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The Journal of Immunology, 2004, 173: 3013-3019.
Copyright © 2004 by The American Association of Immunologists

Constitutive Expression of CCR7 Directs Effector CD8 T Cells into the Splenic White Pulp and Impairs Functional Activity1

Heike Unsoeld*, David Voehringer*, Stefan Krautwald{dagger} and Hanspeter Pircher2,*

* Institute for Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany; and {dagger} Department of Nephrology, University of Schleswig-Holstein, Campus Kiel, Kiel, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antigenic stimulation down-regulates CCR7 on effector T cells. To analyze the importance of CCR7 down-regulation, transgenic (tg) mice constitutively expressing CCR7 were generated. CD8 T cells with defined Ag specificity were obtained by breeding CCR7-tg mice with P14 TCR-tg mice specific for lymphocytic choriomeningitis virus. Transgenic CCR7 expression did not impair proliferation of P14.CCR7 T cells induced by lymphocytic choriomeningitis virus infection, but prevented CCR7 down-regulation. Compared with wild-type P14 effector cells, P14.CCR7 effector cells, expressing the CCR7 transgene, were increased in the spleen, but decreased in blood and peripheral tissues. Moreover, P14.CCR7 effector cells localized almost exclusively in the splenic white pulp, whereas P14 effector cells were excluded from splenic white pulp cords and were found preferentially in the red pulp. Functional experiments further revealed that P14.CCR7 effector cells were impaired in rapid viral clearance and in inducing Ag-specific delayed-type hypersensitivity reactions. Thus, the present study demonstrates that down-regulation of CCR7 during CD8 T cell activation is important to release effector cells from the white pulp of the spleen, and highlights the importance of effector cell localization in providing rapid immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cells enter lymph nodes from the bloodstream via specialized high endothelial venules (HEVs)3 in a complex process that involves rolling, adhesion, and diapedesis. Selectins, integrins, and chemokines play a key role in this process (1, 2). The mechanisms that are involved in lymphocyte migration in the spleen are less well understood (3, 4, 5). The spleen has a complex anatomy with an open-structured red pulp and interwoven branches of white pulp, separated by the marginal zone. The red pulp has a crucial filter function for encapsulated bacteria and senescent RBC, whereas the white pulp is densely populated with T and B cells. HEV-like vessels are not present in the spleen, and lymphocytes from the blood enter the white pulp via the marginal sinus (6). CCR7 has been demonstrated to be essential for migration of naive T cells into lymph nodes and into splenic white pulp cords (7). After T cell activation and differentiation, expression of CD62L and CCR7 is lost on a subset of memory T cells (8, 9). Ag-experienced T cells lacking CCR7 and CD62L have been termed effector memory cells, whereas memory cells expressing these homing receptors were called central memory T cells (8). The concept of central vs effector memory T cells has attracted much attention. Nonetheless, experimental data from in vivo models providing evidence for or against this concept are limited. In addition, lineage relationship and effector cell function of the two memory T cell subsets have been analyzed by several groups with conflicting results (8, 10, 11, 12, 13, 14, 15, 16).

We have shown previously that virus-specific effector and memory CD8 T cells were present in large numbers in the splenic red pulp of lymphocytic choriomeningitis virus (LCMV) immune mice (9). CXCR6 induced on activated CD8 T cells has been postulated to guide T cell movements to the splenic red pulp (17). However, red pulp localization of LCMV-specific CD8 T cells could also be due to down-regulated CCR7 expression on effector memory T cells. To address this issue and to analyze the significance of CCR7 down-regulation during CD8 T cell responses, transgenic (tg) mice were generated that constitutively express CCR7 on LCMV-specific P14 T cells. The present study provides evidence that down-regulation of CCR7 during an antiviral immune response facilitates the release of P14 effector cells from the splenic white pulp, which improves migration of these cells to peripheral sites via the blood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 (B6) mice were obtained from Harlan Winkelmann (Borchen, Germany). P14 TCR-tg mice (line 318) on a B6.Thy-1.1 background have been described previously (18). To generate CCR7-tg mice, CCR7 cDNA from murine thymocytes was amplified with CCR7-specific primers (CCR75'SalI, 5'-TGTGATGTCGACAGCCCCCAGAGCACCATG-3'; CCR73'BamHI,5'-GGAGGGATCCTTGTAGTCCGGGCAGGGGAG-3'). The PCR product was digested with SalI and BamHI, subcloned into the class I promoter expression cassette (19), and injected into fertilized (B6 x CBA)F1 eggs to generate tg mice. Transgenic mice, typed by PCR with H-2K (5'-TCACTTCTGCACCTAACCTG-3')- and CCR7 (5'-ATAGCCAGCATAGGCACTAG-3')-specific primers, were backcrossed more than eight times to B6 mice. P14 TCR x CCR7 double-tg mice were generated by breeding. Animals were kept under conventional conditions and were used for experiments at 8–16 wk of age.

