CD27 Cells Defined by Expression of CCR7 and Stepwise Differentiation

Stepwise Differentiation of CD4 Memory T Cells Defined by Expression of CCR7 and CD27

This information is current as Ruth D. Fritsch, Xinglei Shen, Gary P. Sims, Karen S. of September 28, 2021. Hathcock, Richard J. Hodes and Peter E. Lipsky J Immunol 2005; 175:6489-6497; ; doi: 10.4049/jimmunol.175.10.6489 http://www.jimmunol.org/content/175/10/6489 Downloaded from

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Stepwise Differentiation of CD4 Memory T Cells Deﬁned by Expression of CCR7 and CD271

Ruth D. Fritsch,2* Xinglei Shen,2† Gary P. Sims,* Karen S. Hathcock,† Richard J. Hodes,†‡ and Peter E. Lipsky3*

To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7؉CD27؉, the CCR7؊CD27؉, and the CCR7؊CD27؊ population. In vitro stimulation led to a stepwise differentiation from naive to CCR7؉CD27؉ to CCR7؊CD27؉ to CCR7؊CD27؊. Telomere length in these subsets differed significantly CCR7؉CD27؉ > CCR7؊CD27؉ > CCR7؊CD27؊), suggesting that these subsets constituted a differentiative pathway with) progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations Downloaded from declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7؉CD27؉, whereas IFN-␥ was produced by CCR7؊CD27؉ and to a slightly lesser extent by CCR7؊CD27؊ T cells. IL-4 secretion was predominantly conducted by CCR7؊CD27؊ memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined. The Journal of Immunology, 2005, 175: 6489–6497. http://www.jimmunol.org/ he ability to mount adaptive immune responses is a hall- only minimal capacity to secrete cytokines and to migrate prefer- mark of the immune system and involves the Ag-induced entially to the T cell areas of lymphoid organs, where they can be activation and expansion of naive T cells and their dif- restimulated by Ag. In contrast, CCR7-negative T are thought T EM ferentiation into memory cells. This process is essential for the to have acquired different migratory capabilities and effector func- evolution of an effective immune response because memory cells tions, such as cytokine production or cytotoxicity, which they can acquire altered capacity to produce cytokines, different migratory exert after their migration to inflamed peripheral tissue (5). In line capability, and diminished activation requirements that allow them with this model, an enrichment of TCM was demonstrated in ton- to generate an effective secondary response on rechallenge with sils, whereas most T cells isolated from nonlymphoid tissue such by guest on September 28, 2021 specific Ag. Current understanding of the differentiation pathway as the lung, skin, or intestine lacked the expression of CCR7 and from naive cells through terminally differentiated memory cells is thus were thought to be TEM (6). incomplete. A model has been proposed (1), which distinguishes Notably, however, the use of CCR7 as a marker distinguishing central memory T cells (T )4 from effector memory T cells CM recirculating TCM from cytokine-secreting TEM has been chal- (TEM) by the phenotypic expression of the cysteine chemokine lenged. Thus, it has been reported that the majority of cytokine- receptor CCR7. CCR7 enables cells to home to secondary lym- secreting human T cells reside in the CCR7-expressing subset and phoid organs through interaction with its ligands, CCL19 (also as a result may be capable of lymphoid organ homing (7). Nev- known as EBV-induced molecule 1 ligand) and CCL21 (also ertheless, these authors did find a higher frequency of polarized called secondary lymphoid tissue chemokine), which are predom- Th1 or Th2 cells in the CCR7-negative TEM subset. Furthermore, inantly expressed by high endothelial venules (HEV) of secondary new data indicate the presence of “pre-Th1” and “pre-Th2” cells in lymphoid organs and parenchymal cells of the T cell zones of ␥ the TCM compartment, which secrete low amounts of IFN- or lymph nodes (2–4). TCM, expressing CCR7, are believed to have IL-4, and can be induced to differentiate into Th1 and Th2 effector memory T cells by antigenic stimulation or in response to homeo- static cytokines (8). *Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin In addition to CCR7, phenotypic expression of other cell surface Diseases (NIAMS), †Experimental Immunology Branch, National Cancer Institute, and ‡National Institute on Aging, National Institutes of Health, Bethesda, MD 20892 proteins, especially costimulatory molecules, has been used to Received for publication May 17, 2005. Accepted for publication September 1, 2005. study the differentiation of memory T cells. In this respect, CD27, The costs of publication of this article were defrayed in part by the payment of page a member of the TNFR family, has also been used to delineate charges. This article must therefore be hereby marked advertisement in accordance stages of T cell differentiation (9–11). CD27 is expressed by naive with 18 U.S.C. Section 1734 solely to indicate this fact. CD4 and CD8 T cells as well as by most memory T cells and has 1 This work was supported by a Max Kade Foundation Postdoctoral Research Ex- change Grant (to R.D.F.). been assigned a costimulatory function. Initial up-regulation of CD27 expression upon TCR engagement is followed by irrevers- 2 R.D.F. and X.S. contributed equally to this work. ible loss after repeated antigenic stimulation (12, 13). CD27 ex- 3 Address correspondence and reprint requests to Dr. Peter E. Lipsky, National In- stitute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of pression in CD4 memory T cells distinguishes two populations Health, Building 10, Room 6D47C, Bethesda, MD 20892. E-mail address: whose distinct properties have been characterized (11). The loss of [email protected] CD27 correlates with an increase of IL-4 production. Accumula- 4 Abbreviations used in this paper: TCM, central memory T cell; TEM, effector memory T cell; HEV, high endothelial venule; PI, propidium iodide; FISH, fluorescence tion of these cells has been reported in patients with chronic al- in situ hybridization; Tr1, T regulatory 1. lergic diseases, rheumatoid arthritis, and in aged donors (11, 14).

