Functional re-expression of CCR7 on CMV-specific CD8+ T cells upon antigenic stimulation

Ester M. M. van Leeuwen1,2, Jaap D. van Buul3, Ester B. M. Remmerswaal1, Peter L. Hordijk3, Ineke J. M. ten Berge2 and Rene A. W. van Lier1

1 Department of Experimental Immunology, 2 Department of Internal Medicine, Divisions of Nephrology and Clinical Immunology and Rheumatology, Academic Medical Center Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands and 3 Department of Experimental Immunohematology, Sanquin Research at CLB, Amsterdam, The Netherlands

Correspondence to: E. M. M. van Leeuwen; E-mail: e.m.vanleeuwen{at}amc.uva.nl


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During latency circulating human cytomegalovirus (CMV)-specific CD8+ T cells do not express the chemokine receptor CCR7. We here show that antigen-specific stimulation in vitro with the specific CMV-peptide in combination with CMV-antigen, IL-2 or IL-21 induced re-expression of CCR7 on CMV-specific CD8+ T cells. Although IL-15 induced strong proliferation of peptide-pulsed CMV-specific CD8+ T cells, these cells did not re-express CCR7. CMV-specific cells that re-expressed CCR7 also expressed CD62L and were able to react to specific chemokine stimulation with changes in the cytoskeleton. In addition, activated CMV-specific cells specifically migrated towards a chemokine gradient in a transwell assay, with and without an endothelial cell monolayer. We conclude that antigenic stimulation induced functional re-expression of CCR7 which suggests that the migratory properties of virus-primed T cells are flexible and depend on the presence or absence of antigen and cytokines.

Keywords: CD8+ memory T cells, chemokine receptor, cytomegalovirus, human, migration


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
An efficient adaptive immune response depends on the interactions between lymphocytes and antigen-presenting cells in distinct compartments. The trafficking of these cells is regulated to a large extent by the production of chemokines and the expression of chemokine receptors. The combination of both constitutive and inducible production of chemokines and changes in expression of different chemokine receptors leads to controlled migration of all participants in the immune response (1, 2).

The chemokine receptor CCR7 plays an important role in B cell, T cell and dendritic cell (DC) trafficking (1, 2). The specific ligands for CCR7, CCL19 (ELC, MIP3ß) and CCL21 (SLC, 6Ckine) are constitutively produced by cells of the high endothelial venules and stromal cells within T cell areas of the spleen, lymph nodes and Peyer's patches (1). All naive T cells express CCR7 which enables the cells to re-circulate through the secondary lymphoid organs where the naive cells can be primed by DCs presenting antigen (3, 4). The biological importance of CCR7 is shown in mice lacking expression of CCR7 and in mice homozygous for a spontaneous mutation, plt (paucity of lymph nodes), which lack expression of CCL19 and CCL21 in the lymph nodes. These mice demonstrate disrupted homing of naive T cells to the T cell areas in the lymph nodes and an abnormal distribution of T cells in the secondary lymphoid organs (57).

Upon differentiation, naive T cells can lose expression of CCR7 and antigen-primed cells have been divided into two populations based on the presence or absence of this marker. Human CCR7+ cells have been named central-memory cells (TCM) because they have the ability to migrate to the secondary lymphoid organs, whereas CCR7 cells have been named effector-memory cells (TEM) and are presumed to home to inflamed tissues (3). The concept of TCM and TEM has been confirmed in murine studies where two different populations of memory cells were described based on anatomical localization (8, 9). In addition to the difference in localization, TCM and TEM have been reported to vary in functional terms in that effector cytokines like IFN-{gamma}, IL-4 and IL-5 are predominantly produced by the latter subset (3, 8, 9). However, this functional dichotomy in cytokine production potential has been disputed by other reports (1013). The distinction between different sets of memory T cells based on CCR7 expression relies on the assumption that loss of CCR7 is an irreversible step in the differentiation process of T cells and thereby makes CCR7 a stable marker linking phenotype to function. However, studies from Wherry et al. showed that murine TEM converted to TCM following antigen clearance in vivo (11). Moreover, in vitro antigenic stimulation of human CCR7 virus-specific T cells induced re-expression of this chemokine receptor (1416). Thus, in both mice and humans the expression of CCR7 may vary with the activation state of antigen-specific T cells.

Here we show that virus-specific CD8+ T cells not only up-regulate CCR7 upon antigen-specific stimulation in vitro but also acquire the ability to migrate towards the CCR7 ligands CCL19 and CCL21, implying that re-activated memory T cells can alter their migratory properties in vivo.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
PBMCs
Heparinized peripheral blood samples were collected from healthy volunteers and PBMCs were isolated using standard density gradient centrifugation techniques and subsequently cryopreserved until the day of analysis.

