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The Journal of Immunology, 2004, 172: 857-863.
Copyright © 2004 by The American Association of Immunologists

Dendritic Cell Immunization Route Determines CD8+ T Cell Trafficking to Inflamed Skin: Role for Tissue Microenvironment and Dendritic Cells in Establishment of T Cell-Homing Subsets1

Jan C. Dudda, Jan C. Simon and Stefan Martin2

Clinical Research Group Allergology, Department of Dermatology, University of Freiburg, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effector/memory T cell pool branches in homing subsets selectively trafficking to organs such as gut or skin. Little is known about the critical factors in the generation of skin-homing CD8+ T cells, although they are crucial effectors in skin-restricted immune responses such as contact hypersensitivity and melanoma defense. In this study, we show that intracutaneous, but not i.v. injection of bone marrow-derived dendritic cells induced skin-homing CD8+ T cells with up-regulated E-selectin ligand expression and effector function in contact hypersensitivity. The skin-homing potential and E-selectin ligand expression remained stable in memory phase without further Ag contact. In contrast, i.p. injection induced T cells expressing the gut-homing integrin {alpha}4{beta}7. Although differential expression of these adhesion molecules was strictly associated with the immunization route, the postulated skin-homing marker CCR4 was transiently up-regulated in all conditions. Interestingly, dendritic cells from different tissues effectively induced the corresponding homing markers on T cells in vitro. Our results suggest a crucial role for the tissue microenvironment and dendritic cells in the instruction of T cells for tissue-selective homing and demonstrate that Langerhans cells are specialized to target T cells to inflamed skin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There is accumulating evidence from clinical research and animal models that the effector/memory T cell pool consists of subsets trafficking to distinct tissues (1, 2, 3). As a consequence, immune responses are compartmentalized. Tissue-restricted autoimmune or allergic diseases and efficient defense against pathogens can only take place if effector cells enter their target organ. For this purpose, T cells are equipped with a specific set of chemokine receptors and adhesion molecules (1, 2, 3). Well characterized is the enhanced expression of the integrin {alpha}4{beta}7 together with the chemokine receptor CCR9 by T cells homing to small intestine (4, 5, 6, 7, 8). In contrast, many skin-associated disorders such as atopic dermatitis are driven by a CD4+ T cell subset expressing the E-selectin ligand (ESL)3, cutaneous lymphocyte Ag (3, 9, 10). Generation of E-selectin binding sites by fucosyltransferase VII on the P-selectin glycoprotein ligand-1 backbone is differentially regulated by various factors, e.g., IL-12, TGF-{beta}1, and IL-4 (11, 12, 13), and not completely understood yet. For skin homing of CD4+ T cells as well as for the onset of contact hypersensitivity (CHS) in mice, the essential role of ESL (14, 15, 16) and the chemokine receptors CCR4 together with CCR10 has been demonstrated (17, 18, 19). However, little is known about factors determining tissue-selective T cell homing, and the differential in vivo generation of tissue-selective CD8+ T cells by dendritic cell (DC) immunization as shown in this work has not been reported to our knowledge. In this study, we concentrate on skin homing of CD8+ T cells, which are crucial effectors in skin-specific diseases such as CHS to epicutaneously applied haptens (20, 21, 22, 23).

The basis for our experiments was the observation that intracutaneous (i.c.), but not i.v. injection of hapten-modified bone marrow-derived DC (BM-DC) efficiently sensitized mice for hapten-specific CHS (24). We detected equal induction of cytotoxic CD8+ effector T cells in both cases. Therefore, we hypothesized that Ag presentation in the skin was required to induce skin-homing T cells. In the present study, we investigated factors that may determine the homing characteristics of CD8+ T cells. We analyzed the contribution of the tissue microenvironment using the same BM-DC injected via different routes. In addition, we compared the skin-homing efficiency of these T cells after in vivo activation by i.c., i.v., or i.p. DC injection upon induction of skin inflammation by the contact sensitizer 2,4,6-trinitrochlorobenzene (TNCB) (25) following adoptive transfer of naive CD8+ TCR transgenic P14 T cells (26, 27, 28). Then we analyzed their potential to elicit a peptide-dependent CHS. Furthermore, we addressed a direct role of tissue-specific DC for imprinting of CD8+ T cells for differential expression of homing markers.


