| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Cellular Immunology, German Cancer Research Center, Heidelberg, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
, a smaller fraction first acquires CCR7 expression. We demonstrate that this acquisition of lymph node homing potential is associated with strong proliferation similar to that of activated TCM cells. After proliferation, most of these cells lose CCR7 expression again and acquire effector functions (e.g., perforin production). A small proportion (
6%), however, maintain phenotypic and functional TCM properties over a long time interval. These results suggest that TEM cells provide immediate effector function by a fraction of cells as well as self-renewal by others through up-regulation of CCR7 followed by either secondary peripheral effector function or long term maintenance of TCM-like properties. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
According to the progressive linear differentiation hypothesis (8), differentiation involves a phase of proliferation preceding the acquisition of fitness and effector function. Because T cell stimulation is a stochastic event, not all T cells receive the identical strength of signals. Therefore, primed T cells reach a variety of differentiation stages that contain effector cells as well as cells that have been arrested at intermediate levels of differentiation. These intermediates retain expression of lymph node homing receptors such as CCR7 and CD62L and have initiated but not completed the remodeling events of genes involved in effector function (e.g., IL4, IL-5, IFN-, perforin). They thus retain a flexible gene imprinting. Cells that may survive after the retraction phase of an immune response can be resolved into distinct subsets of either TCM representing cells at intermediate levels of differentiation or fully differentiated memory T cells with effector capacity (TEM). Reactivation of cells arrested at intermediate stages should lead to a progression of gene remodeling and imprinting. Consequently, differentiation of stimulated memory T cells was proposed to follow a linear process from TCM to TEM (1, 8, 9, 10).
There is, however, growing evidence that a differentiation pathway from TEM to TCM may also occur, given that stimulation of sorted HIV-specific CD8+ TEM cells induced their CCR7 expression (11). Similarly, stimulation of CD4+ TEM cells from healthy individuals induced a transient expression of CCR7 (12). This phenotypic change suggests a TCM conversion associated with migration to lymph nodes, but data resolving the functional implications of this change are still missing. Investigations performed in a mouse model analyzing the differentiation process of Ag-specific CD8+ TCM and TEM cells in vivo suggest a differentiation from TEM to TCM cells after adoptive transfer (13).
We decided to isolate memory subsets based on two markers, so that CCR7–CD62L– double-negative cells represent TEM and CCR7+CD62L+ double-positive TCM.
We undertook this study to investigate the potential functional implications of CCR7 up-regulation in TEM cell subsets. We show that human peripheral blood CD8+ and CD4+ TEM cells activated by high signal strength through anti-CD3/CD28 stimulation exhibit a dynamic differentiation. Whereas a majority of the cells immediately produced perforin or IFN- but showed little proliferation, a smaller subset acquired phenotypic and functional characteristics of TCM cells characterized by expression of CCR7 and associated with strong proliferation and little effector potential. After proliferation, most of these cells lost CCR7 expression again and reacquired a TEM-like functional capacity characterized by low proliferation but strong perforin production. A proportion of 6% of the CCR7+ TEM, however, maintained TCM properties (CCR7 expression and high proliferative potential) for the long term.
These data suggest a flexible differentiation of activated TEM cells that in parallel allows execution of immediate effector function, extensive expansion followed by a secondary effector phase, as well as stable maintenance of self-renewing capacity.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Peripheral blood of healthy normal donors was used after informed consent.
Monoclonal Abs
The following mouse anti-human mAbs were used: allophycocyanin-Cy7- or PE-Cy5-CD8 (clone 3B5; Caltag; clone HIT8a, BD Biosciences); allophycocyanin-Cy7-CD4 (clone S3.5; Caltag Laboratories); allophycocyanin-CD45RA (clone MEM-56; Caltag Laboratories); FITC-CD62L (clone Dreg56; BD Biosciences); IFN--FITC (clone 4S.B3; BD Biosciences); biotin-CCR7 (clone 3D12; BD Biosciences) together with streptavidin-PE (BD Biosciences) or streptavidin-PE-Cy7 (Caltag Laboratories); FITC- or PE-Ki-67 (clone B56; BD Biosciences); and FITC- or PE-perforin (clone 
G9; BD Biosciences).