Although the combination of CCR7 and CD27 has been used in Cell cycle progression of T cell subsets after stimulation was examined an attempt to define a maturational pathway of CD8 T cells (15, by DNA staining with propidium iodide (PI). 16), the differentiation steps of CD4 memory T cells characterized by expression of CCR7 and CD27 have not been defined. Telomere length measurement Therefore, the purpose of this study was to characterize the mat- Telomere length of five individuals (age range: 43–72 years; mean: 55.0 Ϯ urational pattern of CD4 memory T cells by expression of CCR7 4.9) was measured by flow-fluorescence in situ hybridization (FISH) (17). ϩ and CD27. Isolated T cells were incubated in PBS 1% BSA. Cells were then incubated in the dark for 15 min in a hybridization mixture containing either 0.3 ␮g/ml FITC-labeled peptide nucleic acid-telomere FISH probe (Ap- Materials and Methods plied Biosystems) or without probe for autofluorescence background mea- Subjects surement. Cells were then heated at 86°C for 15 min and hybridized in the dark at room temperature for 1.5 h. Cell bodies were then gently washed The investigated population consisted of 42 healthy individuals (50% and resuspended. LDS 751 (Exciton), a DNA binding dye, was added to the Ϯ women, age: 40.6 2.5 years), whose blood was provided by the Depart- resuspended cells (10 ng/ml), and fluorescence intensities of the telomere ment of Transfusion Medicine, Clinical Center, National Institutes of probe and LDS were measured by flow cytometry using a FACSCalibur Health. Informed consent was obtained from all subjects. (BD Biosciences). Single lymphocytes were identified by forward scatter PBMC were obtained by centrifugation of heparinized blood over Fi- ϩ and by total DNA content, and these cells were gated for telomere binding coll-Hypaque (Amersham Biosciences). T cells or CD4 T cells were neg- fluorescence. Quantitation of telomere length was performed by interpo- atively selected on a magnetic column using the AutoMACS (Miltenyi lating the fluorescence signal from the peptide nucleic acid telomere probe Biotec) and the respective isolation kits for Pan T cell or CD4 isolation ϩ to a standard curve generated by beads with known fluorescence units (Miltenyi Biotec). In some cases, CD4 T cells were further subjected to (Bangs Laboratories). Variations in hybridization conditions between ex-

positive selection for CD45RO-expressing cells by the CD45RO kit and periments were normalized by comparison to an aliquot of a control cell Downloaded from AutoMACS. All procedures were conducted according to the manufactur- preparation run with each experiment. er’s instruction. The purity of the resultant population was analyzed by Ͼ ﬂow cytometry and was generally 95%. Telomerase activity Flow cytometry Naive CD4 T cells and sorted CD4 memory T cell subsets were stimulated ␮ For phenotypic analysis, T cells were incubated for 30 min at 4°C with a overnight with 10 ng/ml PMA plus 1.34 M ionomycin followed by a 3-day culture period in medium supplemented with IL2. Telomerase ac-

FITC-coupled mAb against CCR7 (clone no. 150503; R&D Systems), PE- http://www.jimmunol.org/ conjugated anti-CD27 mAb (clone no. M-T271; BD Pharmingen), Per- tivity was assayed three days later using the TRAPeze Telomerase Detec- CPCy5.5-coupled anti-CD4 mAb (clone no. SK3; BD Biosciences), and tion kit (Chemicon/Serologicals) following the manufacturer’s instruc- allophycocyanin-conjugated anti-CD45RO mAb (clone no. UCHL-1; BD tions. Telomerase activity in serial dilutions of cell lysates was detected by Pharmingen). Abs of the appropriate IgG isotypes were used as negative its ability to extend telomeric repeats on a substrate, the template strand controls. Naive T cells served as positive control for staining with anti- primer. These telomerase products were then amplified by a two-step PCR CCR7 and anti-CD27. For chemokine receptor analysis, CD4ϩCD45ROϩ for 31 cycles and run on a 12% PAGE gel. DNA bands were visualized T cells were isolated by AutoMACS and stained with the above-mentioned using SYBR-green (Molecular Probes) and a PhosphorImager (Molecular FITC-anti-CCR7, PE-anti-CD27, and PerCPCy5.5-anti-CD4, as well as Dynamics). A competitive internal standard was used to control for the with allophycocyanin-conjugated mAb against CXCR4 (clone no. 12G5; efficiency of the PCR amplification as well as to detect the presence of Taq BD Biosciences) or a biotinylated mAb against CXCR5 (clone no. RF8B2; polymerase inhibitors. Telomerase activity was calculated as the intensity

