Tissue-specific homing of effector and memory T cells to skin and small intestine requires the imprinting of specific combinations of adhesion molecules and chemokine receptors by dendritic cells in the draining lymph nodes. In this study, we demonstrate that CD8+ T cells activated by Ag-pulsed bone marrow-derived dendritic cells were induced to express the small intestine homing receptors α4β7 integrin and chemokine receptor CCR9 in coculture with small intestinal epithelial cells. In contrast, in coculture with dermal fibroblasts the skin-homing receptor E-selectin ligand was induced. Interestingly, the imprinting of gut homing receptors on anti-CD3/anti-CD28 stimulated T cells was induced by soluble factors produced by small intestinal epithelial cells. Retinoic acid was identified as a crucial factor. These findings show that peripheral tissue cells directly produce homing receptor imprinting factors and suggest that dendritic cells can acquire their imprinting potential already in the peripheral tissue of origin.
Upon activation by dendritic cells (DC)3 in skin-draining lymph nodes, T cells up-regulate the skin-homing receptors E- (E-lig) and P-selectin ligands as well as the chemokine receptors CCR4 and CCR10 (1, 2, 3, 4, 5, 6). Activation by DC from mesenteric lymph nodes (MLN) and Peyer’s patches (PP) induces the small intestine homing receptors α4β7 integrin and CCR9 (4, 5, 6, 7, 8, 9). The role of lymph node-resident DC vs DC that immigrated from peripheral tissues has not yet been addressed. DC from MLN and PP express retinal dehydrogenases (RALDH). They produce the vitamin A metabolite retinoic acid (RA) that induces up-regulation of α4β7 and CCR9 (10). Lamina propria-derived CD103+ DC are responsible for the imprinting of gut homing receptors on T cells in PP and MLN (11, 12). Epidermal Langerhans cells were most efficient in the induction of skin-homing receptors on CD8+ T cells in vitro as compared with DC from skin-draining lymph nodes (4, 6). These findings suggest that the DC immigrating into draining lymph nodes from peripheral tissues, rather than the lymph node resident DC, are responsible for homing receptor imprinting. In support of this view, two independent homing phenotypes can be induced on T cells in the same lymph node by DC that immigrated from different peripheral sites (13). Moreover, the majority of DC in skin draining lymph nodes consists of immigrants, i.e., Langerhans cells and dermal DC (14).
For this work, we have studied the role of the peripheral tissue microenvironment in the imprinting of skin and small intestine homing receptors by DC in coculture systems. CD8+ P14 T cells (15, 16), upon activation with Ag-pulsed bone marrow-derived DC (BM-DC), up-regulated the skin-homing receptor E-lig in coculture with dermal fibroblasts or the gut homing receptors CCR9 and α4β7 in coculture with small intestinal epithelial cells (SIEC). Soluble factors such as RA induced the imprinting of the gut homing phenotype, whereas cell-cell contact with dermal fibroblasts was important for the induction of E-lig on T cells. Our findings suggest that peripheral tissue stromal and epithelial cells produce factors that can directly induce homing receptors on T cells. These factors may license DC to also produce such factors and/or allow for the DC to shuttle these imprinting factors to the naive T cells in the regional draining lymph nodes.
Materials and Methods
Media and chemicals
The lymphocytic choriomeningitis virus peptide GP33 (KAVYNFATM) was from Hermann GbR. All-trans RA was from Sigma-Aldrich. 9-cis-RA and RA receptor (RAR) antagonists (RAR and the retinoid X receptor antagonist RXR26, respectively) were provided by B. Homey (Heinrich-Heine University, Dusseldorf, Germany).
Ab and flow cytometry
All Ab were from BD Biosciences unless stated otherwise and used as FITC, PE, or biotin conjugates. Biotin-conjugated Ab were revealed with streptavidin-PE-Cy5. The Ab used are the following: anti-CD16/CD32 (FcγR II/III) (clone 2.4G2); anti-CD90 (Thy1.1) (clone HIS51); anti-CD8α (clone 53-6.7); anti-IAb (clone AF6-120.1); anti-CD11c (clone HL3); anti-CD103 (clone M290); anti-α4β7 (DATK32); E-selectin/human IgG-Fc-chimera (R&D Systems); anti-human-IgG-FITC (DakoCytomation); rat anti-CCR9 (18) provided by R. Förster (Medizinische Hochschule Hannover, Hannover, Germany); mouse anti-rat IgG (H+L) (Jackson ImmunoResearch Laboratories). FACS staining was done as described (4, 6). Data were acquired and analyzed on a FACScan instrument using BD CellQuest Pro software (BD Biosciences). Anti-pan TGF-β was purchased from R&D Systems.
