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In Vivo Differentiated Cytokine-Producing CD4⁺ T Cells Express Functional CCR7¹

Gudrun F. Debes^*, Uta E. Höpken and Alf Hamann^2,*

^* Experimentelle Rheumatologie, Medizinische Klinik, Charité and Deutsches Rheumaforschungszentrum, and Molekulare Tumorgenetik und Immungenetik, Max Delbrück Centrum fur Molekulare Medizin, Berlin, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4⁺ cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4⁺ cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7^-/- mice failed to enter lymph nodes and Peyer’s patches, but did enter a site of inflammation. These findings indicate that CD4⁺ cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Lymphocytes continuously recirculate through lymphoid and nonlymphoid tissue to ensure contact with Ag and provide local protection against pathogens. Chemokines play an important role in this process by guiding lymphocytes from the bloodstream into inflammatory sites or secondary lymphoid organs and into distinct compartments within these tissues.

Ag-experienced Th cells are crucial in the regulation and balance of immune reactions. Upon Ag contact, T cells differentiate into effector/memory cells and develop restricted, polarized patterns of cytokines. Effector CD4⁺ T cells can be defined as cells capable of rapidly mounting effector functions such as secretion of distinct cytokines upon stimulation, and memory cells as long-living, Ag-experienced cells with lowered thresholds of activation compared with naive cells. Whether these two definitions identify overlapping populations or, rather, functionally and phenotypically distinct differentiation stages is a matter of debate (1). Here, we use the term effector/memory cells to identify CD4⁺ cells producing cytokines upon stimulation, which are typical for the fully differentiated, polarized stage. In contrast to naive cells, effector/memory T cells express a variety of receptors for inflammatory chemokines and adhesion molecules, which navigate them into inflammatory sites where they can mediate rapid protective responses.

Secondary lymphoid organs play a key role in bringing together APCs and T cells for initiation of an adaptive immune response. Naive T cells, which express uniformly high levels of L-selectin (CD62L) and CCR7, enter lymph nodes and Peyer’s patches (PPs) via high endothelial venules (HEV) and travel further into the T-zone, where they contact dendritic cells (DC) for recognition of their cognate Ag (2). Memory T cells, heterogeneous in the expression of CCR7 and L-selectin (3, 4), additionally enter these compartments by afferent lymphatics (5).

T cell migration from the bloodstream into lymph nodes and PPs proceeds via a multistep adhesion cascade involving L-selectin and CCR7. The two known CCR7 ligands, CCL21 (secondary lymphoid tissue chemokine) and CCL19 (EBV-induced molecule 1 ligand), are presented by HEV due to expression or transcytosis, respectively (6, 7). In contrast, entry of lymphocytes into the spleen is independent of the above receptors. CCL21 is also expressed by lymph vessels and participates in DC migration into lymph nodes via afferent lymphatics (6, 8); whether CCR7 is also used for memory cell entry via this pathway is not known.

CCL21 and CCL19 are produced by resident stromal cells in the T-zone (9), CCL19 is additionally expressed by T-zone DCs (9, 10). This anatomically defined chemokine expression plays an important role in guiding T cells as well as DCs into distinct compartments within the tissues, such as T-zones of lymph nodes and spleen (6, 9, 11). Accordingly, CCR7 gene-targeted mice and mice homozygous for the spontaneous mutation plt (paucity of lymph node T cells), which lack CCL21 expression in lymphoid organs, have a markedly reduced migration of T cells into lymph nodes and PPs. In these mice T cells accumulate in the red pulp of the spleen and in marginal sinuses and fail to enter or to form T cell-rich zones (11, 12).

