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Prepublished online as a Blood First Edition Paper on February 6, 2003; DOI 10.1182/blood-2002-11-3577.
Blood, 1 June 2003, Vol. 101, No. 11, pp. 4260-4266 Proliferation and differentiation potential of human CD8+ memory T-cell subsets in response to antigen or homeostatic cytokinesFrom the Institute for Research in Biomedicine, Bellinzona, Switzerland.
Four human CD8+ T-cell subsets, naive (CCR7+CD45RA+), central memory (TCM, CCR7+CD45RA–), effector memory (TEM, CCR7–CD45RA–), and CD45RA+ effector memory cells (TEMRA, CCR7–CD45RA+) were compared for their capacity to proliferate and differentiate in response to antigen or homeostatic cytokines. Cytokine responsiveness and interleukin-15 receptor expression were low in naive T cells and progressively increased from TCM to TEM and TEMRA. In contrast, the capacity to accumulate in response to T-cell receptor (TCR) or cytokine stimulation showed a reciprocal pattern and was associated with resistance to cell death and Bcl-2 expression. Whereas all TCR-stimulated cells acquired a CD45RA–CCR7– phenotype, cytokine-stimulated cells maintained their phenotype with the exception of TCM cells, which expressed CCR7, CD45RA, and perforin in various combinations. Single CD8+ TCM cells, but not TEM cells, could be expanded with cytokines, and the obtained clones displayed several distinct phenotypes, suggesting that TCM cells are heterogeneous. Consistently, CCR4 expression in the CD8+ TCM pool discriminated CCR4+ type 2 polarized cells (Tc2) and CCR4–CTL precursors. Finally, ex vivo bromodeoxyuridine (BrdU) incorporation experiments revealed that memory subsets have different in vivo proliferation rates, with CCR4–TCM having the highest turnover and TEMRA the lowest. These results show that human CD8+ memory T-cell subsets have different proliferation and differentiation potentials in vitro and in vivo. Furthermore, they suggest that TEMRA cells are generated from a TCM subset upon homeostatic proliferation in the absence of antigen.
Memory T lymphocytes are heterogeneous and comprise distinct populations that can be distinguished based on surface markers and effector functions, such as cytokine secretion and cytotoxicity. In the CD8+ compartment loss of CD28 identifies a subset of antigen-experienced T cells with high cytotoxic potential and reduced proliferative capacity, which frequently contain in vivo–expanded clones in the elderly or in human immunodeficiency virus (HIV)–infected individuals.1, 2, 3 Van Lier and colleagues defined 2 CD8+ memory T-cell subsets based on the expression of CD45 isoforms and CD27: CD45RO+CD27+ memory cells, which lacked immediate cytolytic function, and CD45RA+ CD27– effector cells with low proliferative capacity and high levels of perforin and cytotoxicity.4 Further, heterogeneity was revealed by the analysis of CCR7, a chemokine receptor for lymph node homing, which discriminated perforin-central memory T cells (TCM, CCR7+ CD45RA–) from tissue homing CCR7– effector memory T cells, which are either perforinlo CD45RA– (TEM) or perforinhi CD45RA+ (TEMRA).5 The differentiation pathways that lead to the generation of these cells, in particular the CCR7–CD45RA+ cells, are uncertain. A progressive and irreversible differentiation from naive to TCM and TEM has been demonstrated for CD4+ T cells and suggested for CD8+ T cells,5 but the precise relationship between the various CD8+ subpopulations is still a matter of debate. Further, complexity is added by the finding that some CD8+ memory T cells share characteristics with Tc2 cells, which are not cytotoxic but produce interleukin-4 (IL-4) and IL-13.6,7 There is considerable interest in understanding the distribution of CD8+ T cells generated in response to viruses or tumors within different subsets.8, 9, 10, 11, 12, 13 HIV-infected patients lack antigen-specific cells of the CCR7–CD45RA+ subset, suggesting that HIV inhibits certain steps of T-cell differentiation as a strategy to subvert immune response.9 Other studies indicated that CD45RA– and CD45RA+ memory T cells are generated in response to different antigens from the same virus and with different kinetics.10, 11, 12, 13 However, it is not clear from these studies whether these phenotypic and functional differences are due to the persistence of antigen, homeostatic mechanisms, or both.
