferative defects, the latter population somes known as telomeres, which are lost with each replicative cycle (23). In the absence of compensatory factors, telomeres shorten by ∼50–100 bases after each round of proliferation until *Division of Infection and Immunity, University College London, London W1T 4JF, a critical point is reached at which the exposed DNA end of the † United Kingdom; Neuroimmunology Unit, Santa Lucia Foundation, Rome 00143, telomere is recognized as a DNA double-stranded break (24). This Italy; ‡Unidade de Imunologia Clinica, Instituto de Medicina Molecular, Lisboa 1649-028, Portugal; xSchool of Medicine, Cardiff University, Heath Park, Cardiff recruits a complex of proteins that are involved with DNA repair CF14 4XN, United Kingdom; and {Unidade de Citometria de Fluxo, Instituto de and is commonly referred to as the DNA damage response (DDR) Medicina Molecular, Lisboa 1649-028, Portugal (23, 25, 26). The development of telomere-dependent senescence Received for publication April 8, 2011. Accepted for publication June 17, 2011. is retarded if cells can upregulate the enzyme telomerase, which This work was supported by the British Biotechnology and Biological Sciences Re- adds telomeres back to the ends of chromosomes (24). Cellular search Council. senescence can also occur when telomeric or nontelomeric DNA Address correspondence and reprint requests to Prof. Arne N. Akbar, Division of is damaged by other means that are independent of telomere Infection and Immunity, Rayne Building, University College London, London WC1E 6JF, United Kingdom. E-mail address: [email protected] shortening (telomere-independent senescence) (23). This includes The online version of this article contains supplemental material. damage by reactive oxygen species, ionizing radiation, chromatin Abbreviations used in this article: CM, central memory; DDR, DNA damage re- perturbation, and activation of p53 and stress pathways (23). In sponse; EM, effector memory; EMRA, effector memory RA expressing T cells; most of these situations, a DDR that is virtually identical to that FISH, fluorescent in situ hybridization; rh, recombinant human; TRAP, telomeric triggered by telomere erosion is induced, and unless the DNA can repeat amplification protocol. be repaired, the cells undergo growth arrest. Senescence-related Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 DNA damage foci, irrespective of whether they arise as a result of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100978 2094 CD4+ EMRA CELLS UNDERGO REVERSIBLE p38 REGULATED SENESCENCE telomere-dependent or telomere-independent DNA damage, can fixed with warm Cytofix Buffer (BD Biosciences) at 37˚C for 10 min. Cells be identified by staining for molecules within the complex such were then permeabilized with ice-cold Perm Buffer III (BD Biosciences) at as the histone protein gH2AX (27). When human CD4+ T cells 4˚C for 30 min and incubated with the anti-p38 Ab (pT180/pY182) (Alexa Fluor 488, clone 36/p38; BD Pharmingen) for 30 min at room temperature. differentiate progressively from a naive to an EM phenotype, they Cells were washed in Stain Buffer (BD Pharmingen). For the detection lose their capacity to upregulate telomerase activity, and this is of gH2AX (pSer139) (Alexa Fluor 488, clone 2F3; BioLegend), purified associated with progressive telomere reduction (28). However, the subsets were activated with 0.5 mg/ml immobilized anti-CD3 and 5 ng/ml telomere length and telomerase activity of CD4+CD272CD45RA+ rhIL-2 for 4 d. Intracellular staining was performed using the BD Phosflow buffers above mentioned. Samples were acquired on a BD LSR II flow (EMRA) T cells have not been investigated previously. cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). In this study, we made the unexpected observation that although CD4+ EMRA T cells have many phenotypic and functional char- Telomere-length measurement by flow-fluorescent in situ acteristics of an end-stage or senescent population, they have hybridization coupled to flow cytometry + 2 significantly longer telomeres than CD27 CD45RA (central Telomere length of CD4+ T cell populations defined by expression of 2 2 memory [CM]) and CD27 CD45RA (EM) cells from the same CD45RA and CD27 was assessed by a modified version of the three-color + 2 flow-fluorescent in situ hybridization (FISH) method that was described donor. This is the first report, to our knowledge, that CD4 CD27 + CD45RA+ EMRA T cells exhibit characteristics of telomere- previously (30, 31). In brief, CD4 cells that were negatively isolated using MACS beads were surface stained, washed in PBS and then fixed in 1 mM independent senescence. This distinguishes them from the EM BS3 (Perbio Science). The reaction was quenched with 50 mM Tris (pH subset that also has senescence characteristics that are associated 7.2) in PBS. After the cells were washed in PBS followed by hybridization with short telomeres. A key observation is that the senescence buffer, they were incubated with the protein nucleic acid telomeric probe characteristics of the CD4+ EMRA T cell population are main- (C3TA2)3 conjugated to Cy5 (Panagene). After the samples were heated for Downloaded from 10 min at 82˚C, they were left to hybridize. Samples were washed in tained in part by active p38 signaling and are reversible. posthybridization buffer, followed by PBS, and analyzed immediately by flow cytometry. All samples were run in triplicate alongside cryopreserved Materials and Methods PBMCs with known telomere fluorescence to ensure consistency of results. Blood sample collection and isolation Kilobase length was determined from mean fluorescence intensity values using a standard curve. The standard curve was constructed using samples Heparinized peripheral blood was collected from healthy volunteers be- of varying telomere length analyzed both by flow-FISH and telomeric tween the ages of 26 and 60 y (median age, 39 y), with approval from the restriction fragment analysis (31). http://www.jimmunol.org/ Ethics Committee of the Royal Free Hospital. PBMCs were isolated by Ficoll-Hypaque density gradient (Amersham Biosciences, Uppsala, Swe- Measurement of telomerase activity + den). CD4 T cells were purified by positive selection using the VARI- Telomerase activity was determined