The proven role of CCR7 in the entry of T cells via HEVs has favored the idea that this receptor serves a constitutive function in the recirculation of naive T cells. More recently, Sallusto et al. (3) have modified and further advanced this view by dividing human effector/memory cells into a CCR7⁺ central memory and a CCR7^- subset of peripheral effector-memory cells. According to this concept, the subset of central memory cells recirculates via CCR7 and L-selectin through secondary lymphoid tissues, whereas the cytokine-producing peripheral effector/memory cells were assumed to migrate preferentially into nonlymphoid tissues (3). In contrast to that, Kim et al. (13) demonstrated by a different CCR7 staining approach that the majority of human cytokine-producing T cells lie within the CCR7-positive fraction. However, validation of these data at the functional level is still lacking. Partially contrasting data were reported by Randolph et al. (14), who found that in vitro differentiated murine IFN--producing Th1 cells exhibited strong reactivity toward CCR7 ligands, whereas Th2 did not respond. As the expression and function of chemokine receptors are highly regulated during activation and differentiation, studies based on receptor expression only or on in vitro differentiated cells might fail to clarify the role of CCR7 on effector cells. Here, we systematically analyzed the responsiveness of polarized CD4⁺ cytokine producers differentiated in their natural environment. The results indicate that the majority of murine as well as human circulating effector cells migrate efficiently toward CCR7 ligands and are able to recirculate through secondary lymphoid tissues, similar to naive T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Animals

Female BALB/c mice were 6–12 mo old and were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany). At this age, between 3 and 10% of total CD4⁺ T cells from the spleen were found to produce IFN-, about 1% produced IL-4, and 1–2% produced IL-10 upon stimulation with PMA/ionomycin. The generation of CCR7^-/- mice has been previously described (12). The CCR7^-/- and their wild-type littermates were on a 129/SvEv x BALB/c mixed background and were bred at the animal facility of the Max Delbrück Center for Molecular Medicine (Berlin, Germany). To exclude phenotypic differences between littermates due to the mixed background, the chemotactic capacity of cytokine-producing cells derived from the founder strain 129/Sv was compared with that of cells derived from BALB/c mice. No differences were found (data not shown). For the model of cutaneous inflammation, 6- to 10-wk-old BALB/c mice were purchased from BgVV (Berlin, Germany). All animal studies were performed according to institutional and state guidelines under specific pathogen-free conditions.

Cell preparation and culture

Single-cell suspensions were prepared from spleens in RPMI 1640 supplemented with 10% FCS. Mononuclear cells were obtained by high-density gradient centrifugation (Histopaque-1083; Sigma-Aldrich, St. Louis, MO). CD4⁺ T cells were isolated by depletion of CD8⁺ cells, B cells, and macrophages, by panning as described using anti-CD8 (53-6.72) and anti-CD11b (M1/70) (15, 16). To increase the frequency of effector/memory cells in the starting population of chemotaxis assays, the majority of naive, CD62L^high cells was additionally depleted in some experiments by inclusion of 1.5 µg/ml anti-CD62L (MEL-14) before panning. The resulting population expresses levels of L-selectin from low to intermediate. Human PBMCs were obtained from normal healthy volunteers after Ficoll-Paque centrifugation (LSM; Organon Teknika, Durham, NC). The frequencies of IFN-- and IL-4-producing cells ranged from 7 to 14% and from 1 to 4% of CD4⁺ T cells, respectively.

Th1 cells from cultures of spleen-derived naive T cells of CCR7^-/- and their wild-type littermates were obtained by standard polyclonal activation under polarizing cytokine conditions as previously described (15, 16). For homing experiments Th1 cells were used on day 5 of culture, and polarization was confirmed by intracellular cytokine staining after PMA/ionomycin stimulation.

Chemotaxis assay

Chemokines were purchased from R&D Systems (Minneapolis, MN) and titrated to identify optimal concentrations. If not otherwise stated, the following concentrations of recombinant chemokines were used: 10 nM murine CXCL12, 100 nM murine CCL21, and 300 nM human CCL19. Spleen cells from 3 (for surface marker analysis) or 5–15 (for cytokine subset analysis) mice were pooled. For human T cells, each donor was independently analyzed, and only cells for the medium control were pooled immediately before migration. The assay was performed as previously described (16). Briefly, 5 x 10⁵ cells in assay medium (RPMI 1640 plus 0.5% BSA) were added to the upper wells of fibronectin (Life Technologies, Paisley, U.K.)-coated, 5-µm pore size, polycarbonate, 24-well tissue culture inserts (Costar, Cambridge, MA) in 100 µl. Chemokine dilution or assay medium (600 µl) was added to the bottom well, and migrated cells were harvested after a 90-min incubation at 37°C. The rate of migration was quantified for each cytokine subset by combined determination of cell number and subset frequency in the input and migrated population. Triplicates of 500-µl aliquots for each chemokine and the medium control were added to a fixed amount of beads (TruCount; BD Biosciences, Mountain View, CA), Abs used for gating (anti-mouse CD4, H129.19 or anti-human CD3, UCHT1 plus CD4, TT1) were added, and the numbers of cells and beads were counted without washing using appropriate gates in the FACS. Subsequently, the frequencies of the different cytokine-producing subsets in the input population and in pools of 5–30 wells of migrated cells were determined as described below.