BD Pharmingen), followed by streptavidin-coupled allophycocyanin. Cells of telomerase products generated divided by the intensity of the internal by guest on September 28, 2021 were analyzed using a FACSCalibur flow cytometer (BD Biosciences). The standard. A heat-inactivated lysate and a buffer-only lysate were used as acquired data were analyzed using Flow Jo software (Tree Star). negative controls, and TSR8 control template and EL-4 lysate were used as a positive control. To control for day-to-day variation, one sample from Cell separation by flow cytometry each experimental group was repeated in a single assay. These values were then used as a reference to normalize the other samples from the same For cytokine analysis, proliferation assays, assessment of telomere length, experimental day. and telomerase activity, cells were sorted using a MoFlo flow cytometer (DakoCytomation). Anti-CCR7-FITC, anti-CD27-PE, anti-CD4-Per- Cytokine analysis CPCy5.5, and anti-CD45RO-allophycocyanin were in some cases supplemented with anti-CD3-PE-Cy7 to avoid purification steps on the Au- FACS-sorted CD4 memory T cell subsets (1 ϫ 104/well) were activated toMACS and the inherent cell loss. Postsort controls of the respective with PMA/ionomycin overnight, and supernatants were collected after sorted populations routinely exhibited Ͼ95% purity. 20 h. Cytokines in supernatants were measured by Cytometric bead array (CBAkit-II; BD Biosciences) according to the manufacturer’s manual us- Cell culture and cell counting assays ing a FACSCalibur (BD Biosciences). To normalize individual variation, the cytokine secretion of CCR7Ϫ/CD27ϩ and CCR7Ϫ/CD27Ϫ was ex- After an initial overnight activation with 10 ng/ml PMA (Sigma-Aldrich) ϩ ϩ and 1.34 ␮M ionomycin (Calbiochem), sorted memory T cell subsets were pressed as a percentage of the secretion observed in the CCR7 /CD27 washed and maintained in 50 U/ml IL-2 (Biological Research Branch, Di- population. vision of Cancer Treatment and Diagnosis, National Cancer Institute) in U-bottom 96-well-plates (Costar). Culture medium consisted of Ultra Cul- Statistical methods ture serum-free medium (BioWhittaker). After 1 wk, cells were stimulated Ϯ with anti-CD3 (64.1: 1 ␮g/ml) and cultured for another week, after which Values are expressed as mean SEM, and for statistical analysis Student’s they were activated again with PMA/ionomycin as described above. Phe- t test was applied. notypic characterization was performed immediately after the last stimulation and after an additional 9 days of incubation in IL-2. Alternatively, Results cells were stained 3 days after each respective stimulation and after an additional resting period of 4 days. Characterization of memory CD4 T cells For analysis of actual cell numbers, 5 ϫ 104 cells from each population Using naive CD45ROϪ CD4 T cells that uniformly express CCR7 tested were stimulated with PMA/ionomycin overnight and then cultured and CD27 as a positive control and isotype-matched control mAb with 50 U/ml IL-2. Cell numbers were assessed by flow cytometry using as a negative control, we could identify three major subsets of SPHERO beads (Spherotech) after an additional 72 h of culture. Dead cells ϩ ϩ Ϫ ϩ were excluded by staining with 7-aminoactinomycin D (Molecular Probes). memory CD4ϩT cell: the CCR7 /CD27 , the CCR7 /CD27 , The proliferative capacity is expressed as the ratio of cells recovered from and the CCR7Ϫ/CD27Ϫ. Within CD4ϩ T cells of healthy individ- ϩ ϩ the various subsets to the cells recovered from the CCR7 /CD27 subset. uals, the majority of cells expressed both CCR7 and CD27 Alternatively, proliferation of T cell subsets was measured after stimulation Ϯ Ϯ with anti-CD3 mAb. A total of 3 ϫ 104 cells of each respective subset were (mean SEM: 69.8 2.1%). Memory T cells that did not express incubated in triplicate with plate-bound anti-CD3 (64.1: see above), and CCR7 but were CD27 positive accounted for 21.5 Ϯ 1.8%, ϩ Ϫ proliferation was measured after 72 h by [3H]thymidine incorporation. whereas only 5.6 Ϯ 0.6% of memory CD4 T cells were CCR7 / The Journal of Immunology 6491