using a modified version of the telo- OMACS system (Miltenyi Biotec), according to the manufacturer’s in- + meric repeat amplification protocol (TRAP) (Oncor, Gaithersburg, MD) as structions. In some experiments, CD4 T cells were further sorted into described previously (31). In brief, purified subsets (Supplemental Fig. 1) CD45RA/CD27 subsets using a FACSAria flow cytometer (BD Bio- were activated with anti-CD3 (0.5 mg/ml) and irradiated APCs for 4 d sciences, San Jose, CA) after staining with CD4, CD45RA, and CD27 Abs (Supplemental Fig. 3). Telomerase activity is only induced after T cell ac- for 30 min at 4˚C in 1% PBS containing 1% BSA (Sigma-Aldrich). tivation. To ensure that we are measuring a telomerase defect and not Cell culture and use of inhibitors a proliferative defect in T cells per se, we measure the proportion of proliferating cells in each isolated T cell subset by staining with the nu- by guest on September 25, 2021 Cells were cultured in RPMI 1640 medium supplemented with 10% heat- clear cell cycle marker ki67 prior to the telomerase assay. Absolute inactivated FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml numbers of CD3+ki67+ T cells in each sample are counted using Trucount gentamicin, and 2 mM L-glutamine (all from Invitrogen) at 37˚C in a hu- tubes, and PCR for telomerase activity is performed with samples adjusted + + midified 5% CO2 incubator. Purified CD4 subsets were activated in the to 500 ki67 T cells/reaction. Therefore, we are biasing the results against presence of anti-CD3 Ab (purified OKT3, 0.5 mg/ml) and PBMCs irradi- ourselves. In populations like the EMRAT cells that do not proliferate very ated with 40-Gy gamma radiation, as a source of multiple costimulatory well, we make extracts from larger numbers of cells so that we get the ligands provided by B cells, dendritic cells, and macrophages found in same number of proliferating cells in each PCR. We have described this these populations. In other experiments, where we were investigating the assay previously (32). Cell extracts from equivalent numbers of Ki67+ cells changes in the level of apoptosis the cells were activated by anti-CD3 Ab were used for telomeric elongation, using a [g-32P] ATP-end–labeled in the presence of recombinant human (rh)IL-2 (5 ng/ml) (R&D Systems) telomerase substrate primer. These samples were then amplified by PCR because irradiation in the APCs induces excessive cell death, which im- amplification, using 25–28 cycles of 30 s at 94˚C and 30 s at 59˚C. The pacts negatively on accurate gating of the appropriate activated popu- PCR products were run on a 12% polyacrylamide (Sigma-Aldrich) gel that lations. In some experiments, the p38 inhibitor BIRB796 was added to the was then exposed to an autoradiography film (Hyperfilm MP; Amersham culture at a final concentration of 500 nM (29). Cells were pretreated with Biosciences). As a negative control, lysis buffer was used in place of cell the inhibitor for 30 min. A solution of 0.1% DMSO was used as control. extract. A control template containing the same sequence as the telomerase substrate primer plus eight telomeric repeats was used as a PCR positive Flow cytometric analysis of cell phenotype control. We have either used radioactivity or real-time PCR analysis to measure the products of the standard TRAP assay with similar results (data Isolated T cells were resuspended in PBS containing 1% BSA and 0.1% not shown). sodium azide (Sigma-Aldrich) and then stained for 10 min at room temperature with the following anti-human mAbs: CD45RA (allophycocyanin, RT-PCR analysis of Bcl-2 mRNA clone MEM56; Abcam); CD27 (PE, clone M-T271; BD Pharmingen); CD4 (PE-Cy7, clone SK3; BD Pharmingen); KLRG1 (Alexa Fluro 488; a gift Expression of Bcl-2 mRNA was analyzed by semiquantitative RT-PCR from Prof. P. Pircher, University of Freiburg). Intracellular staining was amplification. CD4+ cells were cultured with anti-CD3 (0.5 mg/ml) and performed for Bcl-2 (PE, clone Bcl-2/100; BD Pharmingen), Ki67 (FITC, rhIL-2 (5 ng/ml) in the presence or absence of BIRB796 for 3 d. Total clone B56; BD Pharmingen), and p38 (rabbit polyclonal anti-p38 [Cell RNA was isolated using RNeasy kit (Qiagen), and cDNA was synthesized. Signaling Technology]; Alexa Fluor 488 goat anti-rabbit Ig [Invitrogen]). Bcl-2 expression was evaluated by RT-PCR on an ABI PRISM 7500 The intracellular staining was performed using the Foxp3 Staining Buffer (Applied Biosystems) with the following primers: 59-TTGCTTTACGTG- Set (Miltenyi Biotec), according to the manufacturer’s instructions. Apo- GCCTGTTTC-39 (forward) and 59-GAAGACCCTGAAGGACAGCCAT-39 ptosis was assessed using an Annexin V/Propidium Iodide detection kit (reverse). The housekeeping 18S mRNA, used as an external standard, (BD Pharmingen). Samples were acquired on a BD LSR II flow cytometer was amplified from the same cDNA reaction mixture using specific (BD Biosciences) after fixation with 1% formaldehyde (Sigma-Aldrich). primers. The level of Bcl-2 was expressed as a ratio to the level of 18S to Data were analyzed using FlowJo software (Tree Star, Ashland, OR). control for differing levels of cDNA in each sample. Staining of phosphorylated proteins by flow cytometry Western blot analysis The analysis of p38 (pT180/pY182) was performed directly ex vivo. CD4+ T cells were activated with PMA (0.5 mg/ml; Sigma-Aldrich) and Following surface staining for CD45RA, CD27, and CD4, PBMCs were ionomycin (0.5 mg/ml; Sigma-Aldrich) in the presence or absence of The Journal of Immunology 2095

BIRB796. Cells were harvested after 30 min of stimulation, and lysates were obtained by sonicating cells in 50 mM Tris-HCl (pH 7.5), 2 mM EGTA, and 0.1% Triton X-100 buffer. Lysates from 2 3 106 cells were fractionated on SDS-polyacrylamide electrophoresis gels and analyzed by immunoblotting with either anti–phospho-p38 (pThr180/pTyr182; Cell Signaling Technology), anti-pJNK (pThr183/pTyr185; BD Biosciences), or anti–b-actin (Abcam) using the ECL Advanced Western blotting Detection kit (Amersham Biosciences), according to the protocol provided by the manufacturer.

Statistical analysis Statistical analysis was performed using GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA). Data are presented as mean 6 SEM. A p value , 0.05 was considered signiﬁcant.