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

C57BL/6 and transgenic P14 mice (26) expressing a TCR specific for the lymphocytic choriomeningitis virus-derived peptide GP33 (27) containing TCRV{alpha}2/TCR V{beta}8 were provided by the breeding facility of the Max Planck Institute for Immunobiology (Freiburg, Germany). P14.Thy-1.1 mice (28) were kindly provided by H. Pircher, Institute for Microbiology and Hygiene, University of Freiburg. All of the experimental procedures were in accordance with the Max Planck Institute and the University of Freiburg guidelines on animal welfare.

Peptides

The H-2Db-binding peptide GP33 from the glycoprotein of lymphocytic choriomeningitis virus has been described before (27) and was purchased from BioChip Technologies GmbH (Freiburg, Germany).

Media and chemicals

RP-10 consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 25 mM HEPES buffer, 50 µg/ml penicillin-streptomycin (all from Life Technologies, Invitrogen, Karlsruhe, Germany), and 10 µM 2-ME (Sigma-Aldrich, Deisenhofen, Germany). The 2,4,6-trinitrobenzene sulfonic acid (TNBS) was from Sigma-Aldrich, and TNCB was obtained from Department of Chemistry, University of Freiburg and from VeZerf Laborsynthesen GmbH (Idar-Oberstein, Germany).

Abs and flow cytometry

Abs and streptavidin-CyChrome were from BD PharMingen (Heidelberg, Germany) and used as FITC, PE, or biotin conjugates at 0.1–1 µg/1 x 106 cells in 100 µl 2% FCS/PBS, unless indicated otherwise. PE-labeled {alpha}4{beta}7 integrin Ab (clone DATK32) was used, as described elsewhere (29). Staining for ESL on cells ex vivo was done with 2 µg E-selectin/human IgG-Fc chimera (R&D Systems, Wiesbaden, Germany) in 50 µl HBSS (Life Technologies) containing 0.2% BSA (Sigma-Aldrich). Before staining, blood cells were washed with 5 mM EDTA/PBS. Cells from in vitro primings were stained with a lower chimera concentration of 0.5 µg/100 µl. Control stainings were done with naive cells or with secondary Ab only. E-selectin chimera was detected with a FITC-labeled Ab for ex vitro cells (DAKO, Hamburg, Germany) or by anti-IgG biotin and streptavidin-CyChrome on cells ex vivo. Purified CCR4 antiserum (Alexis GmbH, Grünberg, Germany) and the secondary FITC or PE anti-rabbit Ab (Serotec, Eching, Germany) were diluted 1/50–1/100.

Generation of BM-DC

Bone marrow cells were cultured at 7 x 105 cells/ml in the presence of 40 ng/ml GM-CSF (supernatant from producer line X63-Ag8 (30)) and 10 ng/ml rIL-4 (Promocell, Heidelberg, Germany) in 10 ml medium in 10-cm petri dishes (Greiner, Nürtingen, Germany). On day 3, 10 ml of fresh medium containing 40 ng/ml GM-CSF was added. On days 5 and 7, 10 ml of medium was replaced with fresh GM-CSF medium. DC were used on days 7–9 after centrifugation over a 14.5% metrizamide (SERVA, Heidelberg, Germany) gradient (409 x g for 15 min). Purity was ~90%.