Immunofluorescent staining and flow cytometry
PBMCs were washed in PBS containing 0.01% (w/v) NaN3 and 0.5% (w/v) BSA (PBA). A total of 250 000 PBMCs were first incubated with allophycocyanin-labeled HLA-A2.1 tetrameric complexes loaded with the cytomegalovirus (CMV) pp65-derived peptide NLVPTMVATV [kindly provided by K. Tesselaar (Sanquin, Amsterdam, The Netherlands)] for 20 min at 4°C. Thereafter, fluorescent-labeled conjugated mAbs (concentrations according to manufacturer's instructions) were added without washing the cells and incubated for 30 min at 4°C. For analysis of expression of surface markers, the following mAbs were used: CCR7–PE (Pharmingen, San Diego, CA, USA), CD8–peridinin chlorophyll protein and CD62L–PE [both BD Biosciences (BD), San Jose, CA, USA]. Cells were washed in PBA and analyzed using a FACSCalibur flow cytometer (BD) and CellQuest software (BD).

Culture and stimulation of the cells
PBMCs were cultured in culture medium for 4–10 days in 24-well plates at a concentration of 0.5–1 x 106 cells ml–1. CMV pp65-derived peptide (IHB-LUMC peptide synthesis library facility; Leiden, The Netherlands) was added at a final concentration of 1.25 µg ml–1. In addition, CMV-antigen (inactivated whole virus, 10 µl ml–1; Microbix Biosystems, Toronto, Canada), IL-2 (50 U ml–1; Biotest Ag, Dreieich, Germany), IL-15 (3 ng ml–1; R&D Systems, Abingdon, UK) and IL-21 (50 ng ml–1; R&D) were used to stimulate cells. Flow cytometric analysis was performed before and after culture.

Actin-polymerization assay
The actin-polymerization assay was adapted from previously described methods (17). PBMCs stimulated for 5 days with CMV-peptide and CMV-antigen were incubated with tetramers in culture medium at 37°C for 30 min. Then 500 000 cells in a volume of 100 µl medium were transferred to tubes and the stimulating chemokine [CCL19, CCL21 or CCL7; 200 ng ml–1 (Biocarta, Hamburg, Germany)] was added for 15 s to 5 min. At indicated time points cells were immediately fixed with 50 µl buffered formaldehyde acetone solution and subsequently permeabilized by washing with PBA containing 0.1% saponine and 50 mM D-glucose. Cells were then incubated for 30 min with phalloidin–FITC (Sigma Chemical, St Louis, MO, USA) to visualize the F-actin. Cells were washed again in PBA containing 0.1% saponine and 50 mM D-glucose and analyzed by FACS.

Migration assay
Migration assays were performed in transwell plates (Costar, Cambridge, MA, USA) of 6.5 mm diameter with 5-µm pore filters. The filter separating the upper and lower compartments of the transwells was coated overnight at 4°C with 100 µl fibronectin (FN) from human plasma (Sigma) at a concentration of 0.1 µg ml–1. Before adding the cells the next day, the wells were washed three times with PBS and subsequently blocked with assay medium (RPMI 0.5% BSA) for 1 h at 37°C. A total of 500 000 cells in 100 µl of assay medium were added to the upper compartment and 600 µl of assay medium with or without chemokine was added to the lower compartment. The chemokines CCL19, CCL21 and CCL7 were used in a concentration of 100 ng ml–1. A 20-µl sample of cells in assay medium was diluted in 580 µl assay medium in a well without insert to be used as an input control (1/5) for quantitation of the number of migrated cells. All conditions were tested in triplicates. The transwell plates were incubated at 37°C with 5% CO2 for 2 h and then the 600 µl of assay medium, now containing the migrated cells, was collected from the lower compartment. Hundred microliters of the collected assay medium was added to FACS tubes to quantitate the number of migrated cells by counting for 50 s by FACS. When comparing the number of cells migrated towards the chemokine gradient with the spontaneous migration towards only medium and the input control, the percentage of specific migration could be calculated according to the following formula: ((number of cells migrated to chemokine – number of cells migrated to medium)/5 x number of cells in input control) x 100%.

The cells in the remaining 500 µl of assay medium from the lower compartments were washed and stained for CMV-tetramers, CD8 and CCR7 to determine the phenotype of the migrated cells by FACS analysis. These stainings were also combined with the quantification of the cells to calculate the specific migration of the different subsets of cells.