Isolation and preparation of cells
BM-DC were prepared as described (4) but without IL-4. m-ICcl2 cells were differentiated for 6 days on collagen in 24-well plates and cultured as described previously (17). Primary SIEC were isolated as described (19). Dermal fibroblasts were isolated from the skin of 2-day-old mice with 5 U/ml dispase (Invitrogen) for 1 h at 37°C. The dermis was separated from the epidermis, washed in PBS, and incubated in collagenase I (500 U/ml) (Worthington Biochemical) for 45 min at 37°C. Cells were singularized, washed in PBS, and resuspended in medium. Medium was changed after 24 h and cells were cultured until confluent. P14 spleen cells were prepared as described (4, 6). Total splenocytes were used (referred to as P14 cells). CD8α (clone Ly-2) MicroBead (Miltenyi Biotec)-purified CD8+ P14 T cells were used in some experiments to exclude bystander cell effects.
In vitro priming of T cells
BM-DC were harvested on days 7–9 and pulsed with 1 μM GP33 peptide (BM-DC-GP33) (4, 6) or used unpulsed. BM-DC-GP33 (1 × 105/well) were incubated with P14 splenocytes (2 × 105/well) with or without m-ICc12 in 24-well plates (Greiner Bio One) for 4 days in a volume of 2 ml of RP-10/ m-ICc12 medium (1:1; v/v). RAR antagonists (8 μM RAR or 3 μM RXR) were added to these cocultures from the beginning. Cocultures using BM-DC-GP33 (1 × 104/well) were incubated with 2 × 104 splenocytes and 1 × 104 dermal fibroblasts in round-bottom, 96-well plates (Corning Life Science) for 6 days in a total volume of 200 μl of RP-10. Supernatants were collected at day 4 of coculture and stored at −2°C. Cells were analyzed by flow cytometry. Cell culture inserts (1 μm pore diameter; BD Falcon) were used in Transwell experiments. m-ICc12 or dermal fibroblasts were cultured at the bottom of 24-well plates, andDC-GP33 and P14 splenocytes were cocultured in the cell culture inserts. Cell numbers were as described above.
Ab activated splenocytes (soluble anti-CD3ε (clone 145-2C11; 3 μg/ml) and anti-CD28 (clone 37.51; 1.5 μg/ml)) (BD Biosciences) were primed by the addition of all-trans or 9-cis-RA (10 nM and 7.5 μM, respectively).
Isolation of RNA and real-time PCR
Total RNA was isolated from BM-DC, m-ICc12, and BM-DC cocultured with m-ICc12 for 4 days using RNeasy Mini Kit 50 (Qiagen). DC were reisolated with CD11c MicroBeads using an autoMACS following the manufacturer’s instructions or had been separated from m-ICc12 by Transwell culture inserts. cDNA was prepared from 1 μg of template RNA using Qiagen Omniscript reverse transcription kit (Qiagen). RALDH-1-, RALDH-2-, and RALDH-3-specific primers and primers for the housekeeping gene 18S RNA were designed using the Roche Universal Probe Library and were purchased from the TIB MOLBIOL Synthesis Laboratory. The corresponding probes were obtained by Molecular Biochemicals. Real-time PCR was performed on a LightCycler 1.5 (Roche Molecular Biochemicals) using the LightCycler TaqMan Master kit (Roche Molecular Biochemicals). The reaction mixture had a total volume of 20 μl and contained 1 μl of cDNA, 4 μl of Master Mix (TaqMan Master), 1 μl of each primer (forward and reverse, end concentration 0.33 μM, respectively), 1 μl of probe in a final concentration of 0.1 μM, and 12 μl of RNase free water. A negative control was always included and consisted of the same ingredients without any cDNA. The expression of transcripts was related to 18S RNA. Cycle threshold (CT) values for 18S were subtracted from CT values of RALDH 1, 2 and 3, respectively (ΔCT) and normalized to values to ΔCT of DC alone (ΔΔCT). Fold increase was calculated by 2−(ΔΔCT).