FIGURE 1. Percentage of memory CD4 T cell subsets in normal subjects. Memory CD4 T cells were stained for the expression of CCR7 and CD27, and three major populations were assessed by comparison to the negative (isotype control staining) and positive control (i.e., naive T cells), namely CCR7ϩ/CD27ϩ, CCR7Ϫ/CD27ϩ, and CCR7Ϫ/CD27 Ϫ (A). The bars in B are mean (ϮSEM) of 42 samples from different normal subjects. Downloaded from http://www.jimmunol.org/ CD27Ϫ (Fig. 1). The observed patterns of CCR7 and CD27 ex- CD27ϩ and CCR7Ϫ/CD27ϩ CD4 memory T cell subsets (Fig. pression in healthy individuals did not correlate with the propor- 2A). A portion of CCR7ϩ/CD27ϩ memory CD4 T cells (mean Ϯ tion of CD4ϩCD45ROϩ total memory cells and did not correlate SEM: 30.6 Ϯ 4.3%) became CCR7Ϫ/CD27ϩ, whereas approxi- signiﬁcantly with the age of the donors (age range: 8–72 years; mately one-half (mean Ϯ SEM: 46.7 Ϯ 5.0%) became negative for mean age Ϯ SEM: 40.6 Ϯ 2.5). both surface markers. Furthermore, the majority (mean Ϯ SEM: Ϯ Ϫ ϩ ϩ 80.5 6.9%) of the CCR7 /CD27 cell population lost expres- Phenotype of CD4 memory T cells after repeated stimulation sion of CD27 and became CCR7Ϫ/CD27Ϫ after repeated stimula- To examine the relationship between the CD4 memory T cell sub- tion (Fig. 2A). To examine the stability of the phenotypic change, by guest on September 28, 2021 sets, the effect of in vitro stimulation on cell surface phenotype was cells were also assessed after an additional resting period. As can assessed. Repeated stimulation altered the phenotype of CCR7ϩ/ be seen in Fig. 2A, the altered phenotype remained stable during

FIGURE 2. Phenotypic changes of CD4 memory T cell subsets after repeated stimulation. CD4ϩ memory T cells from healthy individuals were sorted according to their phenotypic expression of CCR7 and CD27. Subsets were stimulated three times with anti-CD3 or PMA/ionomycin over the course of 3 wk and subse- quently assessed for their phenotype. After a resting period of 9 days in medium supplemented with IL-2, cells were again stained for the surface expression of CCR7 and CD27. The displayed pattern was seen in six healthy individuals; one representative example is shown in A. B, The mean percentage of CCR7Ϫ/CD27Ϫ cells in the respective populations (CCR7ϩ/CD27ϩ: ϩ/ϩ; CCR7Ϫ/CD27ϩ: Ϫ/ϩ; and CCR7Ϫ/CD27Ϫ: Ϫ/Ϫ) derived from three healthy individuals 3 days after each stimulation. 6492 CD4 MEMORY SUBSET DIFFERENTIATION the resting period of 9 days. CD4ϩ T cells appeared to alter their ulations, except for the CCR7Ϫ/CD27Ϫ subset. Proliferative ca- phenotype in a stepwise manner following activation (Fig. 2B). pacity decreased from naive cells to CCR7ϩ/CD27ϩ (Fig. 3A), Ϫ ϩ After one round of stimulation, the majority of the CCR7 /CD27 further declining in the CCR7Ϫ/CD27ϩ T cells. The lowest cell Ϫ Ϫ converted to a CCR7 /CD27 phenotype (mean: 77.0 Ϯ 1.8%). In recovery was noted in CCR7Ϫ/CD27Ϫ memory T cells (Fig. 3B). ϩ ϩ contrast, Ͼ75% of CCR7 /CD27 cells lost CCR7 after the first Stimulation with anti-CD3 demonstrated a similar pattern, with Ϫ Ϫ stimulation and then progressively converted to CCR7 /CD27 . a significant decrease in proliferative capacity from naive CD4 T ϩ ϩ The bulk of naive cells retained their CCR7 /CD27 phenotype cells to CCR7ϩ/CD27ϩ with the CCR7Ϫ/CD27Ϫ exhibiting the after one stimulation (68.4 Ϯ 9.6%), after which they increasingly least proliferation (Fig. 3C). The difference in proliferation was Ϫ Ϫ became CCR7 /CD27 with additional stimulation (Fig. 2B). Fi- Ͻ Ϫ Ϫ statistically significant between all subsets ( p 0.03). nally, the CCR7 /CD27 subset retained its phenotype despite To gain more insight into the balance of cell death and prolif- repeated stimulation and did not (re)-express either CCR7 or CD27 eration manifested by the subsets, cell cycle analysis was con- (Fig. 2B). These results clearly identify a differentiation pathway ducted. A representative example is shown in Fig. 4A. Immediately of CD4ϩ memory T cells in which CCR7ϩ/CD27ϩ cells mature to Ϫ ϩ Ϫ Ϫ ex vivo, most cells from each subset were in G0-G1, and at most, CCR7 /CD27 cells and finally to CCR7 /CD27 terminally dif- Ϫ Ϫ 2% (CCR7 /CD27 ) were in G -M (Fig. 4B). Upon stimulation, ferentiated memory T cells. 2 each of the subsets progressed into the cell cycle to varying degrees as noted in Fig. 4, C and D. Naive and CCR7ϩ/CD27ϩ ϩ Proliferative capacity and cell survival of the CD4 memory T memory T cells showed a similar pattern of cell cycle progression cell subsets with ϳ30% of the cells in S-G -M and only a small percentage 2 Downloaded from Ϫ ϩ Ϫ The subsets were assessed for the capacity to proliferate after in becoming apoptotic. In contrast, CCR7 /CD27 and CCR7 / Ϫ vitro stimulation. Initially, cells were stimulated with PMA/iono- CD27 subsets exhibited similar cell progression patterns that ϩ ϩ mycin so that intrinsic differences in proliferative capacity could were different from those in naive and CCR7 /CD27 cells. A be assessed separately from potential changes in TCR signaling. rather small percentage of these cells was stimulated to enter