Flow cytometry

Samples were stained with the following Abs: biotinylated, FITC-, indodicarbocyanine (Cy5)-, allophycocyanin-, or PE-conjugated anti-CD4 (GK1.5), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), and anti-CD45RB (23G2). PerCP-conjugated streptavidin was used as second step reagent (BD Biosciences). To prevent unspecific binding, all samples were preincubated with blocking anti-FcRII/III Ab 2.4.G2/75 and purified rat IgG (manufactured by The Jackson Laboratory, Bar Harbor, ME; purchased from Dianova, Hamburg, Germany) or human IgG (Beriglobin; Chiron, Marburg, Germany) Gates were set on viable cells according to propidium iodide staining. Intracellular cytokine detection was performed as previously described (15) by stimulation with 10 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 h with addition of 10 µg/ml brefeldin A (Sigma-Aldrich) for 2 h, staining for CD4 (mouse) or CD4 plus CD3 (human), and subsequent fixation. Cytokines were stained after permeabilization with saponin using the following Abs, conjugated to FITC, PE, Cy5, or allophycocyanin: anti-mouse (m)IL-4 (11B11), anti-mIFN- (AN 18.17.24), anti-mIL-10 (JES5-16E3), anti-human (h)IL-4 (4D9), and anti-hIFN- (4SB3) or appropriate isotype controls. The specificity of mIL-4 allophycocyanin and hIFN- Cy5 staining was verified in control experiments by blocking with unlabeled Abs. Staining Abs were obtained from BD Biosciences, except for PE-labeled anti-hIL-4 (4D9; Hölzel Diagnostik, Cologne, Germany) and biotinylated, PE-, FITC-, and Cy5-labeled or unlabeled anti-m/hCD4 (GK1.5/TT1), anti-hCD3 (UCHT1), anti-m/hIFN-, (AN 18.17.24/4SB3), anti-mIL-4 (11B11), and anti-hCD45RA (4G11), which were provided by H. Hecker and H. Schliemann (Deutsches Rheumaforschungszentrum, Berlin, Germany). Samples were analyzed on a FACSCalibur using CellQuest software (BD Biosciences). Control experiments with incubations in different chemokine concentrations were performed to exclude alteration of the frequency of cytokine producers by effects of the chemokines used. No influence of CCL21 or CCL19 on PMA/ionomycin-induced cytokine production could be detected (data not shown).

To determine CCR7 surface expression of effector/memory CD4⁺ T cells from human peripheral blood or murine spleen, cells were purified using CD4 microbeads (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The resulting population was >98% CD4 T cells, as confirmed by FACS analysis. After stimulation with PMA/ionomycin, human cells were stained with a rat mAb against human CCR7 (3D12) (3, 17), followed by staining with polyclonal goat anti-rat Ig (The Jackson Laboratory) and anti-CD45RA (4G11) before fixation, permeabilization, and intracellular cytokine detection. Murine cells from BALB/c-wt or CCR7^-/- mice were stained in the same way using CCL19-Fc (18), which was a gift from J. Cyster (University of California, San Francisco, CA) and polyclonal donkey anti-human IgG (The Jackson Laboratory). In both procedures saponin treatment led to slightly reduced CCR7 staining. The specificity of the CCL19-Fc staining was controlled by blocking with recombinant murine CCL19 (5 µg/ml).