T-cell homeostasis ensures that the pools of naive and memory T cells are independently maintained at a constant size under changing environmental conditions.14 Studies by Sprent and coworkers established that under steady-state conditions memory T cells turn over continuously at a relatively high rate, whereas naive T cells proliferate poorly.15 However, naive T cells undergo a massive homeostatic expansion when transferred to lymphopenic animals and differentiate to cells that share some characteristics with memory and effector cells.16, 17, 18 It is now well established that both maintenance and homeostatic proliferation of CD8+ T cells depend on cytokines signaling via the common
Comparatively little is known about the homeostasis of human T cells and in particular how the functional heterogeneity of the memory pool is maintained. Based on the length of telomeres and in vivo bromodeoxyuridine (BrdU) incorporation, it was estimated that human memory, but not naive, T cells divide approximately once or twice per year under steady-state conditions, and that they can perform up to 15-30 divisions under lymphopenic conditions.35, 36, 37 We previously reported that human CD4+ memory T cells proliferate in response to IL-7 and IL-15 and that cytokinestimulated TCM cells can differentiate to TEM cells.43 We therefore considered the possibility that the phenotype and function of CD8+ memory T cells may be influenced by homeostatic mechanisms. Consequently, we investigated the proliferation and differentiation potential of human CD8+ T-cell subsets in response to antigen or homeostatic cytokines. The results reported indicate that memory T-cell subsets have different proliferative capacities in vitro and in vivo and that central memory T cells have the unique ability to differentiate in an antigen-independent, but not antigen-dependent, fashion into CCR7–CD45RA+ effector cells.
Antibodies and reagents
Allophycocyanin (APC)– or phycoerythrin (PE)–labeled antibodies specific for IL-7 receptor (R) Cell culture and cloning Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. CD8+ T cells were then isolated by positive selection with anti-CD8–coated magnetic beads (MACS, Miltenyi, Bergisch Gladbach, Germany). The cells obtained were more than 98% CD8+ CD3+. Naive and memory T-cell subpopulations were purified to more than 99% by cell sorting, using anti-CD45RA and anti-CCR7 antibody.5 The distribution between the 4 subsets of 10 donors was the following: naive, 40% +/– 25%; TCM, 15% +/– 12%; TEM, 32% +/– 12%; and TEMRA, 13% +/– 8%. Labeling of T cells with CFSE was performed as described.44 Monocytes were purified by positive selection with anti-CD14 antibodies coupled to magnetic beads (Miltenyi) as described.45 CD14+ cells were cultured for 4 days in RPMI 1640 containing 10% fetal calf serum (Hyclone, Logan, UT), 2 mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 50 µg/mL kanamycin (Gibco, Grand Island, NY), 50 ng/mL granulocyte-macrophage colony-stimulating factor (Novartis, Basel, Switzerland), and 1000 units (U)/mL IL-4. The DCs obtained were stimulated for 24 hours with 100 ng/mL lipopolysaccharide (from Salmonella abortus equi, Sigma, St Louis, MO). T cells were cultured with DCs at a 5:1 ratio in RPMI 1640 containing 100 U/mL IL-2 (Roche, Basel, Switzerland), 5% human serum, 2 mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate, and 50 µg/mL kanamycin. Recombinant cytokines were used at concentrations determined in preliminary dose-response experiments at either 25 ng/mL (IL-7, IL-15, IL-21; R&D, Minneapolis, MN) or 10 ng/mL (IL-6, IL-10, IL-4, IL-12; Pharmingen), unless otherwise indicated. Central and effector memory cells were cloned in U-bottom 96-well plates with a mix of recombinant cytokines (IL-7, IL-15, IL-6, and IL-10) or with 10 µg/mL phytohemagglutinin (PHA, Sigma), 100 U/mL IL-2, and irradiated PBMCs as feeder cells. Flow cytometry