As shown in Fig. 3, A and B, 3 days after in vitro stimulation with S-G2-M, whereas a sizeable portion of the cells become apoptotic PMA/ionomycin, the number of cells detected increased in all pop- (Fig. 4D). http://www.jimmunol.org/

FIGURE 3. Differences in survival and proliferation of CD4 memory subsets. CD4ϩ (CD45ROϪ) naive and (CD45RObright) memory T cells of seven healthy individuals were sorted according to their expression of by guest on September 28, 2021 CCR7 and CD27. Viability and cell number of each subset were assessed 72 h after PMA/ionomycin stimulation by the use of 7-aminoactinomycin D and counting beads and the flow cytometer. Alternatively, proliferation was induced by immobilized anti-CD3 and measured by [3H]thymidine incorporation. A, A typical example of the different proliferation profiles of the subtypes. A total of 5 ϫ 104 cells (dashed line) was stimulated with PMA/ionomycin, and the cell number was assessed by FACS analysis. All subpopulations showed an increased cell number after stimulation with PMA/ionomycin. B, The mean (Ϯ SEM) number of live cells (derived from seven healthy individuals) of the indicated subsets after the 72 h incubation with PMA/ionomycin normalized to the CCR7ϩ/CD27ϩ cell count (naive CD4 T cells: 141.6 Ϯ 37.1%; CCR7Ϫ/ CD27ϩ T cells: 63.4 Ϯ 8.5%; and CCR7Ϫ/CD27Ϫ: 61.4 Ϯ 7.6%). The difference in the number of cells between CCR7ϩ/CD27ϩ and all other subsets reached statistical significance. (૾, p Ͻ 0.01; ૾૾, p Ͻ 0.0003). C, The different degrees of proliferation exhibited by the CD4 memory cell subsets upon stimulation with anti-CD3. A total of 3 ϫ 104 T cells of each subset was stimulated with immobilized anti-CD3 for 72 h, and proliferation was measured by [3H]thymidine incorporation. Proliferation declined from naive CD4 T cells to CCR7ϩ/CD27ϩ and further to CCR7Ϫ/CD27ϩ and was lowest in the CCR7Ϫ/CD27Ϫ subset. The difference in proliferation was statistically significant between all subsets (p Ͻ 0.03). Data from one representative experiment are shown. The Journal of Immunology 6493

FIGURE 4. Cell cycle analysis of memory subsets ex vivo and after in vitro stimulation. A, Cell cycle of the respective memory T cell subsets was assessed immediately ex vivo using PI staining. Subsets from one representative subject ex vivo are shown. B, Analysis of memory subsets from four healthy individuals ex vivo. C, A representative example of changes in cell cycle stage of the different subsets after stimulation with PMA/ionomycin overnight followed by 24 h incubation with IL-2-supplemented medium using PI staining is shown. D, Analysis of memory subsets from four healthy ϩ ϩ Ϫ ϩ individuals after stimulation. The differences in the percentage of cells in G0-G1 and apoptosis between naive or CCR7 /CD27 and either CCR7 /CD27 Ϫ Ϫ ϩ ϩ Ϫ Ϫ or CCR7 /CD27 were all statistically signiﬁcant (p Ͻ 0.01). The difference between CCR7 /CD27 and CCR7 /CD27 was also statistically signiﬁcant Downloaded from ϭ in G2-M (p 0.01).