In vivo homing assay

The assay was performed as previously described (15). Briefly, a cutaneous inflammation was induced by skin painting with 0.5% 2,4-dinitrofluorobenzene in acetone-olive oil on days -21 and -20 and rechallenge on day -1. Th1 cells were labeled with 20 µCi sodium [⁵¹Cr]chromate for 1 h at 37°C. Dead cells were removed on a Nycodenz (Nycomed, Oslo, Norway) density cushion. Cells (1 x 10⁶), resuspended in PBS, were injected into the tail vein. Three hours later mice were killed, and the accumulation of radioactivity in skin pieces of 2.5-cm² size, secondary lymphoid organs, and the rest of the body to determine total radioactivity was measured.

Statistical analysis

Data represent the mean ± SD. Data were considered statistically significant when p < 0.05, as determined by unpaired or, if indicated, paired Student’s t test.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
CCL21 and CCL19 attract resting and activated effector/memory almost as efficiently as naive T cells

Naive T cells express CCR7 and are attracted by the corresponding chemokines CCL21 and CCL19. Whether this also applies to activated and effector/memory subsets remains controversial (4, 6, 10, 19, 20, 21, 22). Therefore, we studied the response of in vivo differentiated effector/memory cells to CCL19 and CCL21. CD4⁺ cells from spleens of untreated mice or the blood of healthy human donors were used as a source of natural effector/memory cells.

First, murine CD4⁺ T cells expressing different activation and memory markers were tested for their chemotactic responsiveness. In accordance with studies by others (19, 23), CCL19 was a more potent attractant for T cells than CCL21 (Fig. 1). As shown in Fig. 1A, the absolute number of migrated naive, CD62L^high CD4⁺ T cells was approximately one-third higher than that of CD62L^low cells, but the sensitivities of the two populations to CCL19 and CCL21 were comparable, as shown by similar half-maximal responses. Small differences in the responsiveness to CCL21 were detected when CD45RB was used to discriminate naive and memory CD4⁺ cells (Fig. 1, B and C). The expression of CD69 on both CD45RB^low and CD45RB^high as a marker for recent activation was associated with a slightly lower (p < 0.01 and p < 0.05, respectively) migration. Under our experimental conditions, the expression of these markers was not altered in the presence of chemokines, as verified in control experiments (data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Chemotactic activity of CCL19/CCL21 on mouse lymphocyte subsets. The chemotactic response of pooled splenic CD4+ cells in an in vitro chemotaxis assay was measured by flow cytometry as described in Materials and Methods. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A, Migration of CD62Llow- vs CD62Lhigh-expressing CD4+ T cells to CCL19 and CCL21. One of three independent experiments is shown. The dotted line indicates the medium control. B, Flow cytometric analysis of input and migrated cells (gated on CD4+). C and D, Migratory properties of CD69- and CD69+ subsets of memory (CD45RBlow) and naive (CD45RB high) CD4+ T cells from three independent experiments. Bars and data points represent the mean ± SD. *, p < 0.05; **, p < 0.01

CCL21 is an effective attractant for in vivo primed CD4⁺ cytokine producers

To further characterize the role of CCR7 ligands in the migration of effector/memory cells, we tested the chemotactic response of those CD4⁺ cells able to produce distinct cytokines or cytokine combinations upon stimulation. CD4⁺ effector/memory cells from spleens of BALB/c mice were enriched for CD62L^low cells to increase the frequency of cytokine producers in the assay and were subjected to an in vitro chemotaxis assay using CCL21 as an attractant. After the migration period, input cells (aliquots of the cells given to the upper well) as well as migrated cells (lower well) were stimulated with PMA/ionomycin and analyzed for intracellular cytokines. Similar frequencies of cytokine producers were detected in the input population and in the population migrated toward CCL21 (Fig. 2A), indicating that CD4⁺ cells, which have the ability to produce effector cytokines upon in vitro restimulation, efficiently respond to the CCR7 ligand.

