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Keywords:

  • Homing;
  • Trafficking pattern;
  • Chemokine receptor;
  • Adhesion molecule;
  • Memory T cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Tissue-selective homing is established during naive T cell activation by the tissue microenvironment and tissue-specific dendritic cells (DC). The factors driving induction and maintenance of T cell homing patterns are still largely unknown. Here we show that soluble factors produced during the interaction of T cells with CD11c+ DC isolated from skin- or small intestine-associated tissues differentially modulate expression of the corresponding tissue-selective homing receptors (E-selectin ligands and α4β7 integrin/CCR9, respectively) on murine CD8+ T cells. Injection of tissue-specific DC via different routes induces T cells with homing receptors characteristic of the corresponding local tissue microenvironment, independent of the origin of the DC. These data indicate an important role for signals delivered in trans. Moreover, DC can reprogram the homing receptor expression on T cells previously polarized in vitro for homing to skin or small intestine. Importantly, skin-homing memory T cells stimulated directly ex vivo can also be reprogrammed by intestinal DC to a gut-homing phenotype. Our results show that tissue-selective homing receptor expression on effector and memory T cells is governed by inductive as well as suppressive signals from both DC and tissue microenvironments.

Abbreviations:
BM-DC:

Bone marrow-derived DC

CCR:

Chemokine receptor

CM:

Conditioned media

E-lig:

E-selectin ligands

FLT3-L:

FMS-like tyrosine kinase 3 ligand

i.c.:

Intracutaneous

LC:

Langerhans cell

MLN:

Mesenteric lymph nodes

M-CM:

Mesenteric DC conditioned media

M-DC:

MLN DC

P-CM:

P-DC conditioned media

P-DC:

Peripheral lymph node DC

PLN:

Peripheral lymph nodes

PP:

Peyer's patch

PP-DC:

Peyer's patch DC

S-CM:

S-DC-conditioned media

S-DC:

Splenic DC

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

T cell homing to different tissues is highly heterogeneous and is determined by the combination of adhesion molecules and chemotactic receptors on the cell surfaces 14. Best characterized among these homing receptors are α4β7 integrin 5, 6 and the chemokine receptor (CCR)9 79 for lymphocyte homing to the small intestinal lamina propria and the mucosal epithelium. In contrast, T cell trafficking to inflamed skin is mediated by E-selectin ligands (E-lig) and P-selectin ligands (P-lig) 1012 as well as chemokine receptors such as CCR4 and CCR10 1315. Expression of the correct access code of homing receptors by T cells has been shown to be crucial for efficient tissue-specific immune responses as well as autoimmune diseases or allergy 14, and interfering with T cell homing proves to be a promising therapeutic strategy 1618. It has been established that different T cell trafficking patterns are rapidly imprinted after priming in different secondary lymphoid tissue microenvironments 1924. Thus, up-regulation of P-lig or α4β7 and acquisition of CCL25 responsiveness of CD4+ T cells following systemic antigen injection was dependent on the site of priming 19. Similar results were found for E-lig and α4β7 expression on CD8+ T cells primed with antigen-pulsed bone marrow-derived DC (BM-DC) via different routes 22, and the in vivo relevance of the immunization route has been shown for contact hypersensitivity 22 and melanoma 25 in mouse models. Moreover, we and others have demonstrated an education of naive T cells by tissue-specific DC in vitro2124. Little is known about the factors that are involved in the polarization of T cell homing, although important roles for cytokines have been suggested from in vitro studies 2629. However, the in vivo relevance of these experiments, which were done in the absence of tissue-specific DC, remains unclear. Moreover, the role of the different cellular constituents of the tissue microenvironment that drive T cell homing (i.e. stromal cells, DC and matrix components) and the molecular mechanisms still have to be defined in detail.