Isolation of TEM and TCM cells
Mononuclear cells from peripheral blood of healthy donors were separated by Ficoll gradient (Biochrom) centrifugation. Endobulin (Baxter)-treated PBMCs were then stained with mouse anti-human mAbs against CD8, CD4, CD45RA, CD62L, and CCR7. TEM cells (CD8+CD4+CD45RA–CD62L–CCR7–) and TCM cells (CD8+CD4+CD45RA–CD62L+CCR7+) were isolated using a FACSVantage SE (BD Biosciences) with CellQuest Pro software (version 4.0.2; BD Biosciences). Sorted memory T cell subsets were subsequently analyzed for CCR7 and CD62L expression without restaining using the same FACS and settings as during cell separation. Only such cell preparations that exhibit a purity of at least 98% were selected.
Separation of CCR7+ TEM cells
Either 2 or 28 days after anti-CD3/CD28 stimulation, cultured TEM cells were stained with mouse anti-human mAbs against CCR7. CCR7+ TEM cells were then isolated using a FACSVantage SE with CellQuest Pro software.
Cell culture
Isolated TEM and TCM cells were transferred into 24- or 48-well plates (TPP and Corning Costar) and cultured in X-VIVO 20 (Cambrex) supplemented with 10% human AB serum (Sigma-Aldrich), 100 U/ml recombinant human IL-2 (Promocell), 50 ng/ml Zienam (MSD), 50 ng/ml Erythrocin (Abbott), 50 ng/ml vancomycin (Abbott), and 2.5 µg/ml amphotericin B (Invitrogen). The medium was changed every 2 days.
Polyclonal stimulation of TEM and TCM cells
Separated memory T cells (4 x 106) were activated by 18 h of incubation with anti-human CD3/CD28 Dynabeads (Dynal Biotech) in a ratio of 1:1–2:1. After stimulation, Dynabeads were extracted from the cells using a magnetic particle concentrator (Dynal Biotech).
Intracellular staining
Intracellular staining with mouse anti-human mAbs against Ki-67, perforin or IFN- were performed after permeabilization and fixation of sorted cells with a Cytofix/Cytoperm Kit (BD Biosciences) according to the manufacturer’s protocol. Isotype controls were performed for all applied intracellular stainings as suggested by the mAb suppliers.
FACS
Sorted TEM and TCM cells (5 x 105) were first treated with human Ig (Endobulin) to block unspecific binding sites. Staining was performed using the above listed mouse anti-human mAbs. Cells were measured by using a FACScan (BD Biosciences) or FACSVantage SE with CellQuest software (version 3.3 or version 4.0.2). Analysis of data was performed using FlowJo software (version 4.3; Tree Star).
CFSE staining
For CFSE staining, cells were washed in PBS (Biochrom) supplemented with 0.1% BSA (Sigma-Aldrich). Afterward, cells were incubated with 2 µM CFSE (Invitrogen) for 10 min at room temperature; then the cells were washed in X-VIVO 20 medium containing 10% AB serum and cultured as described above.
