International Immunology Advance Access published online on October 3, 2007
International Immunology, doi:10.1093/intimm/dxm098
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Abrogation of CCL21 chemokine function by transgenic over-expression impairs T cell immunity to local infections
1 Department of Immunology, Institute of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany
2 Department of Medical Microbiology and Hygiene, University of Freiburg, Freiburg, Germany
3 Department of Immunology, University of Göttingen, Göttingen, Germany
4 Institute for Immunology, Ludwig Maximilian University, Munich, Germany
Correspondence to: Correspondence to: H. Pircher; E-mail: hanspeter.pircher{at}uniklinik-freiburg.de
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Abstract |
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The CC chemokine receptor 7 (CCR7) and its two ligands, CCL21 and CCL19, play an important role in migration of immune cells to lymphoid tissue. To analyze the function of CCR7 in T cell immunity to infectious agents in vivo, transgenic (tg) mice expressing CCL21 in an ubiquitous fashion were generated. These mice contained high amounts of CCL21 in the serum (
0.3 µg/ml that resulted in CCR7 down-regulation and in a strongly impaired migration of T cells toward CCL21 in vitro. Lymph nodes in CCL21-tg mice were reduced in size but with intact microanatomy and normal distribution of T and B cells. CCL21-tg mice showed a significantly decreased CD8 T cell response to lymphocytic choriomeningitis virus after footpad infection, whereas the response after systemic infection was not altered. Likewise, the CD4 T cell response to footpad infection with Leishmania major was considerably lowered and CCL21-tg mice failed to clear parasites from infected skin. Taken together, these data demonstrate the importance of CCR7 in mediating T cell immunity to viral and parasitic pathogens after local infection.
Keywords: cell trafficking, in vivo desensitization, transgenic mice
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Introduction |
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T cells enter lymph nodes (LNs) from the blood stream via specialized high endothelial venules in a complex process that involves rolling, adhesion and diapedesis. The CC chemokine receptor 7 (CCR7) and its two ligands, CCL21 and CCL19, play a key role in this process. This has been demonstrated impressively in CCR7-deficient mice (1) and in plt mutant mice lacking expression of CCL19 and CCL21-Ser (2). In addition to the well established function of CCR7 in lymphocyte migration to lymphoid organs, recent studies also revealed a role for CCR7 in T cell exit from peripheral tissue to afferent lymphatics (3, 4). Apart from chemotaxis, CCL19 and CCL21 are known to regulate various functions in dendritic cells (DCs) including cytoarchitecture (5), rate of endocytosis (6), survival (7) and differentiation (8). CCL19 and CCL21 exhibit similar CCR7 binding affinities and chemotactic potencies in vitro (9, 10) and both chemokines are produced by stromal cells in T cell zones of secondary lymphoid organs (11–13). In addition to stromal cells, CCL19 is also expressed by interdigitating DCs (14–16) and both chemokines can be transcytosed and presented on the lumenal surface of HEVs (17). It is not yet known whether CCL19 and CCL21 are fully redundant in vivo but in vitro studies revealed that CCL19 was more effective in inducing CCR7 phosphorylation (18), CCR7 endocytosis (18–20) and ERK1/2 mitogen-activated protein kinase activation in HEK293 cells (18).
To analyze the function of CCL19 and CCL21 in vivo, several groups have generated transgenic (tg) mice with ectopic expression of these chemokines. These studies showed that tg expression of CCL19 and CCL21 in pancreas led to the formation of LN-like structures (13, 21, 22). In these mice, CCL21 induced larger and more organized infiltrates compared with CCL19 (13). Ectopic CCL21 expression in the thyroid gland led to lymphocyte infiltration (23) and tg mice expressing CCL21 in oligodendrocytes exhibited central nervous system inflammation and neurological disease (24). In contrast, lymphocyte accumulation was not observed when CCL21 was expressed in the skin (21). Tg expression of CCL21 in T cells resulted in defects in CCL19- and CCL21-induced T cell chemotaxis and in abnormal LN architecture with smaller T cell zone and larger B cell follicle (25). However, the capacity of these mice to respond to infectious agents has not been analyzed. Here, we generated tg mice with high systemic levels of CCL21 using an ubiquitous promoter (MHC class I) and analyzed the capacity of these mice to respond to viral and parasitic infections. The results revealed that abrogation of the CCL21 chemokine gradient in vivo by tg over-expression impaired T cell immunity to local but not to systemic infection.