The transendothelial migration assay was performed generally as described before (18). In short, human umbilical vein endothelial cells (HUVEC) were plated at 20 000–50 000 cells per transwell on FN-coated filters. Non-adherent cells were removed after 18 h. The adherent cells were cultured for 2–3 days to obtain confluent endothelial monolayers. The endothelial cells were pre-treated overnight with tumor necrosis factor-{alpha} (PeproTech, Rocky Hill, NJ, USA). Thereafter, the same procedure was followed for the migration assay described above, except that cells were allowed to migrate for 4 h.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CCR7 can be re-expressed on activated CMV-specific CD8+ T cells
During latency the majority of CMV-specific CD8+ T cells do not express CCR7 (14, 19, 20).

As we showed before (15), CMV-specific CD8+ T cells efficiently expanded after stimulation with an immunodominant peptide of CMV pp65 in combination with CMV-antigen, the whole inactivated virus or with the cytokines IL-2, IL-15 or IL-21. Figure 1 shows that a large population of the expanded CMV-specific cells re-expressed CCR7. Upon stimulation with peptide and antigen up to 90% of the CMV-specific cells re-expressed CCR7; this change in phenotype was also seen in >50% of the CMV-specific cells stimulated with peptide and IL-2 or IL-21. An exception was the stimulation with CMV-peptide plus IL-15 where the cells clearly expanded but did not re-express CCR7 (Fig. 1).



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Fig. 1. Antigenic stimulation of CMV-specific cells can lead to re-expression of CCR7. The dot plots show the expression of CCR7 on unstimulated cells or after 5 days of stimulation with CMV-peptide in combination with CMV-antigen, IL-2, IL-15 or IL-21. All dot plots are gated on CD8+ lymphocytes; numbers indicate the percentages in the corresponding quadrants. The data shown are representative of six independent experiments.

 
The kinetics of CCR7 expression was investigated on cells stimulated with peptide and IL-2 or IL-15 during day 4–7 after stimulation. As shown in Fig. 2, the percentage of CCR7+ CMV-specific cells stimulated with peptide and IL-2 increased with time, whereas CCR7 re-expression on cells stimulated with peptide and IL-15 was only seen on a small population on day 7. When cells were cultured for longer periods, up to 10 days, CCR7 expression diminished in all conditions and CMV-specific cells re-acquired the CCR7 phenotype (data not shown). These data show that, depending on the cytokines used to complement the mitogenic signal, antigenic stimulation could induce a transient re-expression of CCR7 on CMV-specific cells.



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Fig. 2. Expression of CCR7 on CMV-specific cells increased with time on IL-2- but not IL-15-stimulated cells. Dot plots show the expression of CCR7 on CMV-specific cells stimulated for 4–7 days with CMV-peptide plus IL-2 or IL-15. All dot plots are gated on CD8+ lymphocytes; numbers indicate the percentages in the corresponding quadrants. The data shown are representative of two independent experiments.

 
Since expression of both CCR7 and CD62L (L-Selectin) is necessary to enable cells to re-enter the lymph nodes (21, 22), expression of the latter was also assessed after antigen-specific stimulation in vitro. Only a low percentage of unstimulated CMV-specific cells expressed CD62L (Fig. 3; 19). After stimulation under the different conditions, the percentage of CMV-specific cells expressing CD62L was enhanced. Again, cells stimulated with peptide and IL-15 differed from the other culture conditions since no up-regulation of CD62L was seen (Fig. 3a). Interestingly, addition of IL-15 reduced the up-regulation of CD62L on cells stimulated with peptide and antigen (Fig. 3b). We did not see such an effect on CCR7 expression (data not shown). These experiments showed that, like for CCR7, CMV-specific cells did up-regulate expression of CD62L after antigenic stimulation in vitro, which was dependent on the cytokines provided.



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Fig. 3. Antigenic stimulation of CMV-specific cells can lead to re-expression of CD62L. (a) Expression of CD62L on unstimulated cells or after 5 days of stimulation with CMV-peptide in combination with CMV-antigen, IL-2, IL-15 or IL-21. All dot plots are gated on CD8+ lymphocytes; numbers indicate the percentages in the corresponding quadrants. The data shown are representative of three independent experiments. (b) Expression of CD62L on CMV-specific cells after stimulation for 5 days with peptide and IL-15, peptide and antigen or peptide, antigen and IL-15.