Immunization and induction of CHS

For standard sensitization, mice were painted with 100 µl of 7% TNCB/acetone on the shaved abdominal skin. Alternatively, DC were modified with 3 mM TNBS/PBS for 7 min at 37°C and washed twice in RPMI/10% FCS and finally in PBS (TNP-modified BM-DC (DC-TNP)). A total of 1 x 106 DC-TNP was injected i.v. in 200 µl PBS or i.c. in 100 µl PBS into two sites of the shaved abdominal skin. Control mice received unmodified DC. On day 5, mice were challenged on both ears with 20 µl of 1% TNCB/acetone, and ear swelling was measured before and 24 h after ear painting using an engineer’s micrometer (Mitutoyo, Leonberg, Germany). For induction of peptide-dependent ear swelling, TNCB was painted on both ears of mice 8 days after adoptive P14 T cell transfer to induce skin inflammation. Ten minutes later, the superficial TNCB was carefully removed by washing ears with 50 µl acetone and rubbing down the ears using a paper towel to minimize potential TNP modification of peptide GP33. Peptide was then applied to ear skin by painting 10 µl of 0.8 mg/ml GP33 in PBS/20% DMSO.

Cytotoxicity assay

Spleen cells after i.v. injection of DC and draining LN cells after i.c. injection were restimulated in 96-well U-bottom plates (Costar, Acton, MA) with irradiated and TNP-modified spleen cells for 3 days. Target EL4 lymphoma cells were labeled with Na251CrO4 (Amersham, Freiburg, Germany) for 90 min, washed three times, and modified with 3 mM TNBS or left untreated. T cell cultures were harvested and washed, and 3-fold serial dilutions were made. The assay was then incubated for 5 h at 37°C/5% CO2. Supernatant (30 µl) was transferred to Luma Plates (PerkinElmer Life Sciences, Rodgau-Jügesheim, Germany). Counts were measured in a TopCount (PerkinElmer Life Sciences), and percentage of specific lysis was calculated.

Adoptive P14 T cell transfer

A total of 1.5 x 106 spleen cells of P14 mice in 200 µl PBS was injected i.v. into C57BL/6 mice. BM-DC were incubated for 1 h in RP-10 with peptide GP33 (1 µM) and LPS (0.3 ng/ml) (Sigma-Aldrich) at 37°C. After washing three times with PBS, DC were used for injection. On days 1 and 3 after P14 T cell transfer, i.c. DC injection was performed into the shaved abdominal skin. A total of 3 x 106 DC in 200 µl PBS was injected at three sites. The i.v. or i.p. injection of 1 x 106 DC in 200 µl PBS was performed on days 2 and 4 after T cell transfer.

Skin-homing assay

On day 7 after adoptive P14 transfer and DC immunization, skin inflammation was induced by painting ears with 20 µl of 1% TNCB/acetone. Epidermal sheets were prepared from ears 24 h later and incubated overnight in RP-10. Emigrated cells were counted, stained with Abs for the expression of CD8{alpha} and the transgenic P14 TCR-V{alpha}2 or Thy-1.1, and analyzed by flow cytometry. Memory T cell homing was investigated using the same protocol on day 20 after transfer.

Isolation of DC and in vitro priming

DC were isolated from peripheral and mesenteric lymph nodes (LN) by CD11c+ AutoMACS bead separation (Miltenyi Biotec, Cologne, Germany) or 15% metrizamide/RP-10 gradient centrifugation to a purity of ~80–95%. Langerhans cells (LC) were isolated from earsheets. Ears were split in dorsal and ventral sheets using forceps. Sheets were incubated for 35 (dorsal) or 40 min (ventral, cartilage-containing half) in 1% trypsin/PBS solution at 37°C, and the epidermal layer was separated from the dermis by scraping off with forceps. After extensive up-and-down pipetting, cells were isolated by passing through a cell strainer (70 µm), followed by metrizamide gradient centrifugation.

DC were incubated with GP33 peptide (1 µg/ml) for 45 min at 37°C and washed three times. A total of 5 x 103 DC and 4 x 104 P14 spleen cells/well was cocultured in 96-well plates (Corning Life Sciences, Wiesbaden, Germany) in 200 µl of RP-10. In some experiments, magnetic bead (Miltenyi Biotec) negatively isolated CD8+ T cells were used and similar results were obtained.