Virus

The WE strain of LCMV and recombinant vaccinia virus expressing LCMV glycoprotein (VVGP) were grown and titered, as described (20, 21). B6 recipient mice of P14 T cells were infected i.v. with 200 PFU of LCMV or with 2 x 106 PFU of VVGP. VVGP-primed mice were challenged with 106 PFU of LCMV.

Adoptive cell transfers

Spleen cells from P14 TCR-tg or from P14 TCR x CCR7 double-tg mice containing 105 P14 TCR+ (V{alpha}2+/V{beta}8+) cells were adoptively transferred (i.v.) into B6 mice, followed by infection with LCMV or VVGP on the same day. For homing studies, spleen cells containing 1 x 107 P14 effector T cells (day 8 postinfection (p.i.)) were adoptively transferred (i.v.) into naive B6 mice. For CCR7 desensitization, P14.CCR7 effector T cells (107/ml) were incubated (37°C for 60 min) with ~5 µg/ml CCL19-Ig before cell transfer. Twenty hours after transfer, localization of P14 T cells was determined by flow cytometry and immunohistology.

Flow cytometry

Lymphocytes were stained in PBS containing 2% FCS and 0.1% NaN3 at a concentration of 106-107 cells/ml, by incubation at 4°C for 20 min with 100 µl of mAb at the working dilution. For isolation of lymphocytes from nonlymphoid tissue, liver and lung were perfused, cut into small pieces, and digested in PBS containing 0.1% collagenase (Sigma-Aldrich, Munich, Germany), 0.01% hyaluronidase (Sigma-Aldrich), and 0.002% DNase I (Sigma-Aldrich) for 30 min at 37°C before forcing through a 100-µm cell strainer. Clumps and undigested material were allowed to settle, and the resulting suspension was underlayered with Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden). The following mAb were used: anti-CD8 (clone 53-6.7), anti-CD62L (clone MEL-14), anti-TCR V{alpha}2 (clone B20.1), anti-TCR V{beta}8 (clone MR5-2), and anti-Thy-1.1 (clone OX-7). All Abs including corresponding isotype control Abs were purchased from BD Pharmingen (San Diego, CA). Cell surface expression of CCR7 was determined by flow cytometry using a chimeric CCL19-Ig fusion protein, as described previously (13). Before analysis of PBL, RBC were lysed using FACS lysing solution (BD Pharmingen). Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), which was calibrated routinely using caliBRITE beads (BD Biosciences), according to the protocol of the manufacturer.

In vitro chemotaxis assay

Chemotaxis assays were performed at 37°C using 106 total cells in 100 µl of tissue culture medium containing 5% FCS per 5 µm Transwell (Corning Costar, Acton, MA). The lower chambers contained 1000 ng/ml CCL19 or 100 ng/ml CXCL12 (R&D Systems, Wiesbaden, Germany). After 3 h, migrated cells were pooled from three wells, counted, and analyzed by flow cytometry using CD8- and Thy-1.1-specific mAb to determine the percentage of migrated P14 T cells.

Immunohistochemistry

Frozen sections (7 µm) of splenic tissue were acetone fixed, washed in 0.1 M Tris buffer, pH 7.5, and incubated for 60 min with biotinylated anti-Thy-1.1 mAb (clone OX-7; BD Pharmingen) in a humidified chamber. After a further wash, the sections were incubated with phosphatase-conjugated streptavidin (DakoCytomation, Hamburg, Germany) for 30 min, washed, and developed with the Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories, Burlingame, CA). Sections were counterstained with Meyer’s hemalum and mounted with Kaiser’s glycerol gelatin for analysis. For double staining, sections were first incubated with biotinylated anti-B220 mAb (clone RA3-6B2; BD Pharmingen) and FITC-coupled anti-Thy-1.1 mAb, followed by polyclonal rabbit anti-FITC Abs (DakoCytomation). Rabbit Abs were detected with goat anti-rabbit-conjugated HRP (Jackson ImmunoResearch Laboratories-Dianova, Hamburg, Germany), and biotinylated Abs with phosphatase-conjugated streptavidin (DakoCytomation). Enzyme reactions were developed with Vector Blue Alkaline Phosphatase Substrate Kit III and 3-amino-9-ethylcarbazole substrate kit for peroxidase (both Vector Laboratories).