Results CD4+ CD272CD45RA+ (EMRA) T cells exhibit phenotypic and functional characteristics of cellular senescence CD4+ T cells can be subdivided into four populations on the basis of their relative surface expression of CD27 and CD45RA mole- Downloaded from cules (Fig. 1A). These subsets are analogous to those identified in other reports in which surface CCR7 together with CD45RA expression were used to distinguish between T cells at different stages of differentiation (4, 33). On the basis of changes in surface receptor expression, functional activity, and telomere length, un- + differentiated populations have been shown to express CD27 http://www.jimmunol.org/ CD45RA+, those that are at an early stage of differentiation are CD27+CD45RA2, whereas highly differentiated CD4+ T cells are CD272CD45RA2 (4, 7). Although the CD4+ EMRA T cells also exhibited characteristics of highly differentiated T cells (7, 34), their relative telomere length or telomerase activity has not been investigated previously. We found that CD4+ EMRA T cells express high levels of surface KLRG1 that identify senescent T cells (Fig. 1B) (35, 36), and this, together with their elevated expression of CD57 (7), supports the possibility that they may be an end- by guest on September 25, 2021 stage population. The CD272CD45RA2 (EM) population also + + 2 + expresses high levels of both KLRG1 (Fig. 1B) and CD57 (7), FIGURE 1. CD4 CD4 27 RA T cells express high levels of KLRG1 g indicating that they also have characteristics of senescence. The and H2AX following activation and are defective for telomerase activity. A, Phenotypic analysis of CD27 and CD45RA expression on CD4+ T cells. phosphorylation of the histone H2AX (gH2AX) can be used to PBMCs stained for CD4, CD27, and CD45RA were analyzed by flow identify DDR foci in senescent fibroblasts (27, 37). We found that 2 2 cytometry. Representative pseudocolor plots are shown. B, Bar graph after TCR activation both of the CD27 CD45RA (EM) and the shows the cumulative data for the percentage of KLRG1 expressed on 2 + CD27 CD45RA (EMRA) T cell populations expressed signifi- CD4+ CD27/CD45RA T cell subsets. Error bars represent the SEM of 16 cantly higher levels of gH2AX than the other subsets (Fig. 1C). donors. Statistical analysis was performed using the two-tailed Student t This was not due to the identification of replicating instead of test (GraphPad Prism). C, Purified CD27/CD45RA CD4+ T cell subsets damaged DNA as we excluded proliferating blast T cells from our were activated with anti-CD3 Ab and rhIL-2. On day 4, expression of analysis (Supplemental Fig. 2). We also found that telomerase the DNA damage marker gH2AX was assessed by flow cytometry. The activity was significantly reduced in the CD272CD45RA+ cells analysis has been performed on nonproliferating lymphocytes (Supple- g compared with the other populations (Fig. 1D), which is an addi- mental Fig. 1, gate A). The bar graph shows the percentage of H2AX- positive cells within each subset. Error bars represent the SEM of three tional characteristic of highly differentiated T cells that are close 2 2 separate experiments. Statistical analysis was performed using the two- to senescence (28, 38). Therefore, both CD27 CD45RA and tailed Student t test (GraphPad Prism). D, Telomerase activity was de- 2 + CD27 CD45RA T cells have the characteristics of T cells that termined by TRAP assay. Purified subsets defined by CD45RA and CD27 are close to senescence; however, these changes were more pro- expression were activated with anti-CD3 Ab and irradiated autologous nounced in the latter population. mononuclear cells as APCs for 4 d. The graph represents telomerase activity normalized for the activity observed in the naive (CD45RA+CD27+) CD4+CD272CD45RA+ EMRA T cells have relatively long subset. Error bars represent the SE from the mean of five separate ex- telomeres periments. Statistical analysis was performed using the two-tailed Student t One prediction from the observation of low telomerase activity in test (GraphPad Prism). Autoradiography of a TRAP assay acrylamide gel CD272CD45RA+ EMRA T cells was that these cells would have from a representative experiment is shown. Control template consists of very short telomeres. The relatively low numbers of these cells PCR mix and telomeric template with no cell extract added. As a negative p , p , in vivo precluded the use of conventional DNA isolation and control, lysis buffer was used instead of cell extract. * 0.05, ** 0.01, ***p , 0.001. electrophoretic methods to analyze their telomere lengths. Instead, we investigated the telomere length of MACS-isolated CD4+ T cells by three-color FISH coupled to flow cytometry using undifferentiated T cells (CD27+CD45RA+) have longer telomeres 2 CD45RA, CD27, and a fluorescence-labeled telomere probe (flow- than the early differentiated, CM-like subset (CD27+CD45RA ), FISH; Fig. 2A, Supplemental Fig. 3). We confirmed that relatively which in turn have significantly longer telomeres than the EM-like 2096 CD4+ EMRA CELLS UNDERGO REVERSIBLE p38 REGULATED SENESCENCE

FIGURE 2. CD4+272RA+ T cells do not have the shortest telomeres. A, Comparison of telomere analysis by flow-FISH technique and Southern blotting. A total of 19 PBMC samples were collected from individuals of different ages ranging from umbilical cord blood samples to individuals .70 y old. Cells were cyropreserved in 10% DMSO for telomere-length analysis by flow-FISH and snap frozen as pellets for Southern blotting. B, Telomere length in CD4+ T cells defined by CD45RA and CD27 expression was determined by three-parameter flow-FISH. Each circle represents one individual with the mean telomere length shown as a horizontal bar. Statistical analysis was performed using the two-tailed Student t test (GraphPad Prism). **p , 0.01.