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Chemotactic response profile of cytokine producing murine CD4+ T cells toward CCL21. The response of pooled splenic CD62Llow CD4+ cells in an in vitro chemotaxis assay was measured by flow cytometry. For cytokine analysis input and migrated cells were stimulated for 4 h with PMA and ionomycin and stained intracellularly for IL-4, IFN-, and IL-10. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A, Flow cytometric analysis of input and migrated cells (gated on CD4). B, Response profiles of the different cytokine subsets. Subsets marked "single" are positive for the indicated and negative for the other analyzed cytokines; nonproducers are negative for IL-10, IL-4, and IFN-. The means ± SD of four independent experiments are shown.

These findings were not restricted to spleen cells, as similar responses were found for cytokine-producing CD4⁺ T cells from mouse blood (data not shown). To exclude differential reactivity of long term memory cells and recently generated effectors, the same analyses were performed with splenic CD4⁺ cells from mice immunized with OVA/alum and challenged 4 days before cell isolation. Under these circumstances, increased numbers of IFN-, IL-4, and IL-10 producers were found. Yet their reactivity to CCL21 was undistinguishable from that found with effector/memory cells from untreated mice (data not shown).

In conclusion, cytokine-producing CD4⁺ effector cells migrate efficiently toward CCR7 ligands. In contrast to findings obtained with in vitro-generated T cells (14), our data do not support the idea of a preferential chemotactic responsiveness of the Th1 vs the Th2 subset toward CCR7 ligands. IL-4 single-positive cells responded as well to CCL21 as IFN- single-positive cells, whereas only IL-10 single-positive cells exhibited a remarkably lower responsiveness toward CCL21. These results suggest that the properties of effector cells generated in vitro under rather extreme cytokine conditions might not always reflect the properties of natural effector cells. In accordance with our data are findings in a TCR-transgenic transfer model, where functionally diverse effector populations, characterized by different levels of P-selectin ligands, were found to migrate toward CCL21, with the subset able to induce delayed-type hypersensitivity and containing the majority of cytokine producers being almost as responsive as naive cells (24).

CCR7 is the responsible receptor for CCL21-mediated chemotaxis of effector/memory T cells

Recently it has been reported that murine CCL21 binds and signals to an alternative receptor besides CCR7. Murine CCL21 was found to bind to CXCR3 (25, 26), a chemokine receptor expressed predominantly on activated T cells and the Th1 subset (20, 27). To clarify this, we compared different subsets of cytokine-producing CD4⁺ T cells derived from CCR7-deficient mice (12) with cells from their wild-type littermates. Effector/memory cells derived from wild-type animals showed a high chemotactic response to CCL21 (Fig. 3A); in contrast, the migration rate of CCR7-deficient T cells was reduced to that in the medium control (Fig. 3B), indicating that CCR7 is the dominant receptor mediating recruitment via CCL21. The CXCR4 ligand CXCL12 (or stromal cell-derived factor-1) was used as a positive control to rule out a general defect in migration of CCR7^-/- cells. CCR7^-/- and wild-type cells were equally able to migrate toward CXCL12 (Fig. 3C). Increasing concentrations of CCL21 and CCL19 up to 500 nM had no chemotactic effect on CCR7^-/- CD62L^high or CD62L^low T cells (Fig. 3D). We conclude that CCL21-dependent chemotaxis of cytokine-producing CD4⁺ T cells is exclusively mediated by CCR7.

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Chemotactic response profile of cytokine-producing CD4+ T cells from spleens of CCR7-/- and CCR7+/+ littermates. The responses of pooled splenic CD4+ cells of each group in an in vitro chemotaxis assay to CCL21 (A), medium alone (B), and CXCL12 (C) were measured by flow cytometry. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A–C, For cytokine analysis input and migrated CCR7-/- and CCR7+/+ cells were stimulated for 4 h with PMA and ionomycin and intracellularly stained for IL-4, IFN-, and IL-10. D, Migration of CD62Llow- vs CD62Lhigh-expressing CCR7-/- CD4+ T cells toward CCL19 and CCL21. The dotted line indicates the medium control. Data represent the mean ± SD of three independent experiments (A–C) or one representative experiment of three performed (D).