Proliferation assay

On day 4 of in vitro priming, 20 µl medium containing 1 µCi [3H]thymidine was added to cultures, and incorporation was measured after 18 h with a TopCount plate reader (PerkinElmer Life Sciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sensitization for CHS by i.c., but not i.v. DC immunization

DC-TNP were injected i.c. or i.v. into C57BL/6 mice, and elicitation of CHS was performed 5 days later by ear challenge with TNCB. A strong ear-swelling response after 24 h was detected only with DC i.c., but not DC i.v. (Fig. 1A). Ear swelling after i.v. injection was similar to that of mice after injection of unmodified control DC.



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  		FIGURE 1. The i.v. application of DC is inefficient in sensitizing mice for CHS, but induces CTLs. A, Mice were injected with unmodified DC (Ctrl.) or DC-TNP i.v. or i.c. on day 0 into two sides of the shaved abdomen and challenged on ears on day 5 with TNCB. Ear swelling was measured 24 h later. Numbers give the mean increase in ear thickness from three mice ± SD. B, Detection of TNP-specific cytotoxicity in a 4-h chromium release assay. Skin-draining LN cells on day 4 after DC i.c. (•) and spleen cells after DC i.v. ({blacktriangleup}) injection were restimulated in vitro for 3 days. Cells were then incubated with chromium-labeled, TNP-modified EL4 cells (filled symbols) or unmodified control targets (open symbols). Numbers give the mean percentages of specific lysis at the indicated E:T ratios from triplicate measurements ± SD in a 4-h chromium release assay. Representative results from one of three experiments.

 
We have previously shown that cytotoxic CD8+ type 1 CD8+ T cells (Tc1) cells are the crucial effector cells in TNP-induced CHS (22, 23), and cytotoxicity is believed to be mandatory for CHS responses (31). To test whether the lack of ear swelling after i.v. immunization was due to defective generation of CTLs, draining LN cells from DC i.c. and spleen cells from DC i.v. sensitized mice were restimulated in vitro with TNP-modified splenocytes and then used in a cytotoxicity assay. Comparable TNP-specific cytotoxic activity was measured, indicating that also i.v. injection of DC efficiently generates CTLs (Fig. 1B). We then addressed the question as to why these cells did not induce skin inflammation.

CHS effector CD8+ T cells express ESL and CCR4

We hypothesized that DC i.v. in contrast to DC i.c. immunization may fail to induce skin-homing CD8+ T cells. Therefore, we first analyzed the expression of the postulated skin-homing markers ESL and CCR4 on CD8+ T cells from skin-draining LN in TNP-induced CHS. Three days after elicitation, T cells from auricular LN were compared with LN cells of unsensitized control mice. In fact, a subpopulation of CD8+ T cells in the CHS LN coexpressed ESL and CCR4 (Fig. 2), suggesting these cells were CHS effector cells.



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  		FIGURE 2. ESL and CCR4 up-regulation on CD8+ T cells in CHS. Mice were painted with TNCB on the shaved abdomen on day 0. On day 5, ear swelling was elicited by painting with TNCB. Seventy-two hours later, pooled auricular and maxillary LN cells from untreated mice (control) or from mice after elicitation of CHS were analyzed for expression of ESL and CCR4 by flow cytometry. Data are gated on CD8{alpha}+ T cells. Representative results from one of three experiments.

 
DC immunization route determines homing marker expression

To analyze a traceable Ag-specific CD8+ T cell population, we adoptively transferred the TCR transgenic P14 Thy-1.1+ T cells (26, 28) into recipient Thy-1.2+ C57BL/6 mice and immunized twice with peptide-pulsed DC i.c., i.v., or i.p. This protocol results in similar expansion rates and kinetics of T cells that peak between day 6 and 8 in blood (data not shown). Up-regulation of the skin-homing marker ESL and the gut-homing integrin {alpha}4{beta}7 on CD8+ P14 T cells was strictly correlated with the DC immunization route. ESL was up-regulated on blood T cells on day 6 after transfer only after DC i.c. (Fig. 3A), whereas {alpha}4{beta}7 was up-regulated only after i.p. injection of DC. P14 T cells were found in mesenteric LN (data not shown) on day 3 after transfer and DC immunization i.p., indicating that T cell priming had occured at this site.