Proliferation assay
For thymidine incorporation assay, cells were activated by incubation on petri dishes coated with 1µg/ml anti-CD3 (clone OKT3; American Type Culture Collection) and 1 µg/ml anti-CD28 (clone 9.3; DKFZ-Heidelberg)mAbs for 18 h. Afterward, triplicates of the cells were transferred into 96-well plates and cultured for 17 h as described above. The medium was supplemented with 1 µCi/well [3H]thymidine. Thymidine incorporation by proliferating cells was measured using a 1205 liquid scintillation counter (PerkinElmer).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Gated CD8+ and CD4+ memory T cells from the peripheral blood of healthy donors were analyzed for CCR7 and CD62L expression to define CCR7–CD62L– TEM and CCR7+CD62L+ TCM cell subsets (Fig. 1, A-C). Blood samples were then sorted for TEM and TCM cell fractions. Only cell preparations of at least 98% purity were used for the experiments (mean purity, 98.9%; Fig. 1A). To ensure that activated CD8+ and CD4+ TEM and TCM cells have access to required survival signals in form of produced cytokines or cell-cell interactions, CD8+ and CD4+ memory T cells were cocultured. Within the CD8+ population, a strong majority of cells belonged to the TEM pool, whereas the proportion of CD8+ TCM cells was low (Fig. 1D). In CD4+ T cells, the proportion of TCM cells was higher than in the CD8+ population. Importantly, significant proportions of memory T cells did not belong to the defined TCM or TEM subsets but were either CD62L+CCR7– or CCR7+CD62L– (Fig. 1D).
|
It has been shown that activated TEM cells acquire CCR7 expression (11) or exhibit a transient expression of this chemokine receptor (12). However, there are as yet no data available concerning the precise kinetics and maintenance of CCR7 expression of separated CD8+ TEM cells of healthy individuals after stimulation. Therefore, we tested the CCR7 expression profile of purified TEM cell populations during a course of 28 days after a single bead-mediated anti-CD3/CD28 stimulation (Fig. 2, A and B). The proportion of CCR7 expressing TEM cells continuously increased, peaked at day 3, and declined to 5% at day 7 after activation. CCR7 up-regulation was induced by TCR stimulation, because nonstimulated TEM subsets showed only little CCR7 induction after 1–2 days (Fig. 2, A and B) or 4–7 days of culture (data not shown). Similar to an up-regulation of CCR7, a concomitant induction of CD62L was detected in 70–80% of CCR7+ TEM cells until day 4 after stimulation (Fig. 2, C and D). The decline of CCR7 expression was due to CCR7 down-regulation rather than to loss of CCR7+ cells, because CCR7+ TEM cells sorted 2 days after activation adjusted the proportion of CCR7+ cells until day 7 to levels similar to those detected in the peripheral blood without major loss of cell numbers (Fig. 2E). A fraction of 6% CCR7+ cells within the TEM population was detectable up to 28 days after stimulation (mean, 5.8 ± 0.6%). Their proportion was similar to that of CCR7+ cells obtained 28 days after activation of purified TCM (mean, 16.4 ± 7.6%; data not shown). These results suggest that fractions of TEM cells acquire a stable CCR7 expression profile after activation.
|
It was shown that TCM cells exhibit a higher proliferative capacity than TEM cells (1, 10, 11). We examined this observation with respect to our experimental setting. After polyclonal stimulation of sorted TCM and TEM cells, we similarly detected a higher proliferative potential of TCM cells. However, TEM cells also showed a marked and prolonged expansion compared with unstimulated populations (Fig. 3) when tested 2 days (Fig. 3A) and 7 days (Fig. 3B) after stimulation. Thus, TEM posses a certain expansive potential. We now compared the proliferative response of CCR7+ TEM cells to anti-CD3/CD28 stimulation with that of CCR7– TEM and TCM cells using the proliferation marker Ki-67 (Fig. 3, C and D) and analysis of CFSE dilution (Fig. 3E). Because activated TCM cells lose CCR7 expression and differentiate into TEM cells (1), gated CCR7+ TCM cells were used for comparison. As shown in Fig. 3, C and D, direct ex vivo analysis revealed that TEM and TCM cells from peripheral blood do not proliferate. After stimulation, the three subsets of isolated memory T cells became positive for Ki-67 on days 2 and 3. The proliferative capacity of CCR7– TEM cells was lower than that of the two CCR7+ memory subsets. At day 5 after stimulation, Ki-67 expression within all three subsets dropped to similar levels marking the end of the proliferation phase. Similarly, CFSE dilution was found predominantly in CCR7+ TCM and TEM subsets starting at day 2–3 after stimulation and decreased until day 7 (Fig. 3E). These results indicate that CCR7 expressing TEM cells acquire a TCM-like proliferative capacity.