(CD272CD45RA2) T cell population (Fig. 2B). Surprisingly, dependent and telomere-independent senescence of fibroblasts 2 + however, we found that CD27 CD45RA EMRA T cells have (27, 44, 45). We found that TNF-a inhibited telomerase activity in Downloaded from significantly longer telomeres than the CM-like CD27+CD45RA2 TCR-activated CD4+ T cells (Fig. 3A), confirming observations and the EM-like CD272CD45RA2 memory subsets but shorter that were made in the CD8+ T cell population. In addition, this telomeres than the undifferentiated/naive CD27+CD45RA+ T cells cytokine induced p38 activation in CD4+ T cells (Fig. 3B), con- (Fig. 2B). This suggested that although both CD272CD45RA2 firming previous reports (41–43). We also showed that TNF-a– EM and CD272CD45RA+ EMRA T cells have the characteristics induced telomerase inhibition was directly mediated by p38 sig-

of end-stage populations, the senescence in the latter population naling by blocking p38 signaling in activated T cells by the ad- http://www.jimmunol.org/ was not associated with excessive telomere erosion. dition of BIRB796 (BIRB), a p38 inhibitor that blocks the activation of all four of the isoforms of p38 (46). This inhibitor was + a Telomerase inhibition in CD4 T cells by TNF- –induced p38 speciﬁc at the concentration used in our experiments, because it signaling blocked the phosphorylation of p38 (pThr180/pTyr182) but not JNK The relatively long telomeres despite low telomerase activity in (pThr183/pTyr185) in activated T cells (Fig. 3C) in accordance with CD4+CD272CD45RA+ EMRA T cells suggested that this enzyme others (46, 47). The addition of BIRB enhanced whereas the ad- may be actively inhibited in these cells. Previous studies have dition of TNF-a inhibited telomerase activity in TCR-activated shown that the cytokine TNF-a can induce loss of CD28 ex- CD4+ T cells (Fig. 3D). However, the addition of BIRB to acti- pression and T cell differentiation (39) and inhibit telomerase vated CD4+ T cells that had been treated with TNF-a abrogated by guest on September 25, 2021 activity in human CD8+ T human T lymphocytes (40). In addition the inhibitory effects of this cytokine. Identical results were ob- this cytokine has been shown to induce the activation of p38 served in a second experiment with a different donor (data not MAPK (41–43) that has an essential role in both telomere- shown). This suggested that a p38 signaling pathway may be

FIGURE 3. TNF-a decreases telomerase activity of total CD4+ cells. A, Total CD4+ T cells were activated with anti-CD3 Ab and irradiated autologous APCs with and without TNF-a for 4 d. Cell extracts from equivalent numbers of Ki67+ cells were used to determine telomerase activity. Error bars represent the SE from the mean of three separate experiments (A, right panel). Statistical analysis was performed using the Wilcoxon-matched pairs test (GraphPad Prism). B, Total CD4+ were stimulated for 20 min with TNF-a, and phosphorylated p38 expression was assessed by flow cytometry. A representative plot from three donors is shown. C, Western blot showing the effects of the p38 inhibitor BIRB796 on p38 and JNK phosphorylation. b-Actin was used as a loading control. D, Total CD4+ T cells were activated with anti-CD3 Ab and irradiated APCs with and without TNF-a or BIRB for 4 d. Cell extracts from equivalent numbers of Ki67+ cells were used to determine telomerase activity. The bar graph shows the relative telomerase activity following treatment with TNF-a and/or BIRB. One of two experiments with similar results is shown. *p , 0.05. The Journal of Immunology 2097 a mechanism for telomerase inhibition in highly differentiated their ability to expand in culture after TCR and IL-2 activation T cells. The reason why BIRB induced telomerase in TNF-a– compared with the other subsets (Fig. 5A). However, the addition untreated cells was probably because of inhibition of endogenous of BIRB to these cells during activation significantly increased the p38 activity because this was a mixed population of CD4+ T cells cell recovery after activation (Fig. 5A). In addition, we showed containing both differentiated as well as undifferentiated T cells. that the reduced ability of CD272CD45RA+ EMRA T cells to 2 expand after activation was due to increased levels of apoptosis CD4+CD27 CD45RA+ EMRA T cells express high levels of (Fig. 5B, Supplemental Fig. 4A) and not decreased capacity to p38 MAPK activity enter cell cycle as identified by staining with ki67 Ab (Fig. 5C). Although several studies have been performed on the role of p38 in The inhibition of apoptosis by p38 blockade was due in part to the the activation and cytokine secretion of T lymphocytes (41–43), upregulation of the antiapoptotic molecule Bcl-2 in these cells its involvement with lymphocyte differentiation is unclear. When (Supplemental Fig. 4B,4C). we examined the levels of either the total p38 or the phosphory- We next questioned whether the low telomerase activity in the + lated form of this molecule in CD4 T cell subsets directly ex vivo, CD272CD45RA+ EMRA T cell population was linked to in- 2 we found that the highest expression of both was in the CD27 creased p38 signaling in these cells. We found that the low telo- + + CD45RA EMRA CD4 T cell population (Fig. 4A,4B). However, merase activity in the CD272CD45RA+ T cell population that was 2 2 the CD27 CD45RA EM subset also showed significantly higher stimulated with anti-CD3 and irradiated autologous APCs was + + levels of this molecule than the naive CD27 CD45RA and significantly enhanced by up to 3.5-fold by blocking p38 with + 2 CD27 CD45RA CM populations (Fig. 4A,4B). Therefore, both BIRB compared with controls (Fig. 5D,5E). Although telomerase 2 2 2 + the CD27 CD45RA EM and CD27 CD45RA EMRA T cells activity in the CD27-CD45RA- EM population was also increased Downloaded from upregulate the p38 MAPK that may be involved in certain se- by blocking p38 signaling, this result was not significant. These nescence characteristics, in particular telomerase downregulation