Cell staining of CFSE-labeled T cells was performed with PE- or APC-labeled antibodies, with the exception of the anti-CCR7 Ab, which was detected either with a mouse-adsorbed PE-labeled antirat antibody or with a biotinylated antirat antibody, followed by incubation with streptavidin-APC (Pharmingen). Expression of the IL-15R ELISA Cytokine-producing capacity was assessed after stimulation of purified cell populations at 5 x 105/mL for 24 hours with 50 nM phorboldibutyrate (PdBu) and 0.5 µg/mL ionomycin or immobilized anti-CD3 and anti-CD28. Cytokine concentrations of supernatants were then assessed by ELISA following a standard protocol and analyzed with the Softmax program (Molecular Devices, Sunnyvale, CA). Ex vivo BrdU labeling The assay was performed as described15,37 with slight modifications. Briefly, fresh PBMCs from healthy volunteers were immediately cultured with or without 10 µg/mL BrdU (Sigma) for 16 hours. In some experiments the superantigen toxic shock syndrome toxin (TSST, 100 ng/mL) and IL-2 (100 U/mL) were added as control. CD8+ T cells were then purified with magnetic beads and stained with anti–CD45RA-PE or anti–Cy-Chrome, anti–CCR4-PE, and anti-CCR7 followed by antirat APC. Cells were fixed and permeabilized with 1% paraformaldehyde in phosphate buffered saline (PBS), 1% Tween-20 for 15 minutes at 37°C, treated with 50 µg/mL DNAse (Boehringer, Mannheim, Germany) in PBS 4 mM MgCl2, pH 5 for 30 minutes, stained with anti–BrdU-fluorescein isothiocyanate or isotype control antibody (Becton Dickinson). At least 106 events were acquired by flow cytometry.
Expansion potential of naive and memory CD8+ T cells activated by antigen or homeostatic cytokines Subsets of human naive and memory CD8+ T cells were purified according to CCR7 and CD45RA expression, labeled with CFSE, and compared for their capacity to proliferate in response to antigen or cytokines implicated in homeostasis (Figure 1A-B). Antigenic stimulation provided by mature allogeneic DCs resulted in massive expansion of naive T cells and TCM cells, but TEM cells and especially TEMRA cells performed fewer divisions and were recovered in much lower numbers (Figure 1C). Similar results were obtained in the absence or presence of exogenous IL-2 and with anti-CD3 stimulation (data not shown). The low expansion potential of TCR-stimulated TEM and TEMRA cells was associated with a high rate of cell death as revealed by propidium iodide staining (Figure 1D). Death of TEMRA cells was not prevented by neutralizing anti–tumor necrosis factor (TNF) or anti–Fas ligand antibodies (data not shown), but was associated with low expression of the antiapoptotic BCL-2 protein (Table 1).
Proliferation in response to IL-7 was comparable and low in all subsets. In contrast, responsiveness to IL-15 was low in naive T cells, intermediate in TCM cells, and high in effector memory T cells (Figure 1A). IL-2 also selectively stimulated memory T cells and boosted responses to IL-7 and IL-15, whereas addition of anti–IL-2 or stimulation with IL-21 had no effect (Figure 1B). As reported for CD4+ T cells,43 addition of DCs or DC-derived cytokines IL-6 and IL-10 boosted cytokine responsiveness of both CD8+ naive and memory cells, leading to extensive and comparable proliferation of TCM cells and TEM cells (Figure 1B).