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Methods |
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Mice
C57BL/6 (B6) mice were obtained from Harlan Winkelmann (Borchen, Germany). To generate CCL21-tg mice, CCL21-serine cDNA was amplified with CCL21-specific primers (5'-ATCCATAAGTCGACCACAATCATG-3 and 5'-CTGGGATCCAGGCGGGCTAC-3'). The PCR product was digested with SalI and BamHI, subcloned into the pHSE3' expression vector (26) and injected into fertilized (B6 x CBA)F1 eggs. Mice were typed by PCR (5'-TCACTTCTGCACCTAACCTG-3' and 5'-CTGGGATCCAGGCGGGCTAC-3') and were backcrossed for 9 generations to B6 mice. P14 TCR-tg mice (B6; D2-Tg(TcrLCMV)327Sdz/JDvsJ) specific for amino acids 33–41 (=gp33 epitope) of the lymphocytic choriomeningitis virus (LCMV) glycoprotein have been described (27). Female or male mice were used at 8–20 weeks of age. Animals were kept under specific pathogen-free conditions. All animal care and use was approved by the Regierungspräsidium Freiburg.
Analysis of CCL21 expression
Northern blotting was performed as described (28) using 20 µg of total RNA per lane and a 400 bp cDNA fragment of CCL21, random primer labeled with 32P-dATP, as a probe. CCL21 protein levels in mouse sera were determined by a sandwich ELISA (DuoSet ELISA Development Kit, R&D Systems, Wiesbaden, Germany).
Flow cytometry
The following mAb were used: anti-CD8 (clone 53–6.7), anti-CD4 (clone L3T4), anti-B220 (clone RA3-6B2), anti-Thy1.2 (clone 30-H12), anti-Thy1.1 (clone OX-7), anti-CD3 (clone 145-2C11), anti-I-Ab (clone AF6-120.1) and anti-CD11c (clone HL3). Antibodies were purchased from BD PharMingen (San Diego, CA). CCR7 expression was determined by anti-CCR7 mAb [clone 4B12 (29)] followed by a PE-conjugated goat anti-rat IgG antibody. Before analysis of peripheral blood lymphocytes, red blood cells were lysed using FACS-Lysing Solution (BD PharMingen). Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software.
Immunohistochemistry
Frozen sections (7 µm) were acetone fixed, washed in 0.1 M Tris buffer, pH 7.5, blocked with Tris-buffered saline containing 5% mouse serum and incubated for 60 min with biotinylated anti-CD8 mAb (clone 53–6.7, BD PharMingen) in a humidified chamber. After a further wash, the sections were incubated with phosphatase-conjugated streptavidin (DAKO, Hamburg, Germany) for 30 min, washed and developed with the Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories, Burlingame, CA). To determine T cell localization after adoptive transfer, splenic Thy1.2+ cells from CCL21-tg and wild-type (wt) mice were purified by magnetic beads (Miltenyi Biotech, Bergisch-Gladbach, Germany), labeled with CFSE (Molecular Probes, Eugene, OR) and transferred (107) into non-irradiated CCL21-tg and wt mice, respectively. Mice were sacrificed 24 h after transfer and CFSE-labeled T cells were detected by rabbit anti-FITC antibody (DaKoCytomation, Glostrup, Denmark) followed by goat-anti-rabbit-conjugated-HRP (Jackson ImmunoResearch-Dianova, Hamburg, Germany). Metallophilic macrophages were stained by biotinylated MOMA-1 mAb (BMA, Augst, Switzerland) followed by phosphatase-conjugated streptavidin (DAKO) and Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories). Sections were counterstained with Meyer's hematoxylin and mounted with Kaiser's glycerol gelatin for analysis.