Delayed-type hypersensitivity (DTH) reaction

To analyze DTH reaction, spleen cells containing 1 x 107 P14 effector T cells (day 8 p.i.) were adoptively transferred (i.v.) into naive B6 mice. Twenty hours after transfer, the frequency of P14 T cells in the blood was determined by flow cytometry, and a DTH response was induced by injecting 1 µg of gp33 peptide in 10 µl of PBS into the footpad. Footpad thickness was measured before and at the indicated time points using a dial-gauge caliper (Mitutoyo, Tokyo, Japan; 7309). Increased foot thickness was expressed as mean (in millimeters) x 10–2± SD.

Statistical analysis

Student’s t test of unpaired data was used to determine the significance of differences in means. A value of p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P14.CCR7 effector cells constitutively expressing CCR7 proliferate normally

CCR7 expression is down-regulated on effector T cells in humans and mice (8, 9). To investigate the importance of CCR7 down-regulation, tg mice constitutively expressing CCR7 cDNA under the control of the H-2Kb promoter were generated. CD8 T cells with defined Ag specificity were obtained by crossing CCR7-tg mice with P14 TCR-tg mice that are specific for the gp33 epitope of LCMV. Mice expressing the CCR7 transgene only on CD8 T cells were produced by adoptively transferring CD8 T cells from P14 TCR x CCR7 double-tg mice (Thy-1.1+1.2+) into B6 recipient mice (Thy-1.2+), followed by LCMV infection. As a control, CD8 T cells from single P14 TCR-tg mice were used. LCMV infection of the recipient mice induced a similar expansion of both P14 and P14.CCR7 effector cells, peaking on day 8 p.i. (Fig. 1a). Cell surface expression of CCR7 was determined by flow cytometry using a chimeric CCL19-Ig fusion protein (13). Naive CD8 T cells from P14.CCR7 mice expressed slightly increased levels of CCR7 (mean fluorescence (MF) 905 vs 768) when compared with naive cells from P14 mice (Fig. 1b). A large fraction of P14 effector cells (day 8 p.i.) displayed down-regulated CCR7 expression. In contrast, down-regulation of CCR7 was strongly inhibited on P14.CCR7 effector cells (Fig. 1, c and d, top). In contrast to CCR7, expression of CD62L was decreased similarly on both types of P14 effector cells (Fig. 1d, bottom). To demonstrate that CCR7-tg expression on P14 effector cells was functional, an in vitro chemotaxis assay was performed (Fig. 1e). The results revealed that P14.CCR7 effector cells were still able to migrate toward CCL19 in contrast to P14 effector cells. Thus, these data indicated that CCR7-tg expression was functional and that constitutive expression of CCR7 did not impair Ag-induced expansion of P14.CCR7 T cells in vivo.



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  		FIGURE 1. P14.CCR7 effector cells constitutively expressing CCR7 proliferate normally. CD8 T cells (105) from P14 TCR-tg (Thy-1.1+, •) or P14 TCR x CCR7 double-tg (Thy-1.1+.1.2+, {circ}) mice were transferred into B6 (Thy-1.2+) mice, followed by LCMV infection. a, Number of P14 T cells in recipient mice determined by Thy-1.1 staining of total spleen. The circles represent values from individual mice. n.d. = Not detectable (<0.1%). b, The histogram shows a representative CCL19-Ig staining of CD8 T cells from naive P14 (solid line) and P14.CCR7 (dotted line) mice. Negative control (filled histogram) and MF intensities are indicated. c, Percentage of CCL19-Ig+ cells of splenic Thy-1.1+ P14 T cells before transfer (week 0) and weeks 1 and 4 after transfer and infection. *, p < 0.001; **, p < 0.2. d, CCL19-Ig staining and CD62L expression on P14 and P14.CCR7 effector cells from the spleen 8 days p.i. e, Chemotactic activity of P14 ({blacksquare}) and P14.CCR7 ({square}) effector cells (day 8 p.i.). Results are expressed as the percentages of P14 cells migrating to the lower chamber of a transwell plate, in the absence of a gradient (left) or in the presence of CCL19.