Cytokine responsiveness correlated with the expression of the relevant cytokine receptors (Table 1). Thus, whereas the IL-7R was expressed on all subsets to a comparable level, IL-15R Altogether, these results show that whereas antigen-dependent expansion, cell viability, and Bcl-2 expression are progressively lost from naive and TCM to TEM and TEMRA cells, IL-15R expression and cytokine responsiveness have a reciprocal pattern and are progressively acquired with differentiation. Generation of TEMRA cells by cytokine-stimulated TCM cells In order to assess the differentiation potential of CD8+ T-cell subsets, CFSE-labeled purified naive and memory CD8+ T-cell subsets were stimulated with either allogeneic DCs or IL-7 and IL-15. On day 7 CCR7 and CD45RA expression was analyzed on cells that had performed the same number of divisions (Figure 2A). Antigenic stimulation of any of the 4 CD8+ subsets resulted in the generation of rather homogeneous CCR7–CD45RA– effector cells. In contrast, whereas naive, TEM, and TEMRA cells proliferating in response to cytokines largely maintained their phenotype, TCM cells gave rise to cells expressing CCR7 and CD45RA in all possible combinations. Thus, some cytokine-stimulated TCM cells maintained their phenotype, whereas others generated cells with a TEM, a TEMRA, or, surprisingly, an apparent naive phenotype.
We next compared the capacity of perforin– naive T cells and TCM cells to acquire perforin following stimulation with antigen or cytokines (Figure 2B). Antigenic stimulation induced perforin in both naive T cells and TCM cells, but the cells were CD45RA–. In contrast, whereas naive T cells proliferating in response to IL-7 and IL-15 remained perforin–, a large fraction of TCM cells acquired perforin under the same condition, including cells that had up-regulated CD45RA. The addition of inhibitory anti–MHC class I antibody had no effect on cytokine-induced proliferation or perforin acquisition (data not shown). Cytokine-stimulated TEM cells, which constitutively express low levels of perforin, further up-regulated perforin expression (data not shown) but failed to acquire high levels of CD45RA (Figure 2A). Thus, cells coexpressing high levels of CD45RA and perforin were generated rather exclusively from cytokine-stimulated TCM cells. Importantly, CD45RA re-expression on dividing cytokine-stimulated TCM cells was largely unaffected by the composition and concentration of the cytokine mix but was in all cases prevented by the presence of anti-CD3 antibody (Figure 2C), showing that CD45RA re-expression requires cytokine-driven proliferation in the absence of TCR stimulation but is not under the exclusive control of a single cytokine. Altogether, these results suggest that cytokine-stimulated TCM cells can self-renew and generate different types of effector cells, including TEMRA cells, in the absence of antigen. Heterogeneity of the TCM pool revealed by cloning with cytokines The generation of multiple fates from cytokine-driven TCM cells raises the question of whether TCM cells are heterogeneous or oligopotent. To address this issue we cloned TCM cells with either PHA plus IL-2 as an antigen surrogate or with a mix of homeostatic cytokines. The cloning mix contained IL-7 and IL-15 as well as IL-6 and IL-10, because the latter mix induced optimal expansion of both TCM and TEM cells (Figure 1B). Cloning efficiency of TCM with PHA exceeded 50%, and approximately 20% of cytokinestimulated TCM cells generated clones of limited size (approximately 102-104 cells), consistent with the slower kinetics of cytokine-driven versus antigen-driven proliferation.43 In contrast, TEM cells had a low cloning efficiency with PHA (< 30%), and no clones were obtained from cytokine-stimulated TEM, consistent with the reduced viability and expansion potential of TEM. The obtained TCM-derived clones were analyzed for CCR7 and CD45RA expression (Figure 3). While in some cytokine-derived clones all cells retained the original phenotype (Figure 3A), in all others a large fraction of the cells either down-regulated CCR7, acquired CD45RA, or both (Figure 3B-E). In contrast, all clones obtained with PHA displayed a CCR7–CD45RA– phenotype (Figure 3F). These results suggest that TCM cells are heterogeneous in that they are programmed to generate different types of effector cells under homeostatic conditions, whereas they acquire the same effector phenotype upon antigenic stimulation.