results also suggest that the low telomerase activity that has pre- of these cells. viously been found in highly differentiated human CD4+ T cells (28) is more pronounced in the CD272CD45RA+ (EMRA) com- p38 MAPK signaling regulates senescence-associated 2 2 functional changes in CD4+CD272CD45RA+ EMRA T cells pared with the CD27 CD45RA (EM) T cell population. Thus, + 2 + + 2 + the decreased capacity of CD4 CD27 CD45RA T cells to ex- http://www.jimmunol.org/ Previous studies showed that CD4 CD27 CD45RA EMRA pand in culture and to upregulate telomerase after activation is T cells have diminished capacity to expand in culture and were mediated largely by p38 signaling. highly susceptible to apoptosis compared with the other subsets (7). We investigated whether this defect was mediated by p38 Discussion signaling. In these particular experiments, we activated the T cell There has been a long-standing interest in the regulation of human subsets with anti-CD3 Ab and IL-2 instead of anti-CD3 Ab and T cell senescence and how this may restrict the maintenance of irradiated autologous PBMCs as APCs to avoid the confusion of immune memory (48–53). Previous work has focused either on the the presence of excessive numbers of apoptotic cells in the cul- role of persistent viruses in shaping the T cell repertoire during tures because of irradiation induced death in the APCs. We con- by guest on September 25, 2021 2 + aging and how this leads to immune dysfunction (12, 13, 17, 53, firmed that CD27 CD45RA EMRA T cells were impaired in 54) or the impact of telomere erosion on restricting the proliferative capacity of memory T cell populations (55). Both ave- nues of investigation were linked by the observation that persistent viruses such as CMV induce the accumulation of large populations of virus-specific CD8+ and CD4+ T cells that are highly differentiated and have very short telomeres (17, 28, 54). In general, telomere erosion in T cells is accompanied by changes in the cell surface phenotype that mark a progressive increase in differentiation (48, 56). However, in this study, we have shown that the correlation between the end-stage phenotype and excessive telomere erosion does not apply to CD4+ EMRA T cells that appear to be close to senescence yet have relatively long telomeres. CD4+ EMRA T cells upregulate gH2AX, a component of the complex of proteins that make up the senescence-related DDR (57). However, it is not clear at present what initiates the senescence program in this population. We showed previously that highly differentiated CD272CD282 T cells in both CD4 (28) and CD8 subsets (31) have short telomeres. CD45RA+CD272 T cells are mainly CD272 CD282 in both CD4 and CD8 subpopulations but not all CD272 CD282 T cells express CD45RA (4). Thus, if telomeres in CD282 2 FIGURE 4. CD4+27 RA+ T cells express higher levels of total and CD272 subsets are investigated, the characteristics of the smaller phosphorylated p38 ex vivo. The median fluorescence intensity of total p38 EMRA population are missed. We previously showed that within (A) and of phosphorylated p38 (B) was assessed ex vivo in PBMCs by CD8 cells that are identified on the basis of CD45RA and CD27 + gating within total CD4 T cells and within each of the CD27/CD45RA expression, the EMRA population, especially in younger donors subsets. Overlays of total p38 (A) and of phospho-p38 (B) within the re- also showed longer telomeres (58), as has been found in the CD4+ spective subsets are shown. The values represent the median fluorescent population in the current study. intensity of p38 within each subset. The bar graphs represent the ex vivo 2 mean fluorescence intensity of total p38 (A) and phospho-p38 (B) nor- We found that p38 signaling, which is increased in the CD27 + 2 2 malized for the levels of expression in the naive population. Error bars CD45RA (EMRA) compared with the CD27 CD45RA (EM) represent the SE (A, n =7;B, n = 10). Statistical analysis was performed T cell populations, is involved in the induction of increased apo- using the two-tailed Student t test (GraphPad Prism). *p , 0.05, **p , 0.01. ptosis and impaired telomerase activity in these populations after 2098 CD4+ EMRA CELLS UNDERGO REVERSIBLE p38 REGULATED SENESCENCE Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 FIGURE 5. p38 inhibition enhances cell recovery by decreasing apoptosis and increases telomerase activity in CD4+CD45RA+CD272 T cells. A, T cells were activated with anti-CD3 Ab and IL-2 for 4 d in the presence or absence of BIRB. The bar graph represents the number of cells recovered normalized for the initial number of cells placed in culture. Error bars represent the SEM. The dashed line represents the initial cell input (n = 3). B, The bar graph shows the percentage of apoptotic cells (Annexin V+propidium iodide [PI]2) within each subset that was activated as in A, in the presence or absence of BIRB. Error bars represent the SEM (n = 3). C, The bar graph shows the percentage of Ki67+ proliferating cells in the presence and absence of BIRB796 in cells that were activated as in A. The results from five experiments are shown. D, An autoradiography of a TRAP assay acrylamide gel from a representative experiment is shown. Telomerase activity was determined by TRAP assay. Purified subsets were activated with anti-CD3 and irradiated APCs for 4 d in absence or presence of BIRB. The bar graph shows the fold change of telomerase activity following treatment with BIRB. As a negative control, lysis buffer was used instead of cell extract. Results have been normalized for the telomerase activity of each population in absence of the inhibitor. Error bars represent the SE from the mean of four separate experiments. Statistical analysis was performed using the two-tailed Student t test (GraphPad Prism). *p , 0.05. activation. It is possible that proinflammatory cytokines such as It would be of particular interest to determine whether the de- TNF-a may induce the p38 expression in T cells in vivo. Indirect fective ability of