 
CCR7 directs P14.CCR7 effector cells to the spleen

Adoptive transfers of P14 cells into uninfected B6 recipients were performed to examine whether constitutive expression of CCR7 altered the homing pattern of these cells. Naive CD8 T cells from P14 and P14.CCR7 mice migrated to blood, spleen, and lymph nodes of B6 recipient mice to a similar extent (Fig. 2a). However, both P14 and P14.CCR7 effector cells failed to home to lymph nodes after adoptive transfer, irrespective of CCR7 expression (Fig. 2b). This finding can be explained by the strongly decreased CD62L expression on both P14 and P14.CCR7 effector cells. Interestingly, P14 effector cells were found with increased frequency in the blood compared with the spleen, whereas P14.CCR7 effector cells were found in both organs at similar frequencies. Moreover, P14.CCR7 effector cells showed a decreased homing capacity to peripheral organs such as liver and lung compared with P14 effector cells (Fig. 2b). To compare the different homing capacities of P14 and P14.CCR7 cells more directly, equal numbers (105) of naive P14 (Thy-1.1+) and P14.CCR7 (Thy-1.1+1.2+) T cells were mixed and transferred into B6 (Thy-1.2+) mice, followed by LCMV infection. Eight days p.i., the distribution of P14 and P14.CCR7 effector cells was determined in the blood and spleen of the same animals. Confirming the results from the adoptive transfer experiments described above, a higher ratio of P14 to P14.CCR7 effector cells in the blood (ratio = 1.2–1.5) than in the spleen (ratio = 0.8–0.9) was observed, indicating preferential homing of P14.CCR7 effector cells to the spleen (Fig. 2c).



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  		FIGURE 2. Homing pattern of P14 and P14.CCR7 cells. a, CD8 T cells (107) from naive P14 and P14.CCR7 mice were adoptively transferred into uninfected B6 recipient mice. After 20 h, donor (Thy-1.1+) cells were determined in the indicated organs. The bars represent the percentage of P14 T cells (Thy-1.1+) of total CD8+ T cells in the organs indicated. Mean ± SD of 4–7 mice per group is shown. b, Adoptive transfer of in vivo generated P14 and P14.CCR7 effector cells (1 x 107, 8 days p.i.) into uninfected B6 recipient mice. c, Equal numbers (105) of naive P14 (Thy-1.1+) and P14.CCR7 (Thy-1.1+.1.2+) T cells were mixed and transferred into B6 (Thy-1.2+) recipient mice, followed by LCMV infection. Eight days p.i., the distribution of P14 (region R1) and P14.CCR7 (region R2) effector cells was determined in the blood and spleen of the same animal. The numbers in the dot plots indicate percentage of P14 and P14.CCR7 effector cells of total viable cells. The ratio of P14 to P14.CCR7 effector cells in PBL (left) and spleen (right) in two individual recipient mice is shown below the dot plots.

 
CCR7 determines localization of P14.CCR7 effector cells within the spleen

Localization of P14 and P14.CCR7 effector cells within the spleen was determined by immunohistology. Because acute LCMV infection is accompanied by destruction of the follicular organization of the spleen, the migration patterns of P14 effector cells (day 8 p.i.) were determined after retransfer into naive B6 mice. The majority of P14 effector cells, being mostly CCR7–, accumulated in the splenic red pulp and did not enter white pulp areas. In striking contrast, P14.CCR7 effector cells, which constitutively expressed CCR7, localized mainly to the periarteriolar lymphatic sheath (PALS) (Fig. 3a). This migration was mediated by CCR7, because desensitization of P14.CCR7 effector cells by preincubation with CCL19-Ig prevented entry into the PALS (Fig. 3b).