In vitro Chemotaxis assay
Chemotaxis assays were performed in 5-µm Transwell chambers (Corning Costar Corp., Acton, MA, USA) using 106 spleen cells in 100 µl tissue culture media containing 1% FCS. The lower chambers contained CCL21 or CCL19 (R&D Systems) at the concentration indicated. After 3 h, migrated cells were pooled from three wells, re-suspended in 500 µl PBS containing 2% FCS and analyzed by flow cytometry using CD3-, CD4- and CD8-specific mAb to determine the percentage of migrated T cells.
Adoptive cell transfers
To assess autocrine or paracrine effects of tg CCL21 expression, spleen cells (3 x 107) from CCL21-tg (Thy1.2+) and B6.Thy1.1 mice were injected intravenously (i.v.) into non-irradiated B6.Thy1.1 and CCL21-tg mice, respectively. After one week, chemotactic activity to CCL21 and CCR7 expression was determined using Thy1.1- and Thy.1.2-specific mAb to distinguish donor and host T cells. To assess T cell migration in vivo, Thy1.2+ cells from CCL21-tg and wt mice, purified by magnetic beads (Miltenyi Biotech), were labeled with CMTMR (Molecular Probes) or CFSE, respectively. Afterward, equal numbers (107) of tg and wt T cells were pooled and were injected i.v. into non-irradiated B6 mice. After 3 or 20 h, mice were killed and peripheral blood and inguinal LNs (ILNs) were analyzed by flow cytometry. Bone marrow-derived DC (BMDCs) from wt and tg mice were generated by culturing bone marrow cells from femurs in Roswell Park Memorial Institute 1640 media (Sigma) supplemented with 10% FCS (Biochrom, Seromed) and mouse granulocyte macrophage colony-stimulating factor (20–50 ng/ml) and recombinant mouse IL-4 (10 ng/ml; Promokine, Heidelberg, Germany) for 8 days. To assess DC migration in vivo, BMDCs were labeled with CFSE and 106 CFSE-labeled BMDCs were injected subcutaneously (s.c.) into footpads of B6 mice and 24–35 h later percentages of CFSE+CD11c+ cells were determined in the draining popliteal LNs (popLNs).
Analysis of LCMV-specific CD8 T cell responses
CD8 T cell priming was determined by transferring (i.v.) 107 CFSE-labeled LCMV-specific P14 TCR-tg T cells (Thy1.1+) into non-irradiated CCL21-tg and wt mice that were subsequently primed (s.c.) with 20 µg of virus-like particles (VLPs) containing the gp33 epitope (VLPgp33). The generation of VLPgp33 (a kind gift from Martin Bachmann, Cytos, Zurich) has been described (30). P14 T cells were analyzed 3 days after cell transfer. To assess the polyclonal LCMV-specific T cell response, mice were infected with LCMV-WE either by i.v. (200 pfu in 200 µl or by footpad injection (104 pfu in 30 µl. Footpad thickness was measured immediately before infection and at the time points indicated using a dial-gauge caliper (Mitutoyo, Aurora, Illinois, USA). Footpad swelling was related to the footpad thickness before infection as the percentage of increase in footpad thickness. Absolute numbers of LCMV gp33-specific CD8 T cells were determined by flow cytometry using PE-conjugated gp33/H-2Db tetramers and cell counting.