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  		FIGURE 3. Differential in vivo induction of homing markers following BM-DC injection. A, P14 T cells were transferred into C57BL/6 mice and primed by injection of DC-GP33 i.v., i.c., or i.p., as described in Materials and Methods. On day 6 after transfer, blood P14 T cells were analyzed for expression of ESL, CCR4, and {alpha}4{beta}7 (filled lines) by FACS. Controls were naive P14 T cells (filled gray). Gate is on CD8+V{alpha}2+ P14 T cells. Representative results from one of three experiments. B, Blood P14 T cells were stained for CCR4 expression every 24 h from day 5 to 9 after transfer and DC injection. Numbers give the mean percentages of CCR4+ naive Thy-1.1+ P14 T cells ({blacksquare}) or after DC injection i.v. (•) or i.c. ({blacktriangleup}) (±SD) from three mice. One representative of three experiments is shown.

 
These data suggest that skin-homing T cells were generated during priming in skin-draining peripheral LN, whereas gut-homing T cells were induced in mesenteric LN draining the gastrointestinal tract and peritoneum. In contrast, i.v. immunization did not efficiently induce these adhesion molecules.

Interestingly, CCR4 was strongly up-regulated on a major subpopulation of blood T cells at the peak of expansion (day 6) independently of the immunization route (Fig. 3A). Monitoring of CCR4 expression from day 5 to 9 revealed a fast down-regulation between day 6 and 8 (Fig. 3B). The expression was transient and activation dependent and was most elevated on freshly activated T cells. In contrast, memory cells lacked CCR4 (data not shown). These data suggest that CCR4 is not an exclusive skin-homing marker and that it may be more important for skin homing of effector rather than memory T cells.

We also injected peptide-loaded, heat-killed DC in some experiments to exclude the possibility of cross-presentation of Ag from dead BM-DC by skin-resident DC. No significant expansion and skin homing of T cells were observed (data not shown).

Stable ESL expression on memory T cells

It has been reported that up-regulation of ESL on T cells in vitro can be simply activation dependent (12). However, we found that i.v. and i.c. injection of DC led to differential expression of ESL in vivo on effector T cells on day 7 (Fig. 3A) and on memory T cells on day 20 after transfer (Fig. 4), respectively. We observed similar expansion kinetics and memory T cell counts in blood (Fig. 5, C and D), indicating a comparable and maximal activation of T cells. These results show that the expression of ESL following T cell priming by DC in vivo is stable and tissue specific, but not simply activation dependent.



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  		FIGURE 4. ESL expression is stable on memory P14 T cells. Blood P14.Thy-1.1 T cells were stained for ESL expression 3 wk after adoptive transfer and DC i.v. (dashed line) or DC i.c. injection (filled line) on days 2 and 4 or days 1 and 3 after transfer, respectively. Control staining (filled gray) was done with biotin-conjugated anti-human IgG secondary Ab and streptavidin-CyChrome only. Gated on Thy-1.1+ cells. One representative of three experiments is shown.

 


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  		FIGURE 5. Induction of skin-homing P14 T cells by i.c. injection of BM-DC. Percentages of P14 T cells in blood were quantified by FACS on day 8 (effector phase (A), upper panel) and day 21 (memory phase (C), upper panel) after adoptive transfer and immunization with peptide GP33-pulsed DC i.v. or i.c., as described in Fig. 4. For induction of skin homing, TNCB was painted on ears on days 7 and 20, respectively, and ear sheets were prepared 24 h later. P14 T cells emigrated overnight were quantified by FACS after staining for CD8{alpha} and TCR V{alpha}2 or Thy-1.1 (A and C, lower panels). Gate was set on CD8{alpha}+ cells in blood and on total live emigrated cells for ear sheets, respectively. Representative dot plots and statistical analyses are shown for one of three independent experiments. Mean cell counts for effector and memory T cells from three experiments ± SD are given in B and D as percentage of P14 T cells of total CD8+ T cells.