Telomere lengths in memory CD4 T cell subsets Inducible telomerase activity in memory CD4 subsets Previous studies have indicated that telomeres shorten signiﬁcantly As telomere length is inﬂuenced by the activity of telomerase,

as T cells divide during in vivo differentiation from naive to mem- inducible telomerase activity was examined in CD4 T cell subsets. http://www.jimmunol.org/ ory cells. To test the lineage relationship between memory CD4 The populations derived from each normal donor showed very subsets suggested by the in vitro experiments, we examined the ex consistent patterns of inducible telomerase activity, which was vivo telomere length of the respective subsets by flow-FISH. As higher in the naive population than any of the memory subsets shown in Fig. 5A, consistent differences in telomere length were ( p Ͻ 0.002; Fig. 6A). Within the CD4 memory subsets, the observed among these populations. Naive CD4 T cells of five CCR7ϩ/CD27ϩ subset had higher activity than either the CCR7Ϫ/ healthy individuals (age range: 43–72 years; mean: 55.0 Ϯ 4.9; CD27ϩ or CCR7Ϫ/CD27Ϫ subsets (both p Ͻ 0.002), both of 60% female) had significantly longer telomeres than any of the which had similarly low telomerase activity levels (Fig. 6B). memory CD4 subsets. Moreover, subsets within the memory population had significant differences in telomere length, with longest Differences in cytokine secretion in CD4 T cell memory subsets by guest on September 28, 2021 telomeres in the CCR7ϩ/CD27ϩ subset, followed by the CCR7Ϫ/ The functional properties of the memory T cell subsets were ex- CD27ϩ subset, whereas CCR7Ϫ/CD27Ϫ cells had the shortest amined by measuring cytokine secretion after stimulation (Fig. 7). telomeres. IFN-␥ and IL-4, the main cytokines associated with mature Th1 Thus, the finding of progressive telomere shortening is consis- and Th2 cells, respectively, showed a differential secretion pattern tent with the developmental sequence suggested by the phenotypic in CD4 memory subsets. The greatest IFN-␥ secretion was ob- changes after in vitro stimulation. served in CCR7Ϫ/CD27ϩ and less in the CCR7ϩ/CD27ϩ and

FIGURE 5. Telomere length in CD4 naive and memory T cell subsets. Telomere length was measured by flow-FISH in each of the sorted populations. A, Representative sample of relative telomere lengths from one do- nor. B, Mean telomere lengths of subsets from five healthy individuals. Samples from the same donor are con- nected by dashed lines. Mean values of telomere length for each subset are represented by the horizontal lines. Telomere length of each subset is significantly different from the others (p Ͻ 0.025). 6494 CD4 MEMORY SUBSET DIFFERENTIATION

FIGURE 6. Telomerase activity in CD4 naive and memory T cell subsets. A, Telomerase activity of (PMA/ionomycin) activated cells is calculated as the intensity of the telomerase product generated (TPG) divided by the PCR ampliﬁcation of the internal control (IC) as shown in a representative experiment. B, Com- parison of inducible telomerase activity between subsets. The telomerase activity in CD4 T cells decreased from naive cells to CCR7ϩ/CD27ϩ to CCR7Ϫ/CD27ϩ and CCR7Ϫ/CD27Ϫ (૾, p Ͻ 0.02). Downloaded from http://www.jimmunol.org/

CCR7Ϫ/CD27Ϫ populations. In contrast, as already reported for shown in Fig. 8, the expression of CXCR4 decreased from CD27Ϫ cells (13), CCR7Ϫ/CD27Ϫ cells showed the highest IL-4 CCR7ϩ/CD27ϩ Ͼ CCR7Ϫ/CD27ϩ Ͼ CCR7Ϫ/CD27Ϫ. As with production of the three populations, whereas the IL-4 secretion of CXCR4, a decrease in CXCR5 expression was observed from the CCR7Ϫ/CD27ϩ subset was comparable to the CCR7ϩ/CD27ϩ. CCR7ϩ/CD27ϩ to CCR7Ϫ/CD27ϩ and CCR7Ϫ/CD27Ϫ. The ex- Secretion of the anti-inﬂammatory cytokine, IL-10, was greatest in pression of CXCR4 and CXCR5 in the CCR7ϩ/CD27ϩ was sig- ϩ ϩ Ϫ ϩ Ͻ Ϫ ϩ

the CCR7 /CD27 population, decreased in CCR7 /CD27 niﬁcantly ( p 0.05) higher than in the CCR7 /CD27 subset. by guest on September 28, 2021 cells, and was even less in the CCR7Ϫ/CD27Ϫ. IL-2 production showed an expected decrease from CCR7ϩ/CD27ϩ to CCR7Ϫ/ CD27ϩ and CCR7Ϫ/CD27Ϫ. Discussion The ability to separate stages of development by expression of the Differences in chemokine receptor expression in CD4 T cell chemokine receptor CCR7, as well as the TNFR family member memory subsets CD27, has provided a more precise delineation of stages of CD4 To investigate the trafﬁcking potential of the CD4 memory T cell memory cell maturation. Based on in vitro studies, telomere length subsets, the expression of CXCR4 and CXCR5 was examined. As measurement, and assessment of changes in functional capacities,