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  		FIGURE 3. CCR7 determines localization of P14 effector and memory cells within the spleen. a, In vivo generated P14 and P14.CCR7 effector cells (1 x 107, 8 days p.i.) were transferred into uninfected B6 recipient mice. Twenty hours after transfer, the localization of P14 T cells in the spleen was determined by immunohistology using Thy-1.1-specific mAb. b, In vivo generated P14.CCR7 effector cells were left untreated (left) or were desensitized (right) by incubation with CCL19-Ig before cell transfer. c, Naive P14 and P14.CCR7 T cells (105) were transferred into B6 recipients (Thy-1.2+), followed by LCMV infection. The localization of Thy-1.1+ cells was determined 3 wk after transfer and infection. d, In vivo generated P14 and P14.CCR7 memory cells (1 x 107, 4 wk p.i.) were transferred into uninfected B6 recipient mice. Localization was determined 24 h after transfer. Objective magnification, a–c, x5, d, x 10.

 
The splenic localization of P14 and P14.CCR7 memory cells was also determined in LCMV-infected B6 recipient mice 3 wk p.i. P14 memory cells were found both in the PALS and in the red pulp. In contrast, P14.CCR7 memory cells almost exclusively localized to the PALS (Fig. 3c). The different localization of P14 and P14.CCR7 memory T cells in the spleen was also observed upon retransfer into uninfected recipients (Fig. 3d). Together, these data demonstrated that the constitutively expressed CCR7 transgene was functional and determined the localization of P14 effector and memory cells in the spleen.

Splenic localization and tissue distribution of P14 memory T cells depend on CCR7 expression levels

The data shown above revealed an almost exclusive localization of P14.CCR7 memory cells in the PALS, whereas P14 memory cells were found both in the white and in the red pulp. Surprisingly, both P14 and P14.CCR7 memory cells isolated 3 wk p.i. could be stained with CCL19-Ig. However, the CCL19-Ig-staining profiles of the two memory cell populations differed (Fig. 4a). CCR7 expression determined by the MF intensity of the CCL19-Ig staining was consistently 1.5- to 2-fold increased on P14.CCR7 memory cells when compared with P14 memory cells (Fig. 4b). A chemotaxis assay was performed to test whether the slightly increased expression level of CCR7 on P14.CCR7 memory cells concurred with increased migration toward CCL19. However, we failed to observe a difference in migration of P14 and P14.CCR7 memory cells in vitro (Fig. 4c). Similarly, no difference in migration toward CXCL12 was observed. Moreover, expression levels of other markers such as CD25, CD44, CD62L, CD71, CD122, Ly-6A/E, and Ly-6C did not differ between P14 and P14.CCR7 memory T cells (data not shown). Thus, these data indicated that a relatively small difference in the CCR7 expression level had a strong impact on splenic migration of memory CD8 T cells in vivo, but not in vitro.



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  		FIGURE 4. CCR7 expression and homing of P14 and P14.CCR7 memory cells. a, The histogram shows representative CCL19-Ig staining of P14 (solid line) and P14.CCR7 (dotted line) memory T cells (~5 wk p.i.). Negative control (filled histogram) and MF intensities are indicated. b, Ratios of CCL19-Ig staining (MF) of P14.CCR7 vs P14 memory cells from several independent experiments are shown as individual dots. As indicated, memory cells from either blood or spleen were analyzed. c, Chemotactic activity of P14 (•) and P14.CCR7 ({circ}) memory cells (week 5 p.i.). Results are expressed as the percentages of P14 memory cells migrating to the lower chamber of a transwell plate, in the absence of a gradient (left) or in the presence of CCL19 (middle) or CXCL12 (right). Dots indicate values from individual experiments. d, Distribution of P14 (•) and P14.CCR7 ({circ}) memory T cells in the organs indicated. The percentages of P14 and P14.CCR7 memory T cells of total CD8 T cells were normalized to the corresponding value obtained in the blood of each mouse analyzed. Dots indicate values from individual mice, and data are representative of at least three experiments. *, p < 0.01; **, p < 0.05.

 
To test whether this small difference in CCR7 expression level also altered tissue distribution, frequencies of P14 and P14.CCR7 memory cells in spleen, inguinal lymph node, liver, and lung were compared. To obtain precise values, percentages obtained in the various tissues were normalized to the corresponding value obtained in the blood of each mouse analyzed. The data showed that P14 memory cells were more frequent in peripheral organs such as liver and lung compared with secondary lymphoid organs such as spleen or inguinal lymph node (Fig. 4d). Direct comparison of P14 with P14.CCR7 memory cells revealed increased frequencies of P14.CCR7 memory cells in spleen and lymph nodes and decreased numbers in liver and lung. Thus, these data indicated that tissue distribution of P14 memory cells was also very sensitive to CCR7 expression levels.