 
Intracutaneous DC immunization induces skin-homing T cells

The skin-homing potential of CD8+ T cells after DC i.c. vs DC i.v. immunization was compared using the contact sensitizer TNCB as an inflammatory stimulus (28) to attract T cells into skin. At the peak of expansion on day 7, TNCB was painted on ears. Blood levels of P14 T cells were measured 24 h later (Fig. 5, A and B, upper panels). Ear skin sheets were prepared and incubated overnight. The emigrated T cells were quantified by flow cytometry (Fig. 5, A and B, lower panels). Cumulative data for cell counts from three experiments are shown in Fig. 5, B and D. In i.c. immunized mice, we found that 10–30% of emigrated cells were P14 T cells, whereas in ear sheets from control mice (transfer without DC immunization) no TCR transgenic P14 T cells, and in i.v. immunized mice 0.1–5% were detected. Interestingly, we could also detect cohoming of endogenous nontransgenic CD8+ T cells, which exerted GP33-specific cytotoxicity (data not shown). This demonstrates that the induction of skin-homing T cells following i.c. DC priming is not restricted to the TCR transgenic P14 system, but seems to be of general relevance. The total number of emigrated cells was ~0.5–2 x 105 per ear. At the peak of expansion at ~day 7, P14 T cells represented up to 55% of total CD8+ T cells. Three days later, a dramatic contraction of the effector T cell pool was observed, with levels below 15% (data not shown), followed by an ongoing decline over the next weeks. Three weeks after P14 transfer and DC injection, Thy-1.1+ memory T cells were quantified in blood by flow cytometry to test the stability of the skin-homing phenotype. We observed similar levels with both immunization protocols (Fig. 5D, upper panels). However, in our skin-homing test, hardly any CD8+ memory T cells entered the site of inflammation after i.v. immunization. In contrast, in i.c. immunized mice, up to 7% of total live emigrated cells were P14 T cells (Fig. 5, C and D, lower panels).

Skin-homing T cells elicit peptide-dependent skin inflammation

The functional relevance of these differences in skin-homing capacity was tested by establishing a peptide-dependent CHS protocol. TNCB was painted on ears of mice 8 days after P14 T cell transfer and i.c. or i.v. DC immunization, followed by epicutaneous application of the antigenic peptide GP33 to attract T cells into skin. Strong ear swelling after 24 h was detected if the mice had been immunized by peptide-loaded DC i.c., but not after i.v. immunization (Fig. 6). Control mice were painted with TNCB and GP33 and then injected i.c. with peptide-free control DC.



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  		FIGURE 6. Skin-homing P14 T cells elicit a peptide-dependent ear-swelling response. On day 8 after adoptive P14 transfer and DC immunization i.v or i.c., TNCB and peptide GP33 were painted on ear skin. Mean increase in ear thickness after 24 h is shown. Data from three mice ± SD. Control mice (Ctrl.) received DC without GP33. Representative results from one of three experiments are shown.

 
Tissue-derived DC induce differential homing marker expression

To address the role of the APC in instructing CD8+ T cells for tissue-selective homing, we performed in vitro priming experiments of P14 T cells with DC isolated from different tissues or with BM-DC. Despite similar T cell activation, as detected by proliferation assay on day 4 (Fig. 7), we observed differential expression of homing markers on day 6 (Fig. 8). Only small differences in T cell proliferation were detected when graded numbers of the different DC populations were used in the assays (data not shown). Most interestingly, LC were usually slightly less efficient in these T cell stimulation assays (data not shown), but were always the most efficient inducers of ESL. This underscores their potent intrinsic capacity to induce this homing marker. Peripheral skin-draining LN-DC (pDC) induced ESL less efficiently. ESL on T cells primed with mesenteric, gastrointestinal tract-draining LN-DC (mDC) were present, but much lower compared with LC-primed T cells, whereas BM-DC did not efficiently up-regulate ESL.



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  		FIGURE 7. Similar T cell activation by DC from different tissues. Proliferation of P14 cells was measured by [3H]thymidine incorporation on day 4 after in vitro priming with peptide GP33-loaded BM-DC or DC isolated from mesenteric (mDC) or peripheral LN (pDC) or skin (LC). Data represent mean values from triplicate measurements ± SD. Representative results from one of two experiments.