naive CD4+ T cells in patients with this disease to evidence for this is that anti–TNF-a therapy in patients with upregulate telomerase activity is related to increased p38 signal- rheumatoid arthritis depletes CD8+ EMRA T cells, suggesting that ing in these cells (61). In addition, CD4+ EMRA T cells that are there may be a role for this cytokine in the generation of these specific for particular immunodominant epitopes of persistent cells in vivo (59). However, TNF-a alone did not induce CD45RA viruses such as CMV accumulate in older humans (7, 62, 63). This expression in CM or EM CD4+ T cells in vitro (N.E. Riddell suggests that CMV infection may either directly or indirectly in- and A.N. Akbar, unpublished observations). Because the EMRA duce a p38-related senescence program in CMV-specific CD4+ population has relatively long telomeres, one possibility is that T cells that does not necessarily depend on telomere erosion. they may be generated directly from the naive T cell pool. This is Although CD4+ EM and EMRA T cells have senescence-related currently under investigation. In contrast, previous studies have proliferative defects, they are multifunctional in terms of cytokine shown that IL-7 can induce CD45RA re-expression in CM CD4+ secretion and express high levels of granzyme B and perforin (7) T cell populations (7, 60); however, it is not known whether this and may be important for protection against certain infections cytokine also induces p38 activation in these cells. It also remains in vivo (59). They are therefore distinct from functionally to be determined whether the process of CD45RA re-expression exhausted T cells that have proliferative defects but also pro- itself is dependent on p38 signaling. The clarification of the in- gressively lose the ability to secrete cytokines and to mediate terrelationship between cytokine signaling and p38-mediated se- cytotoxicity (64–66). Previous studies have shown that T cell nescence will be important to define the role of proinflammatory exhaustion can be regulated by the ligation of surface inhibitory cytokines in inflammatory diseases such as rheumatoid arthritis, receptors such as PD-1 and TIM-3 and that by blocking these where excessive T cell differentiation and senescence occur (20). receptors certain functional responses can be reinstated (reviewed The Journal of Immunology 2099 in Ref. 2). We now show that T cell senescence associated with 15. Akbar, A. N., and J. M. Fletcher. 2005. Memory T cell homeostasis and senescence during aging. Curr. Opin. Immunol. 17: 480–485. DNA damage is reversible at least in part by blocking the p38 16. Moss, P., and N. Khan. 2004. CD8+ T-cell immunity to cytomegalovirus. Hum. pathway. Putative signaling pathways that inhibit of cell cycling Immunol. 65: 456–464. in either exhausted or senescent T cells may be different, which 17. van Lier, R. A., I. J. ten Berge, and L. E. Gamadia. 2003. Human CD8+ T-cell differentiation in response to viruses. Nat. Rev. Immunol. 3: 931–939. raises the question of whether it is safe to block either process to 18. Wills, M. R., G. Okecha, M. P. Weekes, M. K. Gandhi, P. J. Sissons, and enhance immunity in vivo has been discussed extensively else- A. J. Carmichael. 2002. Identification of naive or antigen-experienced human + where (reviewed in Ref. 2). CD8 T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8+ T cell response. J. Immunol. 168: Although p38 signaling has a role in inducing senescence, it also 5455–5464. is involved in the production of proinflammatory cytokines such 19. Faint, J. M., N. E. Annels, S. J. Curnow, P. Shields, D. Pilling, A. D. Hislop, L. Wu, A. N. Akbar, C. D. Buckley, P. A. Moss, et al. 2001. Memory T cells as IL-1b, TNF-a, and IL-6 by T cells (44, 67, 68). An interesting constitute a subset of the human CD8+CD45RA+ pool with distinct phenotypic possibility is that the induction of p38 signaling has a pivotal role and migratory characteristics. J. Immunol. 167: 212–220. in fate determination that governs the generation of short-lived 20. Goronzy, J. J., and C. M. Weyand. 2005. Rheumatoid arthritis. Immunol. Rev. 204: 55–73. effector T cells, because it is responsible for inducing potent 21. Ponchel, F., R. J. Verburg, S. J. Bingham, A. K. Brown, J. Moore, A. Protheroe, functional activity yet increasing the propensity of T cells to un- K. Short, C. A. Lawson, A. W. Morgan, M. Quinn, et al. 2005. Interleukin-7 dergo apoptosis after activation. Although many questions remain, deficiency in rheumatoid arthritis: consequences for therapy-induced lympho- penia. Arthritis Res. Ther. 7: R80–R92. our data clarify a possible link between excessive inflammation 22. Akondy, R. S., N. D. Monson, J. D. Miller, S. Edupuganti, D. Teuwen, H. Wu, and T cell senescence. The fact that p38 signaling in highly dif- F. Quyyumi, S. Garg, J. D. Altman, C. Del Rio, et al. 2009. The yellow fever + + virus vaccine induces a broad and polyfunctional human memory CD8 Tcell ferentiated CD4 T cells is reversible presents the possibility of response. J. Immunol. 183: 7919–7930. modulating upstream and downstream molecules that are involved 23. Campisi, J., and F. d’Adda di Fagagna. 2007. Cellular senescence: when bad Downloaded from in the p38 pathway to counteract the effects of chronic inflam- things happen to good cells. Nat. Rev. Mol. Cell Biol. 8: 729–740. 24. Blackburn, E. H. 2005. Telomeres and telomerase: their mechanisms of action mation that is detrimental during aging (69, 70). and the effects of altering their functions. FEBS Lett. 579: 859–862. 25. d’Adda di Fagagna, F., P. M. Reaper, L. Clay-Farrace, H. Fiegler, P. Carr, T. Von Acknowledgments Zglinicki, G. Saretzki, N. P. Carter, and S. P. Jackson. 2003. A DNA damage checkpoint response in telomere-initiated senescence. Nature 426: 194–198. We thank Mark Bagley (School of Chemistry, Cardiff University) for gifts 26. d’Adda di Fagagna, F. 2008. Living on a break: cellular senescence as a DNA- of BIRB 796 reagents and Terry Davis (School of Medicine, Cardiff Uni- damage response. Nat. Rev. Cancer 8: 512–522. http://www.jimmunol.org/ versity) for useful comments. 