 
CCR7 expression on activated CMV-specific cells is functional
The re-expression of CCR7 (and CD62L) on CMV-specific memory T cells after activation could change the migration pattern of these cells and enable them to re-enter the secondary lymphoid organs. In order to migrate, cells must change their cytoskeleton by polymerization of the F-actin component (23). Phalloidin specifically associates with polymerized actin, the levels of which transiently increase following chemokine stimulation (24). To test whether there-expressed CCR7 on activated CMV-specific CD8+ T cells is functional, we quantitated the increase in F-actin after stimulation with the CCR7 ligands, CCL19 and CCL21, using fluorescent phalloidin. As shown in Fig. 4a, transient actin polymerization was induced within 15 s after stimulation with CCL21. The increase in fluorescence compared with unstimulated cells was similar for the total lymphocyte population and the CMV-specific cells. These data show that the chemokine receptor CCR7, re-expressed on the surface of activated CMV-specific cells, was sensitive for chemokine stimulation, resulting in changes in the cytoskeleton. Comparison of the two chemokines binding to CCR7, CCL19 and CCL21 showed that stimulation with each chemokine gave similar results (Fig. 4b). No effect was seen on phalloidin binding after stimulation with CCL7 (MCP-3), which is a ligand for CCR1 and CCR2, both not expressed by CD8+ T cells (25, 26) (data not shown).



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Fig. 4. Stimulation of activated CMV-specific cells with CCL21 results in actin polymerization. PBMCs cultured for 5 days with CMV-peptide and CMV-antigen were stimulated for 15 s to 5 min with 200 ng ml–1 CCL21 or CCL19. (a) Increase in phalloidin binding (fluorescence of the cells) after CCL21 stimulation compared with unstimulated cells. (b) Increase in phalloidin binding for CCL19 and CCL21 stimulation. The values for total lymphocytes are indicated by triangles and for tetramer+ CMV-specific cells by squares; closed symbols represent cells stimulated with CCL21 and open symbols represent those stimulated with CCL19. The data shown in (a) are representative of three independent experiments.

 
Activated CMV-specific cells specifically migrate towards a chemokine gradient
To test the migration properties of activated CCR7+ CMV-specific cells towards the CCR7 ligands, an in vitro transwell system was used. Preliminary experiments showed that the optimal concentration of CCL21 was 100 ng ml–1 (data not shown). The migration assays were performed using cells cultured for 5 days with CMV-peptide and CMV-antigen. The percentage of specific migration, corrected for the spontaneous migration, is shown. The specific migration of tetramer+ CMV-specific T cells was higher than that of total CD8+ T cells or lymphocytes, reflecting the activated state of the CMV-specific cells (Fig. 5a). Comparison of CCR7+ and CCR7 CD8+ T cells and lymphocytes revealed that indeed the CCR7+ T cells migrated better towards the lower compartment containing CCL21 (Fig. 5a). Transwell migration assays using CCL19 gave similar results, whereas the chemokine CCL7 did not induce any migration (data not shown). Migration resulted in an enrichment of CCR7+ cells (Fig. 5b). CCR7 expression did not change just as a result of stimulation with CCL21 (data not shown). It should be noted that also a certain percentage of CCR7 cells migrated towards the chemokine. This might be caused by soluble factors, such as RANTES, secreted by the migrated cells (27).



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Fig. 5. A high percentage of activated CMV-specific cells specifically migrated to a chemokine gradient. Total PBMCs cultured for 5 days in the presence of CMV-peptide and CMV-antigen were used for a FN-coated transwell migration assay. (a) The percentage of specific migration was calculated for tetramer+ CMV-specific cells (mostly CCR7+), CD8+ T cells and total lymphocytes, the latter two divided in CCR7+ and CCR7 cells. (b) Migration towards a chemokine gradient led to enrichment of the percentage of CCR7+ cells, within tetramer+ CMV-specific cells, CD8+ T cells and total lymphocytes. ‘Medium’ represents the cells that spontaneously migrated to medium alone, ‘input’ represents the starting population and ‘CCL21’ represents the cells that migrated towards the CCL21 chemokine gradient. Error bars represent standard error of the mean from triplicates; differences were not statistically significant (P > 0.05 for all conditions). The data shown are representative of three independent experiments.

 
The ability of the cells to migrate through endothelial cells was tested by using a HUVEC monolayer and the migration time was extended to 4 h. A titration of CCL21 showed that using an endothelial cell layer, there was no difference in the percentage of specific migration of the cells at different concentrations (data not shown). Therefore, we used the same concentration (100 ng ml–1) as for the migration assay with FN. In total, the percentage of specific migration was lower than that with the FN-coated membrane and the activated tetramer+ CMV-specific cells were no longer better in migrating than total CD8+ T cells and lymphocytes (Fig. 6). These data show that activated CCR7+ CMV-specific cells were capable of migrating through an endothelial cell layer towards the ligand of CCR7, CCL21. Like the previous migration assays, the results obtained when using CCL19 were similar as with CCL21, whereas CCL7 did not induce any migration of the cells (data not shown).