CCR4 expression in the TCM pool distinguishes between cytotoxic effector cell precursors and Tc2 cells Although CD8+ TCM cells are not cytotoxic, they produced considerable amounts of IL-4 and IL-13 upon restimulation with phorbol ester and calcium ionophore (Figure 4A) or anti-CD3 and anti-CD28 (not shown). Furthermore, IL-13, but not IL-4, was also produced by cytokine-stimulated TCM cells both in the absence and presence (data not shown) of anti–MHC class I antibody, indicating that IL-13 can be elicited in the absence of TCR stimulation. In contrast, TEM and TEMRA cells produced only low or undetectable levels of type 2 cytokines. These findings suggest that Tc2 memory cells have a TCM phenotype.
To address whether Tc2 cells were a subpopulation of TCM cells, we searched for phenotypic markers of Tc2 cells. To this aim naive CD8+ T cells were primed with allogeneic DCs under type 1 or type 2 polarizing conditions and compared for chemokine receptor expression (Figure 4B). Priming in the presence of IL-12 induced rapid CCR7 down-regulation on dividing cells, and only few cells expressed CCR4. In contrast, upon priming in the presence of IL-4, a large fraction of dividing cells retained CCR7 and up-regulated CCR4 expression. Consistent with the in vitro priming data, CCR4 also was expressed on a variable fraction (35% +/– 24%) of freshly isolated TCM cells, whereas only few TEM cells expressed CCR4 at low levels (Figure 4B).
To investigate if CD8+CCR4+ TCM cells were indeed Tc2 cells, we sorted TCM cells on the basis of CCR4 expression and analyzed their cytokine profiles. Ex vivo–stimulated CCR4+ TCM cells produced high levels of IL-4 but low levels of interferon (IFN)- To determine if the 2 subsets had different capacities to generate cytotoxic effector cells (CTLs) under homeostatic conditions, we stimulated CCR4+ and CCR4–TCM with cytokines for 7 days and assessed CCR7 and perforin expression (Figure 4D). Most cytokinestimulated CCR4–TCM cells became CTLs because they down-regulated CCR7 and acquired perforin at the same time. In contrast, a smaller fraction of CCR4+ TCM cells down-regulated CCR7 expression, and only few CCR7– cells acquired low levels of perforin. Altogether, these results show that CTL precursors and Tc2 cells are distinct populations of the TCM pool that can be discriminated by CCR4 expression. Differential in vivo turnover of CD8+ memory T-cell subsets
Memory T cells slowly turn over under steady-state conditions in vivo.15,37 Cells that passed the G1 cell cycle checkpoint are committed to replicate DNA and complete the cell cycle.46 The in vivo turnover of T cells can therefore be assessed by ex vivo BrdU incorporation.37 Freshly purified PBMCs from healthy donors were cultured in the presence of BrdU, and CD8+ T cells were purified and analyzed for BrdU incorporation in relation to CCR7, CCR4, and CD45RA expression (Figure 5). Although a significant fraction of small CD69–, CD25– T cells spontaneously incorporated BrdU, TSST-activated T cells (CD69+TCRV
We have shown that human naive and memory CD8+ T-cell subsets have different capacities to proliferate and differentiate in response to TCR stimulation or cytokines. In particular, we show that central memory CD8+ T cells can be cloned with cytokines in the absence of TCR stimulation, and some of them give rise to cells with a characteristic CCR7–CD45RA+ phenotype, which is found in vivo following viral infection12,13 but cannot be induced in vitro by antigenic stimulation. We also provide evidence that central memory CD8+ T cells represent a heterogeneous subset comprising both CCR4+ Tc2 memory cells and CCR4–CTL precursors.
Both constitutive and lymphopenia-induced proliferation of mouse CD8+ T cells depend on the homeostatic cytokines IL-7 and IL-15.19, 20, 21, 22, 23, 24 Although the relative contribution of different Memory T cells have an enhanced susceptibility to cell death49,50 and die upon bystander activation,34 although surviving cells may proliferate and repopulate the memory pool. We show that resistance of human CD8+ T-cell subsets to cell death is associated with the expression of the antiapoptotic Bcl-2 protein, which protects T cells from multiple forms of cell death.51 Both antigen and cytokines up-regulate Bcl-2 in all subsets, but the differences in Bcl-2 expression between the subsets are maintained (data not shown), supporting the notion that the high susceptibility to cell death is an intrinsic feature of TEMRA cells. Consequently, TEMRA cells have a low expansion potential in response to both antigen and cytokines in vitro and a low turnover in vivo.