27. Passos, J. F., G. Nelson, C. Wang, T. Richter, C. Simillion, C. J. Proctor, S. Miwa, S. Olijslagers, J. Hallinan, A. Wipat, et al. 2010. Feedback between p21 and reactive oxygen production is necessary for cell senescence. Mol. Syst. Biol. Disclosures 6: 347. The authors have no financial conflicts of interest. 28. Fletcher, J. M., M. Vukmanovic-Stejic, P. J. Dunne, K. E. Birch, J. E. Cook, S. E. Jackson, M. Salmon, M. H. Rustin, and A. N. Akbar. 2005. Cyto- megalovirus-specific CD4+ T cells in healthy carriers are continuously driven References to replicative exhaustion. J. Immunol. 175: 8218–8225. 1. Sallusto, F., J. Geginat, and A. Lanzavecchia. 2004. Central memory and effector 29. Bagley, M. C., T. Davis, M. C. Dix, C. S. Widdowson, and D. Kipling. 2006. memory T cell subsets: function, generation, and maintenance. Annu. Rev. Microwave-assisted synthesis of N-pyrazole ureas and the p38alpha inhibitor Immunol. 22: 745–763. BIRB 796 for study into accelerated cell ageing. Org. Biomol. Chem. 4: 4158–

2. Akbar, A. N., and S. M. Henson. 2011. Are senescence and exhaustion inter- 4164. by guest on September 25, 2021 twined or unrelated processes that compromise immunity? Nat. Rev. Immunol. 30. Henson, S. M., O. Franzese, R. Macaulay, V. Libri, R. I. Azevedo, S. Kiani- 11: 289–295. Alikhan, F. J. Plunkett, J. E. Masters, S. Jackson, S. J. Griffiths, et al. 2009. 3. Akbar, A. N., L. Terry, A. Timms, P. C. Beverley, and G. Janossy. 1988. Loss of KLRG1 signaling induces defective Akt (ser473) phosphorylation and proliferative CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol. dysfunction of highly differentiated CD8+ T cells. Blood 113: 6619–6628. 140: 2171–2178. 31. Plunkett, F. J., O. Franzese, H. M. Finney, J. M. Fletcher, L. L. Belaramani, 4. Appay, V., R. A. van Lier, F. Sallusto, and M. Roederer. 2008. Phenotype and M. Salmon, I. Dokal, D. Webster, A. D. Lawson, and A. N. Akbar. 2007. The function of human T lymphocyte subsets: consensus and issues. Cytometry A 73: loss of telomerase activity in highly differentiated CD8+CD28–CD27– T cells is 975–983. associated with decreased Akt (Ser473) phosphorylation. J. Immunol. 178: 7710– 5. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, 7719. M. R. Klein, and R. A. van Lier. 1997. Phenotypic and functional separation of 32. Reed, J. R., M. Vukmanovic-Stejic, J. M. Fletcher, M. V. Soares, J. E. Cook, memory and effector human CD8+ T cells. J. Exp. Med. 186: 1407–1418. C. H. Orteu, S. E. Jackson, K. E. Birch, G. R. Foster, M. Salmon, et al. 2004. 6. Pilling, D., A. N. Akbar, P. A. Bacon, and M. Salmon. 1996. CD4+CD45RA+ Telomere erosion in memory T cells induced by telomerase inhibition at the site T cells from adults respond to recall antigens after CD28 ligation. Int. Immunol. of antigenic challenge in vivo. J. Exp. Med. 199: 1433–1443. 8: 1737–1742. 33. Harari, A., F. Vallelian, and G. Pantaleo. 2004. Phenotypic heterogeneity of 7. Libri, V., R. I. Azevedo, S. E. Jackson, D. Di Mitri, R. Lachmann, S. Fuhrmann, antigen-specific CD4 T cells under different conditions of antigen persistence M. Vukmanovic-Stejic, K. Yong, L. Battistini, F. Kern, et al. 2011. Cytomega- and antigen load. Eur. J. Immunol. 34: 3525–3533. lovirus infection induces the accumulation of short-lived, multifunctional CD4+ 34. Champagne, P., G. S. Ogg, A. S. King, C. Knabenhans, K. Ellefsen, M. Nobile, CD45RA+CD27– T cells: the potential involvement of interleukin-7 in this V. Appay, G. P. Rizzardi, S. Fleury, M. Lipp, et al. 2001. Skewed maturation of process. Immunology 132: 326–339. memory HIV-specific CD8 T lymphocytes. Nature 410: 106–111. 8. Kimmig, S., G. K. Przybylski, C. A. Schmidt, K. Laurisch, B. Mo¨wes, 35. Ouyang, Q., W. M. Wagner, D. Voehringer, A. Wikby, T. Klatt, S. Walter, A. Radbruch, and A. Thiel. 2002. Two subsets of naive T helper cells with C. A. Mu¨ller, H. Pircher, and G. Pawelec. 2003. Age-associated accumulation of distinct T cell receptor excision circle content in human adult peripheral blood. CMV-specific CD8+ T cells expressing the inhibitory killer cell lectin-like re- J. Exp. Med. 195: 789–794. ceptor G1 (KLRG1). Exp. Gerontol. 38: 911–920. 9. Waller, E. C., N. McKinney, R. Hicks, A. J. Carmichael, J. G. Sissons, and 36. Voehringer, D., C. Blaser, P. Brawand, D. H. Raulet, T. Hanke, and H. Pircher. M. R. Wills. 2007. Differential costimulation through CD137 (4-1BB) restores 2001. Viral infections induce abundant numbers of senescent CD8 T cells. J. proliferation of human virus-specific “effector memory” (CD28–CD45RAHI) Immunol. 167: 4838–4843. CD8+ T cells. Blood 110: 4360–4366. 37. Tanaka, T., H. D. Halicka, F. Traganos, K. Seiter, and Z. Darzynkiewicz. 2007. 10. Dunne, P. J., J. M. Faint, N. H. Gudgeon, J. M. Fletcher, F. J. Plunkett, Induction of ATM activation, histone H2AX phosphorylation and apoptosis by M. V. Soares, A. D. Hislop, N. E. Annels, A. B. Rickinson, M. Salmon, and etoposide: relation to cell cycle phase. Cell Cycle 6: 371–376. A. N. Akbar. 2002. Epstein-Barr virus-specific CD8+ T cells that re-express 38. Weng, N. P., B. L. Levine, C. H. June, and R. J. Hodes. 1996. Regulated ex- CD45RA are apoptosis-resistant memory cells that retain replicative potential. pression of telomerase activity in human T lymphocyte development and acti- Blood 100: 933–940. vation. J. Exp. Med. 183: 2471–2479. 11. Effros, R. B., Z. Cai, and P. J. Linton. 2003. CD8 T cells and aging. Crit. Rev. 39. Bryl, E., A. N. Vallejo, E. L. Matteson, J. M. Witkowski, C. M. Weyand, and Immunol. 23: 45–64. J. J. Goronzy. 2005. Modulation of CD28 expression with anti-tumor necrosis 12. Pawelec, G., and E. Derhovanessian. 2011. Role of CMV in immune senescence. factor alpha therapy in rheumatoid arthritis. Arthritis Rheum. 52: 2996–3003. Virus Res. 157: 175–179. 40. Parish, S. T., J. E. Wu, and R. B. Effros. 2009. Modulation of T lymphocyte 13. Kovaiou, R. D., and B. Grubeck-Loebenstein. 2006. Age-associated changes replicative senescence via TNF-alpha inhibition: role of caspase-3. J. Immunol. within CD4+ T cells. Immunol. Lett. 107: 8–14. 182: 4237–4243. 14. Hislop, A. D., G. S. Taylor, D. Sauce, and A. B. Rickinson. 2007. Cellular 41. Berenson, L. S., J. Yang, B. P. Sleckman, T. L. Murphy, and K. M. Murphy. responses to viral infection in humans: lessons from Epstein-Barr virus. Annu. 2006. Selective requirement of p38alpha MAPK in cytokine-dependent, but not Rev. Immunol. 25: 587–617. antigen receptor-dependent, Th1 responses. J. Immunol. 176: 4616–4621. 2100 CD4+ EMRA CELLS UNDERGO REVERSIBLE p38 REGULATED SENESCENCE