The above data, generated in the mouse system, are in apparent conflict with previous findings in the human system, where cytokine-producing CD4⁺ T cells were only found in the fraction of cells expressing low levels of CCR7 (3). In a more recent study the majority of cytokine-producing cells was found to be CCR7 positive (13). Apparently, differences in the staining procedures or in the gates used to define positive and negative populations resulted in opposing conclusions. This underlines the importance of functional approaches, as used in this work, to delineate the migratory properties of effector/memory populations. To confirm our conclusions for human effector/memory cells, we additionally tested human peripheral blood T cells with the capability of expressing IL-4 and/or IFN- for their capacity to migrate toward the CCR7 ligand CCL19.

Detection of intracellular cytokines after in vitro restimulation revealed similar frequencies of cytokine-producing T cells in the CCL19-responsive cells and in the starting population (Fig. 4A). Again, IFN-, IL-4 single-positive and IL-4/IFN- double-positive CD4⁺ T cells responded as strongly as the nonproducers (Fig. 4B), indicating that human cytokine-producing T cells express CCR7.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Chemotactic response profile of human cytokine-producing CD4+ T cells to CCL19. Human PBMCs from healthy volunteers were subjected to an in vitro chemotaxis assay and analyzed by flow cytometry. For cytokine analysis, input and migrated cells were stimulated for 4 h with PMA and ionomycin and intracellularly stained for IL-4 and IFN-. Results are expressed as the percentage of cells of the respective subset that migrated to the lower chamber. A, Flow cytometric analysis of input and migrated cells (gated on CD4+and CD3+). B, Response profiles of the different cytokine subsets. Each donor was individually analyzed, except the medium control, where equal numbers of cells of each donor were pooled prior to migration. Data represent the mean ± SD of nine volunteers from two independent experiments.

In vivo differentiated cytokine-producing T cells express heterogeneous levels of surface CCR7

To test our hypothesis that cytokine-producing T cells might express low, but functional, levels of surface CCR7, we determined CCR7 expression levels on human and murine naive and memory CD4⁺ T cells. In accordance with previous data (3, 4), FACS analysis showed that human naive T cells (CD45RA⁺) from peripheral blood express uniformly high levels of CCR7 (Fig. 5A). Splenic murine naive (CD62L^high) T cells showed a similar staining pattern using CCL19-Fc (18) to detect surface CCR7, although some CD62L^high T cells bound CCL19-Fc less efficiently. Thus, naive murine T cells are apparently less uniform in the expression of CCR7 (Fig. 5D). For the determination of CCR7 expression on human and murine cytokine producers, sorted CD4⁺ T cells were stimulated for 4 h with PMA/ionomycin and subsequently stained for surface CCR7 using anti-human CCR7 mAb or murine CCL19-Fc before intracellular cytokine detection. In both species short-term stimulation did not lead to increased CCR7 surface expression; rather, a slight receptor down-modulation was detectable (Fig. 5, B and E). CD45RA expression was not significantly influenced by stimulation with PMA/ionomycin (data not shown). Human (Fig. 5C) as well as mouse (Fig. 5F) cytokine producers were found to express, in the majority, detectable levels of CCR7, yet expression was, in general, lower and more heterogeneous than that in naive cells. To some extent, the level of CCR7 expression appeared to be negatively correlated with cytokine staining. These findings are consistent with the chemotaxis assays, which demonstrated that the majority of cytokine producers express functional CCR7. It can be concluded that in both mice and humans the expression of functional levels of CCR7 and the production of effector cytokines are not mutually exclusive.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Flow cytometric analysis of CCR7-surface expression of naive and effector/memory CD4+ T cells from human peripheral blood and murine spleen. A–C, MACS-sorted CD4+ T cells from peripheral blood of healthy volunteers were stained for CD45RA and CCR7. A, Expression of CCR7 on unstimulated naive and memory T cells. B, Effect of PMA/ionomycin stimulation on CCR7 expression. CD4+ T cells were stimulated for 4 h with PMA and ionomycin or were mock-treated and stained for CCR7 and CD45RA, fixed, and permeabilized. C, CCR7 expression and intracelular cytokine staining after stimulation, separately given for CD45RA- (upper panels) and CD45RA+ (lower panels) cells. D–F, Murine CD4+ T cells from BALB/c wild-type or CCR7-deficient animals were stained (D) or MACS-sorted (E and F) for CD4, and binding of CCL19-Fc was tested. D, CCL19-Fc binding and CD62L expression of CD4+T cells from spleens of BALB/c mice. E, CD4+ T cells from CCR7-wild-type and -deficient animals were stimulated for 4 h with PMA and ionomycin or mock-treated, stained with CCL19-Fc, fixed, and permeabilized and (F) additionally stained intracellularly for IFN-. Data are representative for eight individually analyzed donors in three independent experiments (A–C) and three independent experiments with pooled spleen cells of two or three animals per group (D–F).