 


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  		FIGURE 8. Differential in vitro induction of homing markers with DC from different tissues. P14 T cells were analyzed for expression of ESL, CCR4, and {alpha}4{beta}7 on day 6 of in vitro culture after priming with GP33-loaded BM-DC, mDC, pDC, or LC, as described in Fig. 7. As control (filled gray), T cells from LC-priming cultures were stained with FITC-conjugated anti-human IgG only (control for ESL staining) or isotype control Abs. Data are gated on CD8{alpha}+ cells with activated phenotype in forward/side scatter. Representative results from one of three to eight experiments.

 
CCR4 was most elevated in priming experiments with pDC and LC, but to a lesser extent also present on P14 T cells primed by BM-DC and mDC. As suggested by the in vivo results (Fig. 3A), CCR4 expression does not seem to have a strong tissue-specific correlation. Expression of the gut-homing integrin {alpha}4{beta}7 on P14 T cells was most elevated in in vitro priming cultures with mDC (Fig. 8) or with DC from Peyer’s patches (data not shown), whereas T cells in cultures with LC and pDC were negative. Interestingly, BM-DC also significantly up-regulated {alpha}4{beta}7 expression in vitro. These results suggest that mDC promote a gut-homing T cell phenotype, whereas DC derived from other tissues fail to do so (Fig. 8).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results reveal the induction of three different CD8+ T cell-homing subsets in dependence of the DC injection route: DC given i.c. primed T cells that up-regulated ESL and efficiently homed to inflamed skin, while, in contrast, DC injected i.v. failed to do so. Most importantly, only these skin-homing T cells efficiently elicited a skin-specific immune response, i.e., CHS to epicutaneously applied hapten or peptide. Furthermore, DC given i.p. induced a gut-homing T cell phenotype by up-regulation of integrin {alpha}4{beta}7, but not ESL. It has to be considered that i.c. injected DC activate T cells in skin-draining LN, i.v. mostly in the spleen, whereas i.p. injected DC meet T cells most probably in mesenteric/peritoneal LN (24, 32, 33).

Our finding that the same BM-DC differentially induce homing markers in vivo points out an important role for the specific tissue microenvironment that may directly provide trafficking information to T cells. This is supported by recent studies demonstrating a role for the local microenvironment in the differential regulation of T cell migration and effector functions (34, 35). Campbell and Butcher (34) demonstrated the rapid up-regulation of skin- vs gut-homing markers on CD4+ T cells in the corresponding LN after systemic peptide injection. Our findings suggest that this is also true for CD8+ T cells after in vivo priming by DC injection via different routes, and that the skin-homing potential remains stable in the memory phase without Ag contact at the effector site.

As we know about the multiple functions of DC to shape the immune response, we now also propose a role for the priming DC in the instruction of T cells for tissue-specific homing. Due to its functional plasticity (36), the injected semimature BM-DC may, after acquisition of characteristics of a tissue-specific DC, confer trafficking information to T cells. Thus, LC as specialized skin-resident DC are most effective in up-regulating ESL on T cells in vitro, compared with DC from other tissues, whereas, in turn, mDC induced the gut-homing integrin {alpha}4{beta}7. Similarly, recent data (29) revealed that mDC, but not pDC, induce {alpha}4{beta}7 on T cells activated in vitro by anti-CD3 Ab. In similar experiments, Mora et al. (37) found that CD8+ P14 T cells primed in vitro with Peyer’s patch DC expressed {alpha}4{beta}7 and CCR9. Upon in vivo transfer, these T cells preferentially homed to small intestine. The important role for CCR9 in gut-specific homing of CD8+ T cells was also demonstrated by others (8, 38). We have now extended these findings by demonstrating an Ag-specific in vivo induction of {alpha}4{beta}7 on CD8+ P14 T cells after DC i.p., but not i.v. or i.c. injection. These data show that DC can up-regulate this homing marker without Ag-specific contact, and also raise the possibility that in our DC injection experiments, endogenous DC may influence the generation of T cell-homing subsets in trans.