Statistics and statistical analysis
Statistical analysis was conducted using Student’s t test or ANOVA (nonparametric) for the PCR data. Differences were statistically significant at p < 0.05.
Results and Discussion
Small intestinal epithelial cells induce α4β7 and CCR9 in T cell-BM-DC cocultures by soluble factors
It is conceivable that the Ag-loaded DC immigrating from peripheral tissues confer the information about their tissue of origin to T cells in local draining lymph nodes.
To analyze the role of the peripheral tissue microenvironment in homing receptor imprinting, TCR-transgenic P14 splenocytes (P14 cells), used as a source for GP33 specific CD8+ T cells, were cocultured with BM-DC-GP33 in the presence or absence of the SIEC line m-ICC12. Cells were analyzed by flow cytometry on day 4 of coculture (Fig. 1,A). Almost all CD8+ T cells expressed both α4β7 integrin and CCR9 in the presence of m-ICc12 when compared with controls. A role for soluble factors was shown in Transwell experiments separating cocultures of P14 splenocytes and BM-DC-GP33 from m-ICc12 cells. The extent of homing receptor up-regulation was comparable to that in the cultures without Transwells (Fig. 1,A, left column). E-lig was not detected (Fig. 1,A, right column). Similar results were obtained with freshly isolated primary SIEC (Fig. 1 B) or when using purified CD8+ P14 T cells instead of splenocytes (data not shown).
Supernatants from cocultures were used as conditioned medium (CM). P14 cells activated by anti-CD3ε and anti-CD28 and incubated with CM from cocultures that contained either m-ICc12 or primary SIEC significantly up-regulated α4β7 integrin and CCR9 compared with CM from P14 cells only (Fig. 1,C). Interestingly, even CM from m-ICc12 cells alone and, to a lesser extent, from primary SIEC alone were able to induce gut homing receptor expression on CD8+ T cells (Fig. 1 C).
These findings indicate that SIEC release soluble factors that can induce small intestine homing receptors on T cells. It remains to be determined whether the difference between primary SIEC alone and SIEC plus DC (Fig. 1 C) is indicative of an imprinting of DC by SIEC.
Retinoic acid is involved in homing receptor imprinting in SIEC cocultures
Both isoforms of the vitamin A metabolite RA, all-trans and 9-cis-RA, efficiently induced α4β7 integrin and CCR9 on P14 T cells activated with anti-CD3 and anti-CD28 (data not shown) as previously reported (10). All-trans RA binds to RAR only, whereas 9-cis-RA binds to RAR and RXR. Both receptors mainly function as heterodimeric, ligand-inducible transcription factors (20).
To test a potential role of RA, mICc12 were cocultured with P14 cells and BM-DC-GP33 in the presence or absence of RAR antagonists. The up-regulation of CCR9 was completely blocked in the cocultures by both RAR and RXR antagonists whereas the expression level of α4β7 was only reduced (Fig. 2 A). In contrast to the antagonists used in this study, the inhibition of RALDH by citral or that of RAR by the antagonist LE135 efficiently suppresses RA- or MLN-DC-induced α4β7 up-regulation (10). It has been reported that TGF-β is a potent regulator of α4β7 (21). Neutralization of TGF-β in the presence or absence of RAR and RXR antagonists had no effect on the expression of α4β7 in our cocultures (supplementary Fig. S1).4
These results suggest that SIEC play an important role in the induction of gut homing receptors on T cells by the production of imprinting factors such as RA. RA production by human intestinal epithelium has been reported (22) and seems to be crucial for gut homing receptor induction.