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Fig. 6. Activated CMV-specific cells specifically migrated over an endothelial cell layer. PBMCs cultured for 5 days with CMV-peptide and CMV-antigen were used for a transwell migration assay over an endothelial cell monolayer. The percentage of specific migration was calculated for tetramer+ CMV-specific cells, CD8+ T cells and total lymphocytes, the latter two divided in CCR7+ and CCR7 cells. Error bars represent standard error of the mean of triplicates; differences were not statistically significant (P > 0.05 for all conditions). The data shown are representative of two experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Here we show that in vitro-activated CD8+ CMV-specific cells re-expressed CCR7 and CD62L and were then capable of migrating towards a chemokine gradient, which implies that activation of these cells in vivo during CMV re-activation could change their migratory capacity. This up-regulation of CCR7 was dependent on the cytokines present since in contrast to the other stimulatory conditions, stimulation with CMV-peptide in combination with IL-15 did hardly provoke re-expression of CCR7, although proliferation of the cells was induced. What makes IL-15 an exception is that it is a pleiotropic cytokine, produced by all kind of cells but not by T cells. This contrasts the other stimulatory conditions since stimulation with CMV-antigen causes activation of CD4+ T cells and both IL-2 and IL-21 are helper cell-derived cytokines. This suggests that the destiny of activated memory T cells is partially regulated in vivo depending on which cells are activated and which cytokines are produced during the immune response that is mounted to a specific pathogen. Also, the location in the body where the activation of memory T cells takes place may play a role. One can envisage that CMV-specific cells will be activated near endothelial cells which contain, together with myeloid cells, the reservoir of CMV during latency. These endothelial cells are capable of producing IL-15 and memory cells activated in this condition will remain CCR7 and CD62L negative and stay at the site of infection. CCR7 up-regulation on memory T cells may serve different goals. First, the fact that memory T cells re-acquire CCL19 and CCL21 responsiveness may lead to their migration to the lymph nodes where they can come in contact with antigen-bearing DCs. In the lymph nodes, the T cells may receive additional competence signals that might be important for optimal secondary immune responses. This would be in analogy with the recent findings that naive CD8+ T cells can be fully activated for a primary response without help but do need an extra signal to be able to mount an efficient memory response (2830). Next to this, CCR7+ memory T cells in the lymph nodes might play a role in the regulation of other T cells by the secretion of cytokines. Concerning this, IFN-{gamma} might be of particular importance through its ability to up-regulate not only MHC class I and II but also the co-stimulatory molecules CD80 and CD86 (31, 32). Alternatively, CCR7+ memory T cells could be recruited to peripheral tissues since CCL21 is also expressed in the endothelium of small vessels in several organs like the intestine and the lungs (33). Homing of additional CCR7+ T cells to the site of infection would accordingly amplify the inflammatory response. In this respect, the expression of two different forms of CCL21 in lymphoid and non-lymphoid tissues might play a role in balancing the central and peripheral immune responses (34).

As mentioned before, it has been debated that stable functional differences in cytokine production ability exist between TCM and TEM (3, 8, 9, 11, 12, 35). Do our data reconcile the differences between these various observations? Data from our group and other studies on human CD8+ T cells (14, 15) and findings on murine virus-specific T cells (11) show that CCR7 loss is not an irreversible differentiation event. Consequently, the quantity and function of CCR7+ and CCR7 T cells within the circulation will (minimally) depend on concurrent viral infections, being acute, recently resolved or latent. Especially in humans, immune activation, which is shown to regulate CCR7 expression, cannot be accurately determined. Therefore, applying CCR7 as a subset marker for human memory T cells is of limited use.


    Abbreviations
 
CMV   cytomegalovirus
DC   dentric cell
FN   fibronectin
HUVEC   human umbilical vein endothelial cells
PBA   PBS containing 0.01% (w/v) NaN3 and 0.5% (w/v) BSA
TCM   central-memory cells
TEM   effector-memory cells

    Notes
 
Transmitting editor: C. Terhorst

Received 8 July 2004, accepted 5 March 2005.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Articles by van Leeuwen, E. M. M.
Articles by van Lier, R. A. W.
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Articles by van Leeuwen, E. M. M.
Articles by van Lier, R. A. W.