In the present study, we therefore investigated whether homing receptor polarization is mediated by soluble factors produced by tissue-specific DC upon interaction with T cells. We observed that conditioned media (CM) generated by co-culture of DC isolated from different tissues with CD8+ P14 T cells 30, 31 was able to polarize the homing patterns of anti-CD3-stimulated T cells. In contrast, the same DC failed to induce their corresponding T cell homing phenotype in an alternative tissue in vivo following injection via different routes. These data indicate that signals from tissue-specific DC can be overridden or modified by the local microenvironment. Finally, we tested the ability of DC to reprogram established homing patterns of effector and memory T cells polarized for homing to the skin or small intestine in vitro and in vivo, respectively. We found a DC-driven switch of homing receptor expression corresponding to the tissue origin of the DC used for stimulation.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Regulation of T cell homing receptor patterns by tissue-specific DC

The tissue homing phenotype of T cells is established by tissue-specific DC during T cell priming in vitro 2124. In contrast to our previous study 22, here we used tissue-specific DC from mice injected with B16 melanoma cells expressing FMS-like tyrosine kinase 3 ligand (FLT3-L) 32 for in vitro priming of CD8+ P14 T cells (Fig. 1A). Similar to our previous results 22, we found a clear polarization towards E-lig expression using Langerhans cells (LC) or peripheral lymph node DC (P-DC) and towards α4β7/CCR9 expression with mesenteric lymph node DC (M-DC) and Peyer's patch DC (PP-DC) on day 4. Interestingly, E-lig levels on T cells were lower with M-DC from FLT3-L-treated mice as compared to DC isolated from untreated mice 22, while polarization to a skin-homing phenotype with skin-associated DC was more pronounced.

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Figure 1. DC or soluble factors regulate expression of tissue-specific homing receptors. (A) Purified CD8+ P14 T cells were activated in vitro with CD11c+ DC isolated from the indicated tissues of C57BL/6 mice treated with B16 melanoma cells expressing FLT3-L. Expression of E-lig, α4β7 integrin and CCR9 was determined by flow cytometry on day 4. (B) CD8+ P14 T cells were activated in vitro with anti-CD3 mAb in the absence or presence of CM from 48 h co-cultures of CD8+ P14 T cells with DC isolated from PLN (P-CM), MLN (M-CM) or spleen (S-CM). On day 4, expression of E-lig or α4β7 integrin and CCR9 was measured by flow cytometry. CCR9 measurements in (A) are from a different experiment. Numbers give the percentage of the CD8+/homing receptor-positive cells. Data are representative of eight (A) and three (B) independent experiments. Staining controls were as described in Materials and methods.

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Soluble factors mediate DC-driven homing receptor polarization on T cells

In order to determine whether soluble factors are involved, naive CD8+ P14 T cells were primed in vitro with anti-CD3 mAb in the absence or presence of CM, i.e. supernatants from co-cultures of CD11c+ DC from P-DC (P-CM), M-DC (M-CM) or splenic DC (S-DC, S-CM) with CD8+ P14 T cells. Homing receptor expression was determined by flow cytometry on day 4 (Fig. 1B and Table 1). E-lig were not efficiently up-regulated on CD8+ P14 T cells by anti-CD3 in the absence of CM, while α4β7 was strongly expressed. Moderate CCR9 levels were detected. In contrast, E-lig were strongly induced on T cells primed with anti-CD3 mAb in the presence of P-CM, while α4β7 and CCR9 levels were down-regulated in comparison to the medium control and M-CM. M-CM promoted a gut-homing phenotype (Fig. 1B). These results suggest a suppression of α4β7/CCR9 and induction of E-lig by P-CM. M-CM also induced E-lig, however less efficiently. In the presence of S-CM, we observed intermediate E-lig and low α4β7/CCR9 levels, indicating suppressive effects on the gut-homing receptors. Similar results were obtained when phorbol 12-myristate 13-acetate (PMA)/ionomycin was used for T cell stimulation (data not shown).

Table 1. Homing receptor polarization by soluble factorsa)
E-ligα4β7CCR9
  1. a) Experiments were performed as described in Fig. 1B. Numbers indicate the mean percentage of homing receptor-positive, Thy1.1+ cells ± SD from three independent experiments. Differences in the mean values among groups are statistically significant at p<0.001 for E-lig and α4β7 and p=0.014 for CCR9.