Infection with Leishmania major
Promastigotes of the L. major stain (MHOM/IL/81/FE/BNI) were grown from amastigotes isolated from skin lesions of BALB/c mice and propagated in vitro. Mice were infected into both hind footpads with 1 x 105 or 3 x 106 stationary-phase L. major promastigotes of a low in vitro passage (<5) in 50 µl PBS. The footpad swelling was measured with a dial-gauge caliper and related to the footpad thickness before infection. Number of parasites in the tissue was determined by limiting dilution analysis applying Poisson statistics as described (31). Differences were assumed to be significant when the 95% confidence intervals did not overlap. IFN-
production was determined by culturing lymphocytes from popLNs (106/ml) from L. major-infected mice with Leishmania antigen (freeze thaw lysates of promastigotes; parasite: cell ratio = 1:1) for 48 h. IFN- in the culture supernatant was measured by a sandwich ELISA (DuoSet ELISA Development Kit, R&D Systems). To determine the absolute number of CD11c+ I-Ab+ DCs in popLNs, single cell suspension of popliteal lymphocytes was underlayed with medium containing 14.5% Histodenz (Sigma). After centrifugation at 800 g for 17 min, cells of the interphase were harvested, washed twice and stained for flow cytometry.
Statistical analysis
Student's t test of unpaired data was used to determine the significance of differences of mean. A value of P < 0.05 was considered to be statistically significant.
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Generation of tg mice with systemic CCL21 expression
To achieve CCL21 expression in an ubiquitous fashion in vivo, murine CCL21-Ser cDNA was inserted into the pHSE3' expression cassette (26) that is driven by the H-2Kb promoter. Several tg founders were obtained and one high-expressing line was selected for further analysis. CCL21-tg mice appeared healthy and were fertile. Northern blot analysis revealed that tg CCL21 mRNA (
600 bp) was expressed most prominently in lymphoid organs of CCL21-tg mice such as thymus, mesenteric LNs (MLN) and spleen (Fig. 1A). In contrast, both in CCL21-tg and in wt mice (Fig. 1A, most right lanes), endogenous CCL21 mRNA (900 bp) was barely detectable under these exposure conditions. Lower levels of tg CCL21 mRNA were also found in non-lymphoid organs including liver, lung, heart and kidney but not in the small intestine, muscle or brain. The serum of CCL21-tg mice contained 190–570 ng/ml of CCL21, whereas the values obtained with wt sera were below detection levels (<10 ng/ml) (Fig. 1B).
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ILNs and MLNs from CCL21-tg mice contained about 10 times less total lymphocytes compared with age-matched wt mice (Fig. 2A). Despite the difference in total lymphocyte counts, the proportion of CD4, CD8 and B cells was comparable in CCL21-tg and wt mice (Fig. 2B). In addition, immunohistology of ILNs (Fig. 2C) and MLNs (Fig. 2D) from wt and CCL21-tg mice revealed a similar localization of B cell follicles in the outer cortex and of T cells in the paracortical regions of both strains of mice. Similarly, no alterations were observed in the spleen of CCL21-tg mice and total thymocyte numbers and thymocyte subset distribution in tg mice did not differ from non-tg littermates (data not shown). To further analyze the migration behavior of CCL21-tg T cells, adoptive transfer experiments were performed. These experiments showed that both the transferred wt and CCL21-tg T cells became localized to the same extent in the paracortical T cell zone of LNs from wt or CCL21-tg mice, respectively (Fig. 2E). Thus, LNs from CCL21-tg mice were significantly reduced in size and cellularity, but showed an intact microanatomy with normal distribution of T and B cells.
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Systemic CCL21 expression down-regulates CCR7 and inhibits chemotactic activity of T cells to CCL21
T cells from CCL21-tg and wt mice were analyzed by flow cytometry using CCR7-specific mAb. The mean fluorescence of CCR7 staining on CD4 and CD8 T cells from blood and spleen of tg mice was reduced about 3–4 fold compared with wt T cells (Fig. 3A). Since binding of CCL21 to CCR7 does not affect CCR7 recognition by this particular mAb, this result demonstrates that tg CCL21 expression led to a significant down-regulation of the corresponding chemokine receptor. In vitro chemotaxis assays further revealed that CD3 T cells from CCL21-tg mice were strongly impaired in their capacity to migrate toward CCL21. Despite CCR7 down-regulation, migration of tg T cells toward CCL19 was surprisingly not impaired when saturating amounts (1 µg/ml) of CCL19 were used (Fig. 3B, top). The strongly impaired migration of CD4 and CD8 T cells from CCL21-tg mice to CCL21 was confirmed in chemokine titration experiments (Fig. 3B, bottom left). These experiments also revealed that migration of CD4 and CD8 T cells from tg mice toward CCL19 was also decreased when lower chemokine concentrations were used (Fig. 3B, bottom, right).