White pulp localization of P14.CCR7 effector cells impairs rapid LCMV clearance

To examine whether the differential localization of P14 and P14.CCR7 effector cells in the spleen influenced antiviral activity, naive P14 and P14.CCR7 T cells were transferred into B6 mice, followed by priming with VVGP. Because the architecture of the spleen is not impaired by vaccinia virus infection, the distribution of these P14 effector cells could be determined directly in situ. Similar to the LCMV model, down-regulation of CCR7 was prevented by the CCR7 transgene (data not shown), and P14 and P14.CCR7 effector cells exhibited a different localization pattern. Most of the P14.CCR7 effector cells induced by VVGP were found in the PALS, whereas P14 effector cells were localized mainly in the marginal zone and the red pulp, and to a smaller degree to the PALS (Fig. 5a). To compare antiviral activity under these two conditions, mice were challenged with a high dose of LCMV. After 24 h, viral titers were determined in the spleen. Fig. 5b shows that P14 effector T cells provided a slightly better control of LCMV replication in the early phase of the infection than P14.CCR7 effector T cells.



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  		FIGURE 5. White pulp localization and impaired LCMV clearance of P14.CCR7 effector T cells induced by VVGP priming. Naive P14 and P14.CCR7 T cells (105) were transferred into B6 recipients, followed by VVGP priming. Seven days after priming, splenic localization (a) of P14 T cells was determined by two-color immunohistology. Thy-1.1+ cells appear in brown, and B220+ B cells in blue. Objective magnification, x10. b, In parallel, VVGP-primed B6 recipient mice of P14 and P14.CCR7 T cells were challenged with a high dose (2 x 106 PFU) of LCMV. Dots represent LCMV titers in the spleen 24 h after challenge in individual mice. The control shows viral titers from unprimed mice, and the dotted line corresponds to the detection limit of the virus plaque assay. *, p < 0.05 by Student’s t test.

 
Impaired DTH response by P14.CCR7 effector cells

Finally, we compared the ability of P14 and P14.CCR7 effector cells to induce a DTH response. For these experiments, the same number (107) of in vivo generated P14 and P14.CCR7 effector cells was adoptively transferred (i.v.) into uninfected B6 mice. One day after transfer, a DTH response was induced in the recipient mice by injecting the gp33 peptide into the footpad. As shown in Fig. 6a, recipients of P14.CCR7 effector cells exhibited a clearly less potent DTH reaction compared with mice that had received P14 effector cells. The decreased DTH reaction in these mice correlated with the lower frequency of P14.CCR7 effector cells in the blood (Fig. 6b). Importantly, this was not due to a lower number of transferred cells or impaired survival because P14.CCR7 effector cells were present at increased numbers in the spleen (Fig. 6c). Thus, these data indicated that CCR7-tg expression impaired the release of P14.CCR7 effector cells from the spleen to blood, and thereby impaired the rapid induction of a DTH response in the periphery.



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  		FIGURE 6. Decreased DTH response by P14.CCR7 effector cells. In vivo generated P14 and P14.CCR7 effector cells (1 x 107, 8 days p.i.) were transferred into uninfected B6 recipient mice. One day after transfer, the number of donor P14 cells in the blood of the recipient mice was determined (b). Immediately afterward, DTH reaction induced by injection of the gp33 peptide (1 µg) into the footpad of the recipient mice. Mean ± SD of three mice per group are shown (a). Six days after the DTH reaction, the number of donor P14 cells was determined in the spleen (c).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Homing of naive T cells to lymph nodes has been shown to depend on the expression of CD62L and CCR7 (7, 22, 23). Memory T cells migrating through peripheral tissues are also thought to enter lymph nodes via afferent lymphatics by a mechanism for which CD62L expression is not required (23, 24). Our data show that both P14 and P14.CCR7 effector cells, being CCR7–CD62L– or CCR7+CD62L–, respectively, failed to migrate into lymph nodes. This result implies that entry into lymph nodes via afferent lymphatics plays a minor role for effector CD8 T cells induced by a viral infection in mice.