|
A characteristic attribute of CD8+ TEM cells is their ability to express perforin. In contrast, CD8+ TCM cells show marginal perforin expression levels (1, 11). To characterize the effector capacity of CCR7+ TEM cells, their perforin expression after anti-CD3/CD28 stimulation was compared with that of CCR7– TEM, and CCR7+ TCM cells (Fig. 4, A and B). The ex vivo analysis of TEM and TCM cells from peripheral blood indicated that TEM cells, in contrast to TCM cells, express prestored perforin granula. The perforin expression of CCR7– TEM cells showed a strong increase at days 1 and 2 and then slightly decreased to 60% at day 7 after activation. In contrast, the perforin expression of CCR7+ TEM cells did not rise after stimulation but dropped to 15% at day 7 (Fig. 4A). As shown by the dot blot of Fig. 4B, the majority of perforin-expressing TEM cells were CCR7– at day 1 after stimulation. Interestingly, CCR7 up-regulation was detected in both perforin– TEM and perforin+ TEM cells. (Fig. 4B). This suggests that CCR7 expression is regulated independently from the presence or absence of prestored perforin granules. Similar to CCR7+ TEM cells, CCR7+ TCM cells showed a marginal perforin expression with a maximum proportion of 11% at day 7 after stimulation.
|
with respect to CCR7 expression. Interestingly, early cytokine secretion was restricted to low proportions of CCR7– cells in both populations (Fig. 4C) and exerted by CD4+ as well as CD8+ T cells (Fig. 4D). The results indicate that CCR7-expressing TEM cells exhibit a weak effector capacity, which is characteristic for TCM cells. CCR7+ TEM and TCM acquire functional capacity characteristic for TEM cells on loss of CCR7 expression
Consistent with the model of progressive differentiation, fractions of activated TCM cells lose CCR7 expression after proliferation and differentiate into TEM cells (1, 8). Similarly, when we analyzed the perforin expression of CCR7+ or CCR7– TCM cells 7 days after anti-CD3/CD28 stimulation, cells that had lost CCR7 exhibited a significantly higher perforin expression than CCR7+ TCM cells (Fig. 5A). These data indicate that TCM cells losing their CCR7 expression acquire effector capacity. As already shown, CCR7+ TEM cells exhibit a weak effector capacity, which is characteristic for TCM cells. CCR7 expression of TEM cells peaks at day 3, and most of these cells lose their chemokine receptor expression after proliferation. To analyze whether CCR7+ TEM cells losing their CCR7 expression acquire effector capacity, CCR7-expressing TEM cells were separated 2 days after anti-CD3/CD28 stimulation (cells referred to as d2-TEMCCR7+). Seven days after activation, we measured the perforin expression of CCR7+ or CCR7– d2-TEMCCR7+ cells (Fig. 5B). Similar to TCM cells, CCR7– d2-TEMCCR7+-derived cells showed significantly higher perforin expression than CCR7+ d2-TEMCCR7+-derived cells. This suggests that CCR7+ TEM cells losing their chemokine receptor expression acquire effector capacity.