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wt) or vice versa (wt The decrease in CCR7 expression and the impaired CCL21 migration of tg T cells could be due to autocrine and/or paracrine effects of tg CCL21 expression. To address this point, tg and wt T cells were adoptively transferred into wt and tg mice and 1 week afterward chemotactic activity and CCR7 expression of the transferred T cells was analyzed. As shown in Fig. 3C, tg T cells adoptively transferred into wt recipient mice migrated more efficiently toward CCL21 and displayed increased CCR7 expression compared with tg T cells without transfer. However, migration and CCR7 expression of the transferred tg T cells was still considerably lower than wt T cells. On the other hand, adoptive transfer of wt T cells into tg mice lowered CCL21 responsiveness and decreased CCR7 cell surface expression levels. Taken together, these data indicated that both autocrine and paracrine effects of tg CCL21 expression contributed to CCR7 down-regulation and impaired CCL21-induced migration of T cells in CCL21-tg mice.
To determine the homing capacity of tg T cells in vivo, T cells from wt and tg mice were labeled with different dyes and were co-transferred at equal numbers into B6 mice. After 20 h, blood and ILNs were collected and the frequencies of the transferred T cells were determined. These experiments revealed that tg T cells were present in the blood at higher frequencies than in LNs, whereas similar frequencies of wt T cells were found in blood and LNs (Fig. 3D, top). This result could be due to a reduced entry of tg T cells into LNs or due to a more rapid exit of the cells from the organ. Therefore, short-term (3 h) adoptive transfers were also performed (Fig. 3D, bottom). Under these experimental conditions, tg T cells were also present in the blood at higher frequencies than in LNs, indicating that tg T cells were impaired in migration to LNs. This effect was, however, more pronounced in the CD4 than in the CD8 T cell subset.
Systemic CCL21 expression impairs the LCMV-specific CD8 T cell responses to local but not to systemic infection
To determine the effect of CCL21 desensitization on CD8 T cell immunity, CFSE-labeled P14 TCR-tg T cells (Thy1.1+) specific for the gp33 epitope of LCMV were adoptively transferred into CCL21-tg or wt mice that were subsequently primed s.c. with VLPs containing the gp33 epitope (VLPgp33). After 3 days, P14 T cell proliferation in blood, spleen and draining LN was assessed by flow cytometry. In all organs examined, VLPgp33-induced P14 T cell proliferation, as determined by dilution of the CFSE label, was strongly decreased in CCL21-tg compared with wt mice (Fig. 4A). Next, induction of the polyclonal LCMV-specific CD8 T cells after s.c. footpad and i.v. LCMV infection was analyzed. After footpad infection, CCL21-tg mice showed a significantly decreased footpad swelling reaction (Fig. 4B) and 10-fold lower numbers of LCMV gp33 tetramer+ cells in popLNs (Fig. 4C, left) than in wt mice. In contrast, after systemic LCMV infection (i.v.), numbers of gp33 tetramer+ cells in spleen were comparable in both types of mice (Fig.4C, right). Lack of efficient CD8 T cell priming in CCL21-tg mice after local antigen challenge could be due to impaired migration of DCs to draining LNs. Indeed, this proved to be the case. BMDCs from CCL21-tg mice, when injected into footpads of B6 mice, showed strongly impaired migration to popLNs (Fig. 4D). Together, these data indicate that the CD8 T cell response to local antigen challenge was significantly reduced in CCL21-tg mice, most likely due to the impaired migration of DCs into draining LNs.