FIGURE 7. Characterization of cytokine secretion in memory subsets. Subsets from eight individuals were examined. Secretory capacity is expressed as percentage of the secretion observed in the CCR7ϩ/CD27ϩ subset. The signiﬁcant differences (p Ͻ 0.05) between the respective subsets are indicated by ૾. The Journal of Immunology 6495

marked decrease in proliferative capacity appeared to be characteristic of the entire CCR7-/CD27- subset, and not a unique prop- erty of the CD28- fraction. Progressive decrease in telomere length from naive CD4 T cells to the CCR7ϩ/CD27ϩ memory CD4 T cell population and further to the CCR7Ϫ/CD27ϩ subset and the CCR7Ϫ/CD27Ϫ CD4 memory cells substantiated the model of stepwise CD4 T cell maturation and expanded upon the known correlation of telomere shortening and maturation from the naive to memory stage (23) in CD4 T cells. These results have clearly delineated the CCR7Ϫ population into two subsets based on the expression of CD27 and also established the lineal relationship between the previously desig- ϩ Ϫ nated CCR7 TCM and the previously designated CCR7 TEM. Previous data have not clearly demonstrated whether TCM and TEM are independent subsets or stages of maturation along a con- tinuum of differentiation. The current data clearly support the latter conclusion. The intrinsic in vitro proliferative capacity of the memory sub-

sets differed. This was demonstrated initially with PMA/ionomy- Downloaded from cin stimulation to bypass possible variations in TCR signaling and conﬁrmed by stimulation with anti-CD3. In previous reports, memory CD45RO T cells have been shown to exhibit a higher turnover rate and a relatively short lifespan compared with naive T

cells (24–26); furthermore, recently TEM of healthy individuals

were found to manifest a higher in vivo turnover rate compared http://www.jimmunol.org/

with TCM and naive CD4 T cells (27). These results are consistent

with the telomere length analysis conducted here. If TEM had an increased in vivo turnover than TCM, it would be anticipated that FIGURE 8. Characterization of chemokine receptor expression in mem- they would have shorter telomeres, as we found in the CCR7Ϫ ory subsets. Subsets from four healthy individuals were examined for their subsets, indicating that the T had undergone more rounds of in expression of the chemokine receptors, CXCR4 and CXCR5. Significant EM vivo proliferation than T . However, the high turnover of T (p Ͻ 0.05) differences in chemokine receptor expression between the re- CM EM spective subsets are indicated by ૾. shown in the aforementioned study differed from the diminished in vitro proliferative capacity of both CCR7Ϫ subsets shown here. These results suggest that there might be in vivo constraints on by guest on September 28, 2021 ϩ including cytokine secretion, we propose a model of the develop- proliferation by CCR7 TCM, perhaps related to increased require- 3 ϩ ϩ ment from naive CD4 T cells CCR7 /CD27 memory CD4 T ments for productive activation. Alternatively, TEM may have cells 3 CCR7Ϫ/CD27ϩ 3 to the terminally differentiated greater in vivo exposure to Ag presented by the appropriate APC. CCR7Ϫ/CD27Ϫ CD4 memory T cells. Importantly, the in vivo experiments did not examine the potential In normal individuals, the majority (70%) of memory T cells proliferative capacity as was examined here by cell cycle analysis, found in blood bear both CCR7 and CD27 on their surfaces, ϳ20% cell counting by FACS beads and thymidine incorporation. To- express only CCR7, and only a very small proportion (5%) of gether, these data are most consistent with the conclusion that pro- memory cells do not express either markers. An even lower per- liferating TCM eventually give rise to TEM with diminished in vivo centage (3.0 Ϯ 0.3%, data not shown) exhibited the CCR7ϩ/ and in vitro proliferative potential. The endogenous signals that CD27Ϫ phenotype. We did not investigate this subset and its func- may contribute to the somewhat increased in vivo proliferation of tion is currently unknown. TEM remain to be delineated. In vitro culture experiments demonstrated a stepwise conversion The shortening of telomere length in dividing cells is counter- of CCR7ϩ/CD27ϩ into a CCR7Ϫ/CD27ϩ population and the pro- acted by telomerase, an enzyme that is able to extend telomeres by gression of the CCR7Ϫ/CD27ϩ into a CCR7Ϫ/CD27Ϫ subset. Be- synthesis of terminal telomeric repeats. Although regulation of te- cause the induced phenotypes were maintained after a resting pe- lomerase activity in lymphocytes is not completely understood, riod, a transient change of expression of either marker by recent decreased induction of telomerase activity has been observed with stimulation (10, 18) seems unlikely. The conclusion that there is a both repeated in vitro stimulation, as well as in vivo differentiation stepwise down-regulation of CCR7 and then CD27 during CD4 from naive to memory cells, and in parallel with telomere length memory T cells maturation is consistent with the finding that stim- shortening (28, 29). In line with these findings, we observed de- ulated naive CD4 T cells rapidly down-regulated CCR7, whereas creases in induced telomerase activity in the CCR7Ϫ/CD27ϩ and CD27 only declined at a later round of proliferation (19). A similar CCR7Ϫ/CD27Ϫ subsets with shorter telomeres. However, as te- finding was recently observed in bulk cultures of alloreactive lomerase activity might be influenced by stimulation and subse- CD45ROϩ memory T cells (20). quent entry into the cell cycle (27), the lower proportion of in As the down-regulation of CD27 exhibited similarities to the vitro-stimulated cycling cells in the CCR7Ϫ/CD27ϩ and CCR7Ϫ/ loss of CD28 with replicative senescence (21), we examined the CD27Ϫ compared with naive or CCR7ϩ/CD27ϩ memory cell sub- expression of CD28 by the various CD4ϩ memory T cell subsets. sets could account at least in part for the reduced telomerase ac- In accordance with previous findings (22), the CD28- cells were tivity of the CCR7Ϫ subsets. confined to the CCR7-/CD27- subset, constituting about half of Another feature of maturation and differentiation of cells is the this population (data not shown). Thus, the CD28- memory cells acquisition of certain functional properties such as cytokine secre- represent a portion of the CCR7-/CD27- subset. Notably, the tion. In this respect, a difference in cytokine secretion between 6496 CD4 MEMORY SUBSET DIFFERENTIATION