|
TEM cells that show stable CCR7 expression >28 days after activation were further tested for their capacity to express perforin after renewed activation. For this, CCR7+ TEM cells were isolated 28 days after anti-CD3/CD28 stimulation (cells referred to as d28-TEMCCR7+) and compared with ex vivo isolated memory subsets. Because fractions of activated TCM cells lose CCR7 expression after proliferation and differentiate into TEM cells (1), the perforin expression profile of d28-TEMCCR7+, CCR7+ TEM and CCR7+ TCM as well as of CCR7– TEM cells was analyzed 7 days after secondary (in case of d28-TEMCCR7+ cells) or primary anti-CD3/CD28 stimulation, respectively (Fig. 6A). Isolated, d28-TEMCCR7+ cells showed no changes in their perforin expression profile after reactivation (compare perforin expression of d28-TEMCCR7+ and CCR7+ TEM cells). The additional comparison with CCR7+ TCM and CCR7– TEM cells indicated that TEM cells with stable CCR7 expression exhibit a perforin expression profile characteristic for TCM cells. We also tested the proliferative capacity of d28-TEMCCR7+ cells (Fig. 6B). For this purpose, the cells were labeled with CFSE and analyzed for loss of CFSE due to proliferation, 7 days after activation. d28-TEMCCR7+ cells maintained a strong proliferative potential. In contrast, sorted and activated d28-TEMCCR7- cells showed no proliferative potential (Fig. 6B). Taken together, the results demonstrate that TEM cells with long term stable CCR7 expression maintain TCM-like functional capacity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
Studies analyzing the maintenance of CCR7 expression after activation of separated TEM cells as well as the functions of CCR7+ TEM cells are important for the characterization of the differentiation potential of TEM cells. On the basis of the expression of CCR7 and CD62L, we isolated TEM cells of high purity and analyzed their phenotype and function during a period of up to 4 wk after polyclonal stimulation in vitro.
Our results show that on activation of TEM cells, fractions of at least 6% acquire stable CCR7 expression for at least 4 wk (Fig. 2). This change to stable CCR7 expression is unlikely to be an in vitro artifact, because the proportions of peripheral blood TCM cells in vivo are also not much higher (Figs. 1, B and C, and 2, A and B), and proportions of CCR7+ cells 28 days after activation are similar in cultures of sorted TCM and TEM cells (our unpublished observations). Additionally, the functional properties of d28-TEMCCR7+ are very similar to those of freshly isolated TCM cells (Fig. 6). It should therefore be taken into consideration that studies on human TCM cells can deal with mixed populations of both primary TCM cells and secondary TCM-like cells derived from activated TEM cells.
A comparison of CCR7+ TEM and TCM cells revealed that both subsets possess a high proliferative capacity after stimulation, which declines at day 5, marking the end of the proliferation phase. In contrast, TEM cells that remain CCR7– proliferate only marginally (Fig. 3). These data indicate that CCR7+ TEM cells acquire a proliferative capacity that is characteristic for TCM cells, most likely representing cells at intermediate stages of differentiation.
Almost 30% of CD8+ TEM cells, tested directly ex vivo, expressed perforin. On activation, TEM cells showed diverse differentiation processes. Fractions of perforin–CD8+ TEM cells became perforin+. These cells remained CCR7–. In contrast, in stimulated TEM cells that acquired the expression of CCR7, perforin expression dropped to a TCM-like level 7 days after activation (Fig. 4A). These data indicate that fractions of stimulated TEM cells acquire a functional capacity that is characteristic for TCM cells.
When TEM cells were activated, during the first days CCR7– cells increased expression of perforin or IFN-, thus indicating immediate effector capacity, whereas a fraction of cells first became CCR7+ (peak at day 3), then expressed Ki-67, and acquired proliferative activity as seen by CFSE dilution. Until day 7, many of the CCR7+ TEM cells reverted to a CCR7– phenotype with effector function, whereas 6% remained CCR7+ with TCM properties. Thereby, activation of TEM cells provides a sequence of two effector phases: an immediate phase provided by a limited number of nonproliferating TEM cells; and a second phase provided by a high number of clonally expanded TEM cells. The acquisition of TCM-like phenotypic, proliferative, and functional characteristics of fractions of activated TEM cells indicates that not only TCM cells as proposed (8) but also TEM cells contain populations at intermediate stages of differentiation. The acquisition of a stable CCR7 expression profile and a TCM-like functional capacity of fractions of activated TEM cells could assure a self-renewal potential and long term maintenance of TEM cells.