T effector cells are functioning as local actors in inflammation and defense. Accordingly, they express various inducible adhesion and chemokine receptors, which enable them to enter inflamed tissues. Recently, CCR7 ligands were found to be up-regulated in chronic inflammation (30, 31). Thus, CCR7 could play a dual role in homeostatic recirculation as well as entry into inflammatory sites. To test this we compared the migration of in vitro-generated Th1 cells from CCR7^-/- and wild-type mice into a cutaneous site of inflammation. No difference in the number of T cells entering inflamed skin was detectable between wild-type and CCR7-deficient Th1 cells, indicating that CCR7 is not a prerequisite for entry into a site of acute inflammation (Fig. 6). This does not exclude the possibility that CCR7 ligands play a role in chronic inflammation where organized lymphoid structures with HEV-like vessels expressing CCR7 ligands might develop (31). Previous data and Fig. 6 show that Th1 cells are able to enter secondary lymphoid tissues, although the rate of recirculation is lower for Ag-experienced cells than for naive cells (14, 15, 32). CCR7-deficient effector cells were strongly impaired in their capacity to enter lymph nodes and PPs compared with wild-type cells, indicating that CCR7 plays a major role in guiding effector cells into these compartments (Fig. 6). In line with previous reports (11, 12), accumulation in the spleen in total was not affected by the lack of CCR7.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Trafficking of in vitro-generated Th1 cells from CCR7-/- mice and CCR7+/+ littermates into lymphoid organs and inflamed skin. Th1 cells on day 5 of culture were labeled with [51Cr]chromate and injected into tail veins of BALB/c mice that were locally sensitized with 2,4-dinitrofluorobenzene. The percentage of labeled cells in the skin and lymphoid organs 3 h after injection of six animals in each group is shown. Bars represent the mean ± SD. Differences between groups are significant (p < 0.01). One of two experiments is shown. PLN, peripheral lymph nodes; MLN, mesenteric lymph nodes.

Whether the expression of CCL21 in lymphatic endothelium (6) points to an additional role of CCR7 in the migration of effector/memory cells into and out of lymph nodes via lymphatic vessels remains to be shown.

Why should fully differentiated effector/memory cells travel through lymphoid tissues? Cytokines produced by CD4⁺ effector cells regulate diverse functions, such as B cell help and Ig switching, cross-talk with DCs, and regulation of T cell differentiation; all these occur largely within secondary lymphoid tissue. CCR7 is required to enter some of these sites, but it also targets regulatory effector cells within these tissues toward DCs and into T-zones. The data from this study demonstrate that the majority of cytokine producers are well equipped for travel to lymphoid destinations.

	Acknowledgments

 
We are indebted to J. Cyster for the kind gift of CCL19-Fc. We are grateful to Dr. M. Lipp, K. Bonhagen and R. Manz for helpful discussions, and to U. Haubold for technical assistance. We thank H. Hecker and H. Schliemann for providing Abs.

	Footnotes

 
¹ This work was supported by the Deutsche Forschungsgemeinschaft (SFB 421).

² Address correspondence and reprint requests to Dr. Alf Hamann, Experimentelle Rheumatologie, c/o Deutsches Rheumaforschungszentrum, Schumannstrasse 21/22, 10117 Berlin, Germany. E-mail address: hamann{at}drfz.de

³ Abbreviations used in this paper: DC, dendritic cell; h, human; HEV, high endothelial venule; m, mouse; PP, Peyer’s patch.

Received for publication December 14, 2001. Accepted for publication March 19, 2002.

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