TCM and TEM has been postulated (1) but also disputed in human expression in the more mature stages of CD4 memory T cells as well as murine models (5, 7), whereas the loss of CD27 is might again represent a decreased inclination of the majority of commonly associated with higher IL-4 production (13, 30). We these cells to circulate through lymphoid tissue as these cells dif- found a clear pattern of cytokine secretion of the CD4ϩ T cell ferentiate and is in accordance with recently published findings (8). subsets. We observed increased IFN-␥ and decreased IL-2 secre- Altogether, the data presented in this study lead to a more de- tion by CCR7Ϫ/CD27ϩ and CCR7Ϫ/CD27Ϫ cells compared with tailed view of human CD4 memory T cell maturation. We propose the CCR7ϩ/CD27ϩ population. This result is consistent with the a model of differentiation, in which the sequential stages can be conclusion that there is an association of “effector” cytokine se- distinguished by the phenotypic expression of CCR7 and CD27. ϩ cretion with the loss of CCR7 expression. However, the TEM pop- The results clearly indicate a stepwise conversion of CCR7 / ulation, as defined by CCR7Ϫ only, was not homogeneous, and the CD27ϩ T cells via the stage of CCR7Ϫ/CD27ϩ to a terminally subdivision into CCR7Ϫ/CD27ϩ and CCR7Ϫ/CD27Ϫ cells re- differentiated CCR7Ϫ/CD27Ϫ state. The relationship between vealed different cytokine secretion patterns. Whereas the produc- these subsets was demonstrated by the stepwise unidirectional dif- tion of IL-2 and IFN-␥ was somewhat greater in the CCR7Ϫ/ ferentiation in vitro, as well as differences in telomere length, cell CD27ϩ compared with the CCR7Ϫ/CD27Ϫ cells, IL-4 production cycle analysis, and proliferative capacity. Furthermore, as the cells was predominantly conducted only by CCR7Ϫ/CD27Ϫ memory T mature, they acquire different functional properties illustrated by a cells. change in cytokine secretion and chemokine receptor expression. These findings are consistent with previous results that IFN-␥ Thus, the defined subsets represent different maturational stages of secretion is initiated early after activation and then increases with CD4 memory T cells with unique functional properties successive cell cycles, whereas IL-4 production only appears after Downloaded from several cell divisions (31). The current data indicate that the Acknowledgments CCR7Ϫ/CD27Ϫ population constitutes the most terminally differ- We thank James Simone, Derek Hewgill, and Liusheng He of the Flow entiated state of memory cells, enriched in IL-4-producing cells Cytometry Facility of the National Institute of Arthritis and Musculoskel- and IFN-␥ secretors. The CCR7Ϫ/CD27ϩ subset represents an ear- etal and Skin Diseases Office of Science and Technology for carrying out lier maturational stage consisting of cells that have already been the cell sorting. biased toward Th1 cells but also of cells that are not yet irrevers- Disclosures http://www.jimmunol.org/ ibly committed and might become Th2 cells in the following mat- The authors have no financial conflict of interest. urational step to CCR7Ϫ/CD27Ϫ cells. Interestingly, the pattern of IL-10 secretion was dissociated from IL-4 production, as produc- References tion of this anti-inflammatory cytokine was predominantly con- 1. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two fined to the CCR7ϩ/CD27ϩ cells. subsets of memory T lymphocytes with distinct homing and effector functions. ϩ ϩ Nature 401: 708–712. The enhanced IL-10 production by CCR7 /CD27 cells might 2. Baekkevold, E. S., T. Yamanaka, R. T. Palframan, H. S. Carlsen, F. P. Reinholt, indicate that a pool of T regulatory 1 (Tr1) cells resides within the U. H. von Andrian, P. Brandtzaeg, and G. Haraldsen. 2001. The CCR7 ligand elc CCR7ϩ/CD27ϩ subset. Tr1 cells suppress immune responses and (CCL19) is transcytosed in high endothelial venules and mediates T cell recruit-

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