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Systemic CCL21 expression abolishes parasite elimination after L. major infection
Infection of mice with L. major is a well established experimental model to assess CD4 T cell immunity. Leishmania major promastigotes were injected at an intermediate (1 x 105) or high dose (3 x 106) into the hind footpads of CCL21-tg and wt mice and the course of the infection was monitored by weekly measurements of the thickness of the infected footpads. After intermediate dose infection, the footpad swelling in CCL21-tg mice was clearly reduced during the entire course of the infection (Fig. 5A, top). At day 113 post-infection, when the footpad swelling reaction had declined to baseline levels in both types of mice, parasite numbers were determined. Strikingly, parasite numbers in the infected footpads of tg mice were more than 103-fold higher than in wt mice (Fig. 5B). Moreover, IFN-
production by lymphocytes from popLN induced by L. major antigen stimulation in vitro was 5-fold lower in tg than in wt mice (Fig. 5C). A similar picture with increased parasite burden in infected skin lesions of tg mice and reduced IFN- production by lymphocytes from popLNs was observed after high dose infection (Fig. 5B and C, bottom). However, in contrast to the low dose infection experiments, the decline in footpad swelling reaction was delayed in tg mice compared with wt mice (Fig. 5A, bottom). The failure of CCL21-tg mice to control L. major infection could be due to impaired migration of skin-derived DCs to draining LNs. We thus determined the absolute numbers of DCs in draining LNs of L. major-infected tg and wt mice. As shown in Fig. 5D, the numbers of the induced CD11c+ I-Ab+ cells in popLNs of tg mice were indeed considerably lower than in wt mice. Taken together, these data indicated that CCL21-tg mice were severely impaired to mount a L. major-specific CD4 T cell response and to control the infection most likely due to impaired migration of skin-derived DCs.
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Discussion |
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The present study was designed to analyze the function of CCR7 in T cell immunity to viral and parasitic infections. Mice with a disrupted CCL21 chemokine gradient were generated by systemic tg expression of CCL21 using a strong ubiquitous promotor. Systemic expression of CCL21 resulted in CCR7 down-regulation and impaired T cell migration toward CCL21 in vitro. Size and cellularity of LNs from CCL21-tg mice were reduced but distribution and localization of T and B cells was not altered. CCL21-tg mice showed a significantly decreased CD8 T cell response to LCMV after footpad infection, whereas the response after systemic infection was not altered. Moreover, the CD4 T cell response to footpad infection with Leishmania major was also decreased and CCL21-tg mice failed to clear parasites from infected skin.
The CCL21-tg mice studied here differ from tg mice described previously that expressed CCL21 under the control of the lck promoter in T cells (25). In the latter mice, CCL21 desensitization of T cells was also observed in chemotaxis assay in vitro, but a decrease in LN cellularity was not reported. In our mice, the nodal architecture was not affected, whereas in the CCL21-lck mice, smaller T cell zones and larger B cell follicles were noted. Since different promoters (T cell-specific versus ubiquitous MHC class I) were used to express CCL21 cDNA, differences in expression levels and sites are likely to account for the different phenotypes of these mice. CCR7 expression, CCL21 content in serum, in vivo migration capacity and functional activity after antigen stimulation or infection were not analyzed in the lck-CCL21-tg mice.
T cells from CCL21-tg mice exhibited a strongly reduced capacity to migrate toward CCL21 in vitro, whereas their migration toward CCL19 was clearly less affected. This observation was surprising in view of previous data demonstrating similar CCR7 binding affinities and chemotactic potencies of CCL19 and CCL21 (9, 10). It is, however, noteworthy that CCR7 expression on T cells from CCL21-tg mice was 4-fold lower compared with wt T cells. Thus, our data indicated that under conditions of reduced chemokine receptor expression, CCL19 was more potent than CCL21 in attracting T cells in chemotaxis assays in vitro. This finding complements previous data showing that CCL19 was also more potent than CCL21 in inducing CCR7 phosphorylation (18), CCR7 endocytosis (18–20) and ERK1/2 mitogen-activated protein kinase activation in HEK293 cells (18). The normal localization of T cells in the paracortical regions of LNs from CCL21-tg mice is in contrast to plt mutant and CCR7–/– mice that lack T cell-rich areas in the paracortex of LNs (1, 2). This suggests that the residual chemotactic activity of CCL21-tg T cells to CCL19 or CCL21 in vivo was sufficient for directing T cells to paracortical regions.
