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 References This article cites 33 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/175/10/6489.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *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 © 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 mem- ory 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). Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6490 CD4 MEMORY SUBSET DIFFERENTIATION 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 in- cubated 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).