42. Dodeller, F., and H. Schulze-Koops. 2006. The p38 mitogen-activated protein 57. von Zglinicki, T., G. Saretzki, J. Ladhoff, F. d’Adda di Fagagna, and kinase signaling cascade in CD4 T cells. Arthritis Res. Ther. 8: 205. S. P. Jackson. 2005. Human cell senescence as a DNA damage response. Mech. 43. Li, C., P. Beavis, A. C. Palfreeman, P. Amjadi, A. Kennedy, and F. M. Brennan. Ageing Dev. 126: 111–117. 2011. Activation of p38 mitogen-activated protein kinase is critical step for 58. Plunkett, F. J., O. Franzese, L. L. Belaramani, J. M. Fletcher, K. C. Gilmour, acquisition of effector function in cytokine-activated T cells, but acts as a neg- R. Sharifi, N. Khan, A. D. Hislop, A. Cara, M. Salmon, et al. 2005. The impact of ative regulator in T cells activated through the T-cell receptor. Immunology 132: telomere erosion on memory CD8+ T cells in patients with X-linked lympho- 104–110. proliferative syndrome. Mech. Ageing Dev. 126: 855–865. 44. Iwasa, H., J. Han, and F. Ishikawa. 2003. Mitogen-activated protein kinase p38 59. Bruns, H., C. Meinken, P. Schauenberg, G. Ha¨rter, P. Kern, R. L. Modlin, defines the common senescence-signalling pathway. Genes Cells 8: 131–144. C. Antoni, and S. Stenger. 2009. Anti-TNF immunotherapy reduces CD8+ T cell- 45. Haq, R., J. D. Brenton, M. Takahashi, D. Finan, A. Finkielsztein, S. Damaraju, mediated antimicrobial activity against Mycobacterium tuberculosis in humans. R. Rottapel, and B. Zanke. 2002. Constitutive p38HOG mitogen-activated pro- J. Clin. Invest. 119: 1167–1177. tein kinase activation induces permanent cell cycle arrest and senescence. 60. Geginat, J., F. Sallusto, and A. Lanzavecchia. 2001. Cytokine-driven pro- Cancer Res. 62: 5076–5082. liferation and differentiation of human naive, central memory, and effector 46. Bagley, M. C., T. Davis, P. G. S. Murziani, C. S. Widdowson, and D. Kipling. memory CD4+ T cells. J. Exp. Med. 194: 1711–1719. 2010. Use of p38 MAPK Inhibitors for the Treatment of Werner Syndrome. 61. Fujii, H., L. Shao, I. Colmegna, J. J. Goronzy, and C. M. Weyand. 2009. Telo- Pharmaceuticals 3: 1842–1872. merase insufficiency in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 106: 47. Davis, T., M. A. Bachler, F. S. Wyllie, M. C. Bagley, and D. Kipling. 2010. 4360–4365. Evaluating the role of p38 MAP kinase in growth of Werner syndrome fibro- 62. Khan, N. 2007. The immunological burden of human cytomegalovirus infection. blasts. Ann. N.Y. Acad. Sci. 1197: 45–48. Arch. Immunol. Ther. Exp. (Warsz.) 55: 299–308. 48. Akbar, A. N., P. C. Beverley, and M. Salmon. 2004. Will telomere erosion lead to 63. Weekes, M. P., M. R. Wills, J. G. Sissons, and A. J. Carmichael. 2004. Long- a loss of T-cell memory? Nat. Rev. Immunol. 4: 737–743. term stable expanded human CD4+ T cell clones specific for human cytomeg- 49. Hodes, R. J., K. S. Hathcock, and N. P. Weng. 2002. Telomeres in T and B cells. alovirus are distributed in both CD45RAhigh and CD45ROhigh populations. J. Nat. Rev. Immunol. 2: 699–706. Immunol. 173: 5843–5851. 50. Effros, R. B. 2004. Replicative senescence of CD8 T cells: effect on human 64. Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, and ageing. Exp. Gerontol. 39: 517–524. R. Ahmed. 2003. Viral persistence alters CD8 T-cell immunodominance and 51. Goronzy, J. J., H. Fujii, and C. M. Weyand. 2006. Telomeres, immune aging and tissue distribution and results in distinct stages of functional impairment. J. Virol. Downloaded from autoimmunity. Exp. Gerontol. 41: 246–251. 77: 4911–4927. 52. Rufer, N., A. Zippelius, P. Batard, M. J. Pittet, I. Kurth, P. Corthesy, 65. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. C. Cerottini, S. Leyvraz, E. Roosnek, M. Nabholz, and P. Romero. 2003. Ex J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of vivo characterization of human CD8+ T subsets with distinct replicative history activated T cells without effector function. J. Exp. Med. 188: 2205–2213. and partial effector functions. Blood 102: 1779–1787. 66. Kuchroo, V. K., V. Dardalhon, S. Xiao, and A. C. Anderson. 2008. New roles for 53. Nikolich-Zugich, J., and B. D. Rudd. 2010. Immune memory and aging: an TIM family members in immune regulation. Nat. Rev. Immunol. 8: 577–580. infinite or finite resource? Curr. Opin. Immunol. 22: 535–540. 67. Rincoń, M., and G. Pedraza-Alva. 2003. JNK and p38 MAP kinases in CD4+ and +

54. Akbar, A. N., and M. Vukmanovic-Stejic. 2007. Telomerase in T lymphocytes: CD8 T cells. Immunol. Rev. 192: 131–142. http://www.jimmunol.org/ use it and lose it? J. Immunol. 178: 6689–6694. 68. Zarubin, T., and J. Han. 2005. Activation and signaling of the p38 MAP kinase 55. Weng, N. P., A. N. Akbar, and J. Goronzy. 2009. CD28– T cells: their role in the pathway. Cell Res. 15: 11–18. age-associated decline of immune function. Trends Immunol. 30: 306–312. 69. Fulop, T., A. Larbi, J. M. Witkowski, J. McElhaney, M. Loeb, A. Mitnitski, and 56. Appay, V., P. R. Dunbar, M. Callan, P. Klenerman, G. M. Gillespie, L. Papagno, G. Pawelec. 2010. Aging, frailty and age-related diseases. Biogerontology 11: G. S. Ogg, A. King, F. Lechner, C. A. Spina, et al. 2002. Memory CD8+ T cells 547–563. vary in differentiation phenotype in different persistent virus infections. Nat. 70. Weyand, C. M., J. W. Fulbright, and J. J. Goronzy. 2003. Immunosenescence, Med. 8: 379–385. autoimmunity, and rheumatoid arthritis. Exp. Gerontol. 38: 833–841. by guest on September 25, 2021