Our results demonstrate that upon activation CD8+ TEM cells exhibit a dynamic differentiation. Fractions of TEM cells acquire phenotypic and functional characteristics of TCM cells. After proliferation, most of these cells lose CCR7 expression and acquire effector capacity. In contrast, a proportion of at least 6% maintained TCM properties, suggesting that fractions of TEM cells retain a flexible imprinting of genes characteristic of cells at intermediate stages of differentiation possibly associated with a self-renewal potential of these cells.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 Address correspondence and reprint requests to Dr. Philipp Beckove, Department of Cellular Immunology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail address: p.beckhove{at}dkfz.de 
2 Abbreviations used in this paper: Cy, cyanlne; TCM, central memory T cells; TEM, effector memory T cells.
Received for publication February 1, 2005. Accepted for publication May 18, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Hao, Y. Liu, J. Yuan, X. Zhang, T. He, X. Wu, Y. Wei, D. Sun, and J. Xiang Novel Exosome-Targeted CD4+ T Cell Vaccine Counteracting CD4+25+ Regulatory T Cell-Mediated Immune Suppression and Stimulating Efficient Central Memory CD8+ CTL Responses J. Immunol., September 1, 2007; 179(5): 2731 - 2740. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Bohler, M. von Au, N. Klose, K. Muller, B. Coulibaly, F. Nauwelaers, H. P. Spengler, G. Kynast-Wolf, and H.-G. Krausslich Evaluation of a Simplified Dual-Platform Flow Cytometric Method for Measurement of Lymphocyte Subsets and T-Cell Maturation Phenotypes in the Population of Nouna, Burkina Faso Clin. Vaccine Immunol., June 1, 2007; 14(6): 775 - 781. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-Z. Liu, L.-B. Fang, P. Hjelmstrom, and X.-G. Gao Increased CD8+ central memory T cells in patients with multiple sclerosis Multiple Sclerosis, March 1, 2007; 13(2): 149 - 155. [Abstract] [PDF]
|
|
|
|
 |
|
 J. Carrasco, D. Godelaine, A. Van Pel, T. Boon, and P. van der Bruggen CD45RA on human CD8 T cells is sensitive to the time elapsed since the last antigenic stimulation Blood, November 1, 2006; 108(9): 2897 - 2905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xia, S. Hao, and J. Xiang CD8+ Cytotoxic T-APC Stimulate Central Memory CD8+ T Cell Responses via Acquired Peptide-MHC Class I Complexes and CD80 Costimulation, and IL-2 Secretion. J. Immunol., September 1, 2006; 177(5): 2976 - 2984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Meng, H. Harlin, J. P. O'Keefe, and T. F. Gajewski Induction of Cytotoxic Granules in Human Memory CD8+ T Cell Subsets Requires Cell Cycle Progression J. Immunol., August 1, 2006; 177(3): 1981 - 1987. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), TEM (
), and other memory subsets (CD62L+CCR7–, or CD62L–/CCR7+; 
) among CD8+ or CD4+ memory T cell populations from PBMCs ex vivo displayed as means and SD of 14 healthy donors.
3). C and D, CD62L expression by stimulated TEM 4 days after stimulation. C, One representative dot plot; D, Cumulative data of three donors. E, CCR7+ TEM cells were isolated by FACS 2 days after anti-CD3/CD28 stimulation (blue-outlined square; cells referred to as d2-TEMCCR7+). Cumulative data of CD8+, CCR7+, and CCR7– d2-TEMCCR7+ cells 7 days after stimulation are shown. Proportions of CCR7+ and CCR7– d2-TEMCCR7+ cells are shown as means and SD of healthy donors (n = 2). To check the purity of isolated memory T cell subsets, CD8+ TEM cells were analyzed for CCR7 expression immediately after cell separation (day 0). SSC, Side scatter.