In contrast to lymph nodes, the spleen lacks HEV-like vessels, and migration of T cells to the spleen is independent of CD62L (22, 25, 26). Therefore, P14 effector cells lacking CD62L could efficiently home to this organ. The strikingly different localization of P14.CCR7 effector cells in the splenic white pulp, in contrast to wild-type P14 effector cells, impressively demonstrates the crucial role of CCR7 in guiding effector and memory CD8 T cells within this organ. This important finding of our study appears to be in contrast to the notion that CXCR6 and its ligand, CXCL16, are responsible for the accumulation of activated CD8 T cells in the red pulp (17). However, CD8 T cell migration in the spleen may be controlled by a balanced responsiveness to chemoattractants made in the PALS (CCL19/CCL21) as well as in the red pulp (CXCL16). According to this view, the final splenic localization could be determined by the expression levels of the corresponding chemokine receptors on the responding T cells. Below a certain threshold of CCR7 expression, CXCR6 could direct migration of activated CD8 T cells to the red pulp, whereas in the presence of high CCR7 expression, migration to the PALS would be dominant. This idea of finely tuned migration of CD8 T cells toward chemokines is also supported by our finding that a relatively small difference (2-fold) in CCR7 expression levels between P14 and P14.CCR7 memory T cells had a significant impact on their splenic localization and tissue homing.

Our data argue against a recent report (27) claiming that lymphocyte migration to the splenic white pulp does not involve common homing receptors including CCR7. In this particular study, mildly trypsinized lymphocytes failed to enter splenic white pulp cords despite reduced, but still detectable reactivity to CCL19 in chemotaxis assays in vitro. To explain this result, we would argue that analyzing responsiveness toward a single chemokine with chemotaxis assays in vitro may not allow valid conclusions about the efficiency of lymphocyte migration toward multiple chemokine gradients in vivo.

Data about effector cell functions of central and effector memory T cells are controversial. Some studies showed that effector memory cells are able to perform effector cell functions, such as cytokine production and cell-mediated lysis, more rapidly than central memory cells (8, 10, 11). In contrast, other reports found that both memory T cell subsets were equally efficient in acquiring effector cell functions (12, 13, 14, 15, 16). P14 and P14.CCR7 effector T cells did not differ in IFN-{gamma} secretion and killing ability upon stimulation with Ag in vitro (data not shown). Nonetheless, P14 effector T cells lacking CCR7 controlled LCMV in the spleen more efficiently in the early phase of the infection when compared with P14.CCR7 effector T cells. This difference in immediate anti-LCMV activity could be explained by the different localization of the two effector cell populations in the spleen. LCMV replicates predominantly in marginal zone macrophages. At this site, P14 effector cells were found at a higher frequency than P14.CCR7 effector T cells. Thus, CCR7 down-regulation may allow optimal positioning of effector and memory CD8 T cells in the spleen, where invading blood-borne pathogens are first encountered. Our data further revealed that P14 effector cells were more potent than P14.CCR7 effector cells to induce an Ag-specific DTH reaction. The inferior ability of P14.CCR7 effector cells to induce a DTH reaction is most likely due to a lower number of these cells in the blood because the two types of P14 effector cells did not differ in cytokine secretion, CTL activity, or expression of any homing markers, except CCR7.

In conclusion, the present study demonstrates that constitutive CCR7 expression on effector CD8 T cells overrules chemokine responsiveness in the red pulp and directs effector and memory T cells to the splenic white pulp. In addition, it highlights the importance of CCR7 down-regulation to release effector cells from the splenic white pulp to provide rapid immunity in the periphery.


   Acknowledgments
 
We thank Marlies Rawiel for excellent technical assistance; Peter Aichele and Stephen Batsford for comments on the manuscript; and Theresa Treuer, Sonja Wagenknecht, Rainer Bronner, and Thomas Imhof for animal husbandry.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by European Union Grant QLK2-CT-1990-01321. Back

2 Address correspondence and reprint requests to Dr. Hanspeter Pircher, Institute for Medical Microbiology and Hygiene, Department of Immunology, Hermann-Herder-Strasse 11, University of Freiburg, D-79104 Freiburg, Germany. E-mail address: pircher{at}UKL.uni-freiburg.de Back

3 Abbreviations used in this paper: HEV, high endothelial venule; DTH, delayed-type hypersensitivity; LCMV, lymphocytic choriomeningitis virus; MF, mean fluorescence; PALS, periarteriolar lymphatic sheath; p.i., postinfection; tg, transgenic; VVGP, recombinant vaccinia virus expressing LCMV glycoprotein. Back

Received for publication March 26, 2004. Accepted for publication June 25, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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