Naive T cells expanding in lymphopenic mice differentiate, acquiring some characteristics of effector/memory cells.16, 17, 18 Similarly, naive cytokine-stimulated CD4+ cells43 and CD8+ (data not shown) cells acquire the capacity to secrete low levels of IFN-
Tc2 cells can be generated by in vitro stimulation of naive CD8+ T cells in the presence of IL-4. Tc2 cells normally are not cytotoxic and produce IL-4 and IL-13 but not IFN- CD45RA+ memory cells (TEMRA) are a dynamic and enigmatic population of the CD8+ memory pool. Various lines of evidence, such as the loss of CD28, CD27, and CCR7; the low proliferative capacity; the high susceptibility to apoptosis; and the presence of high levels of perforin and Fas ligand indicate that they represent the most differentiated type of memory cells.4,5,11 This notion is not inconsistent with a recent report showing that Epstein-Barr virus–specific effector cells that proliferate in response to persistent antigen have an even lower expansion potential and are CD45RA–.13 TEMRA cells have been reported to appear after the acute phase of viral infection, contain cells specific for lytic but not latent Epstein-Barr virus antigens,12,13 and are absent in persistent HIV infection.9,10 Intriguingly, we found that although TEMRA cells cannot be generated by antigenic stimulation, cells with a phenotype corresponding to TEMRA, that is, CD45RA+, CCR7–, and perforin+, are generated rather exclusively by a TCM subset upon cytokine stimulation. Importantly, TEMRA generation is largely independent of the cytokine combination and concentrations that drive proliferation but is completely prevented by TCR stimulation (Figure 2C), suggesting that TEMRA generation requires homeostatic proliferation in the absence of antigen, but not a particular cytokine environment. We also observed that some cytokine-stimulated TCM cells acquire CD45RA expression without losing CCR7, that is, an apparent naive phenotype, raising the possibility that circulating CCR7+CD45RA+ cells contain few antigen-experienced revertants. In summary these findings show that CD45RA re-expression on antigen-experienced CD8+ T cells is inhibited by antigen and promoted by homeostatic cytokines, consistent with the selective and late appearance of TEMRA cells in viral infections. Altogether, our results indicate that CD8+ memory T-cell subsets have different capacities to proliferate, differentiate, and resist cell death in response to antigen and homeostatic cytokines. We suggest that TCM cells, due to their high expansion and differentiation potential and in vivo turnover, play an important role in maintaining the heterogeneous memory pool.
We thank D. Jarossay for cell sorting, G. Bosshard for technical assistance, E. Traggiai and M. Manz for discussion, and A. Gett for critical reading and comments.
Submitted November 26, 2002; accepted January 16, 2003.
Prepublished online as Blood First Edition Paper, February 6, 2003; DOI 10.1182/blood-2002-11-3577.
Supported in part by the European Community (contract no. QLK-CT-201-0105) and by the Swiss National Science Foundation (grant no. 31-63885). A.L. is supported by the Helmut Horten Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jens Geginat, Institute for Research in Biomedicine, Via Vincenzo Vela 6, CH-6500 Bellinzona, Switzerland; e-mail: jens.geginat{at}irb.unisi.ch.
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Abstract
Introduction
chain (
and CD45RA were purchased from Beckman Coulter (Marseille, France). All recombinant cytokines (IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, and IL-15), PE-labeled antibodies specific for perforin, BCL-2, CCR4, IL-2/15R
and the common 
) or IL-7 and IL-15 (
). Mean of 4 independent experiments.
) central memory CD8+T cells following stimulation with PdBu and ionomycin. Mean of 3 independent experiments. (D) Cytokine-stimulated CCR4+ and CCR4–CD8 TCM cells were analyzed on day 7 for the acquisition of perforin and CCR7 expression. One representative experiment of 3 is shown.