BM-DC show a gut-associated phenotype after coculture with m-ICc12
CD103+ DC isolated from MLN were the most potent in inducing a gut-homing phenotype on T cells compared with CD103− MLN DC (11, 12). They express RALDH and can produce the imprinting factor RA (10). Furthermore, almost all lamina propria DC, but only a subpopulation of DC from MLN, express CD103 (αE chain of the integrin αEβ7). These CD103+ lamina propria DC produce RA (23). To evaluate whether BM-DC adopt a gut DC phenotype in the presence of SIEC in vitro, BM-DC were stained for CD11c, I-Ab, and CD103 before and after coculture with m-ICc12. At day 4 of coculture, only a slight but reproducible up-regulation of CD103 could be detected compared with BM-DC cultured alone (Fig. 2,C). We also observed the induction of RALDH1 in DC after coculture with mICc12 (Fig. 2 B). These data show a bias toward a gut-associated phenotype of the BM-DC induced by SIEC. This implies that DC may acquire the ability to produce RA in the tissue microenvironment of the lamina propria. Positive feedback loops for the production of RA have been defined (24, 25). Thus, interactions of DC with peripheral tissue cells may induce a differentiation program resulting in the tissue-specific phenotype of the DC (4, 5, 6, 7, 8, 9). It is also possible that DC shuttle soluble factors produced by peripheral tissue cells, in this case SIEC, to the lymph nodes as suggested for RA (22).
BM-DC induce E-selectin ligands in the presence of dermal fibroblasts
To test whether dermal fibroblasts from mouse skin have a similar impact on the imprinting of skin-homing receptors on T cells, cocultures with dermal fibroblasts, BM-DC-GP33, and P14 cells were set up and analyzed after 6 days. The skin-homing receptor E-lig was induced in these cocultures, in contrast to cultures without dermal fibroblasts (Fig. 3 left column). Induction of skin-homing receptors was not observed when cell culture inserts were used to separate DC and P14 cells from the fibroblasts (Fig. 3, left bottom panel). These results suggest that cell-cell contact is necessary for the induction of skin-homing receptors on T cells, although IL-12 can induce E-lig in vitro (26). Most likely this cell interaction takes place between DC and skin cells, as naive T cells do not have access to dermal fibroblasts before they are primed for skin homing in the lymph node in vivo. We did not see any effects on E-lig expression by the vitamin D3 metabolite calcitriol (data not shown), which helps to induce CCR10 expression on human T cells (27). CCR4 was up-regulated in an activation-dependent manner (data not shown) (4). Similar results were obtained in all of these settings when purified CD8 T cells were used instead of splenocytes, excluding the bystander effects of non-T cells. Expression of the gut homing receptors α4β7 integrin or CCR9 were not observed in these experiments (Fig. 3, right column).
Our findings demonstrate an instructive role for peripheral tissue cells in the imprinting of skin and small intestine homing receptors on T cells. It has been reported that stromal cells can influence DC differentiation (28, 29, 30) (reviewed in Ref. 31). For Langerhans cells that differentiate in the skin from monocyte precursors (32, 33), a role for both contact-dependent mechanisms and soluble factors in the acquisition of tissue-specific characteristics has been described (34, 35). In summary, we suggest that the peripheral tissue microenvironment conditions DC to shuttle topographical information to the lymph node in a bimodal fashion. As reported, DC may themselves be induced to produce factors for homing receptor imprinting (10) and, in addition, may transport factors produced by peripheral tissue cells to the draining lymph nodes. This would license the DC to induce tissue-specific homing receptors and thereby pass the information about their tissue of origin and the site of Ag entry on to the T cells.
We are grateful to Dr. Alain Vandewalle, INSERM U773, Paris, France, for providing the m-ICl2 cell line and to Cindy Reinhold for technical assistance.
The authors have no financial conflict of interest.
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.
This work was supported by Deutsche Forschungsgemeinschaft Grants MA1567/8-1 (to S.F.M.) and HO2236/5-2 (to M.H.), Swedish Research Council Grant K2003-31P-14792 (to M.H.), and Thyssen Foundation Grant AZ 10.05.2.173 (to M.H.).
Abbreviations used in this paper: DC, dendritic cell; BM-DC, bone marrow-derived DC; CM, conditioned medium; CT, cycle threshold; E-lig, E-selectin ligand; MLN, mesenteric lymph nodes; PP, Peyer’s patch; RA, retinoic acid; RALDH, retinal dehydrogenase; RAR, RA receptor; RXR, retinoid X receptor; SIEC, small intestinal epithelial cell.
The online version of this article contains supplemental material.