Interestingly, we observed much higher levels of ESL on T cells in vitro than in vivo, and also priming with BM-DC and mDC induced ESL to some extent. This up-regulation in vitro is most likely influenced by the absence of negative regulatory factors such as IL-4 and an accumulation of inflammatory cytokines such as IL-12, which has been shown to be a potent inducer of ESL (12, 39). However, levels induced by LC were 10- to 100-fold higher despite similar T cell proliferation. These results suggest a tissue-selective instruction of T cells directly by the priming DC, and for the first time provide evidence that LC possess the intrinsic ability to induce a skin-homing phenotype on CD8+ T cells.

The specialization for skin vs gut homing observed in this study is restricted to the differential expression of the adhesion molecules ESL and integrin {alpha}4{beta}7, but not CCR4 on CD8+ T cells. Recently, differential expression of ESL/cutaneous lymphocyte Ag on human T cells specific for skin tropic HSV-2, but not the nonskin-tropic herpes viruses, has been reported (40). These data underline the crucial role of ESL in a skin-selective specialization of CD8+ T cells.

A tissue-specific chemokine pattern has been reported to match a specialized chemokine receptor expression of effector T cells trafficking to skin vs small intestine (1, 2, 3). However, in our experiments, CCR4 was up-regulated independently of the DC immunization route. We therefore suggest that CCR4 is an important, but not an exclusive marker of skin-homing CD8+ T cells. CCR4-deficient T cells have been shown to retain their skin-homing capacity (19). Furthermore, an involvement of this chemokine receptor in T cell homing to the lung has recently been reported (41, 42, 43, 44, 45). The observed down-regulation of CCR4 in the late effector phase and its absence on memory T cells as judged by flow cytometry (data not shown) may point to a more important role in orchestrating the early immune response as a receptor for DC-derived CCL22 (46) rather than in skin homing of memory CD8+ T cells. However, it is also possible that CCR4 is reinduced on memory CD8+ T cells, e.g., when they enter inflamed skin. Alternatively, CCR4-negative memory cells may use other chemokine receptors such as CCR6 (47) and CCR10 (1, 18, 19, 48, 49) for this purpose.

In summary, we have shown that the T cell-homing phenotype is influenced by the tissue microenvironment and also by the tissue-specific Ag-presenting DC. Future work should identify the nature of the factors that define the influence of the tissue microenvironment and of the inductive signals that target T cells to different peripheral tissues. Finally, these results point out a crucial role for the application route of in vitro generated DC in immunotherapies. In this context, the recent observation that tumor surveillance by effector/memory CD8+ T cells is governed by the DC immunization route is of interest (50). Interference with T cell trafficking will lead to the development of more specific immunotherapies for organ-restricted autoimmunity, cancer, and allergies as encouraged by several recent in vivo studies in mice (18, 19, 51, 52) and humans (53, 54).


   Acknowledgments
 
We thank Dr. Hanspeter Pircher for kindly providing P14 Thy-1.1 mice and for critical reading of the manuscript, and also Dr. Bernhard Homey for helpful discussions. We also thank Annalisa Lembo for proofreading of the manuscript.


   Footnotes
 
1 This work was supported by German Ministry for Research and Education Grant BMBF FKZ GC 0001. Back

2 Address correspondence and reprint requests to Dr. Stefan Martin, Clinical Research Group Allergology, Department of Dermatology, University of Freiburg, Hauptstrasse 7, D-79104 Freiburg, Germany. E-mail address: martin{at}haut.ukl.uni-freiburg.de Back

3 Abbreviations used in this paper: ESL, E-selectin ligand; BM-DC, bone marrow-derived DC; CHS, contact hypersensitivity; DC, dendritic cell; DC-TNP, TNBS-modified DC; i.c., intracutaneous; LC, Langerhans cell; LN, lymph node; mDC, mesenteric LN-DC; pDC, peripheral LN-DC; TNBS, trinitrobenzene sulfonic acid; TNCB, trinitrochlorobenzene; TNP, trinitrophenyl. Back

Received for publication July 8, 2003. Accepted for publication October 29, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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