Our data further demonstrated that induction of LCMV-specific CD8 T cells after local (footpad), but not after systemic (i.v.) infection, was strongly impaired in CCL21-tg mice. In addition, we found that migration of adoptively transferred tg DCs to draining LNs was almost absent in these mice. Similar to T cells from CCL21-tg mice, CCR7 cell-surface expression on tg DCs was also significantly decreased (data not shown). These data indicated that CCL21-mediated migration of DCs was required for induction of a normal LCMV-specific CD8 T cell response after local, but not after systemic LCMV, infection. LCMV-induced footpad swelling reactions have been examined previously in CCR7-deficient and in plt mutant mice with different outcome. CCR7-deficient mice showed a decreased response (32) similar to the CCL21-tg mice analyzed here, whereas plt mutant mice showed a nearly unimpaired response (33), presumably due to the remaining leucine isoform of CCL21 expressed in lymphatic vessels.
Infection of mice with L. major is a well-established experimental model of cutaneous leishmaniasis. Langerhans cells and DCs in lesional skin are thought to initiate the parasite-specific T cell response by transporting L. major antigen to draining LNs (34–37). This view, however, has been recently challenged by the finding that LN resident rather than skin-derived DCs initiate the specific T cell response after L. major infection (38). Infection of DCs with L. major or treatment with parasite lysate is known to induce CCR7 expression and to enhance CCL21-induced migration (39), but the importance of CCR7 ligands in L. major infection has not yet been examined in vivo. Our data using CCL21-tg mice demonstrated that parasite elimination in infected skin lesions required CCL21 activity. The reduced footpad swelling and the lower IFN- production in draining LNs further indicated that induction of the parasite-specific T cell response was impaired in CCL21-tg mice. Thus, our data support the view that CCR7-dependent migration of skin DCs to draining LNs is required for induction of L. major-specific T cell response that leads to parasite elimination in infected feet. Our data complement a recent study in plt mice that examined the immune response to L. donovani infection that represents a model for visceral leishmaniasis (40).
In conclusion, we have established a novel mouse model to analyze the role of CCR7 in T cell immunity to infectious pathogens in vivo. The results revealed that abrogation of the CCL21 chemokine gradient resulted in an impaired T cell response to local LCMV infection and in a failure to clear L. major from infected skin.
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Deutsche Forschungsgemeinschaft (DFG Pi 295/5 to H. P., DFG Bo 996/3-3, SFB 620 Project A9 to C. B. and U.S.).
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Acknowledgements |
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We thank Martin F. Bachmann (Cytos Biotechnology, Zurich) for providing VLPs, Peter Aichele for helpful comments on the manuscript and Theresa Treuer, Sonja Wagenknecht, Rainer Bronner and Thomas Imhof for animal husbandry.
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Abbreviations |
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| B6, C57BL/6 |
| BMDC, bone marrow-derived DC |
| CCR7, CC chemokine receptor 7 |
| DC, dendritic cell |
| ERK, extracellular signal-regulated kinase |
| i.v., intravenously |
| ILN, inguinal LN |
| LCMV, lymphocytic choriomeningitis virus |
| LN, lymph node |
| MLN, mesenteric LN |
| p.i., post-infection |
| popLN, popliteal LN |
| s.c., subcutaneously |
| SEM, standard error of the mean |
| tg, transgenic |
| VLP, virus-like particle |
| wt, wild type |
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Notes |
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Transmitting editor: Dr. H. Robson Mac Donald
Received 19 April 2007, accepted 10 August 2007.
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References |
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