Medium3.2±3.483.4±9.735.9±14.3
P-CM51.6±15.516.7±12.84.7±2.4
M-CM20.3±9.364.5±6.962.7±27.9
S-CM32.9±2.522.0±17.016.0±13.1

Up-regulation of α4β7 in late skin DC/P14 T cell co-cultures

The gut-homing integrin α4β7 is up-regulated in vitro by M-DC or PP-DC around day 4 of culture 2224, whereas levels stay low with skin-associated P-DC 22, 23. Monitoring of α4β7 expression in priming cultures beyond day 6 revealed that P14 T cells activated with P-DC or LC also became α4β7+ as observed for co-culture with M-DC and PP-DC (Fig. 2A). Interestingly, α4β7 up-regulation was less pronounced in cultures primed by LC. This further underlines the strongest skin-specific polarization of T cells by LC 22. We also observed such a general up-regulation of α4β7 upon priming of CD4+ DO11.10 cells with P-DC, M-DC or PP-DC from untreated BALB/c mice (Fig. 2B) or CD11c+ DC from C57BL/6 × BALB/c F1 mice treated with FLT3-L expressing melanoma cells 32 (data not shown). Tissue-specific differences could still be observed on day 5, whereas the expression of α4β7 was similarly induced in all cultures on day 7. These results may be explained by an active suppression of α4β7 by skin-associated DC, which is absent following DC apoptosis around day 4 24.

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Figure 2. Late up-regulation of α4β7. (A) CD8+ P14 T cells were primed with the indicated tissue-specific DC as described in Fig. 1A. Flow cytometry was performed on day 7 of culture. (B) CD4+ DO11.10 T cells were activated in vitro with DC isolated from the indicated tissues of untreated BALB/c mice. On days 5 and 7 of antigen-specific activation, expression of α4β7 was measured by flow cytometry. Numbers give the percentage of CD8+/α4β7+ (A) or CD4+/α4β7+ (B) cells. Data are representative of five (A) and two (B) independent experiments. Isotype control was as described in Materials and methods.

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Dominance of tissue microenvironment over DC signals

The contribution of tissue microenvironments at the site of T cell priming, which beside the antigen-presenting DC might play a role in the induction of homing patterns, had not yet been investigated. We were interested in whether tissue-derived DC retain the ability to confer their specific homing information to T cells when placed in a ‘foreign’ tissue microenvironment. Therefore, we injected GP33-loaded P-DC or M-DC i.v., intracutaneously (i.c.) or i.p. and measured the homing receptor polarization on adoptively transferred P14 T cells after isolation from the corresponding secondary lymphoid tissues (Fig. 3, Table 2) where priming occurred. Analysis of T cells on day 3.5 revealed that up-regulation of E-lig was restricted to skin-draining peripheral lymph nodes (PLN) after i.c. injection of P-DC or M-DC, with greater efficiency upon P-DC injection (Fig. 3A), while high α4β7 expression was only observed in mesenteric lymph nodes (MLN) upon i.p. injection (Fig. 3B), irrespective of the tissue origin of the injected DC. Interestingly, efficient up-regulation of neither α4β7 nor E-lig was observed in the spleen, where most of the injected DC will be found after i.v. injection. These data demonstrate a general functional dominance of the secondary lymphoid tissue microenvironment over DC signals for the programming of homing phenotypes.

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Figure 3. Secondary lymphoid tissue microenvironment is dominant over tissue-specific DC. CD8+ P14 T cells were adoptively transferred and activated in vivo with P-DC or M-DC administered via i.v., i.c. or i.p. injection. On day 3.5, expression of E-lig (A) and α4β7 (B) was measured on Thy1.1+ cells from the indicated secondary lymphoid tissue by flow cytometry. Numbers give the percentage of the indicated cell population gated on CD8+ cells. Data are representative of three independent experiments. Staining controls were as described in Materials and methods.

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Table 2. Dominance of lymphoid tissue microenvironment over tissue-specific DCa)
E-ligα4β7
  1. a) Experiments were performed as described in Fig. 3. Numbers indicate the mean percentage of homing receptor-positive, Thy1.1+ cells ± SD from three independent experiments. Differences in the mean values among groups are statistically significant at p<0.001.

i.v. spleeni.c. PLNi.p. MLNi.v. spleeni.c. PLNi.p. MLN
P-DC3.1±1.5462.8±10.85.9±2.417.6±5.12.3±1.049.2±14.6
M-DC3.5±2.351.8±16.54.6±1.516.9±5.13.6±1.351.6±12.2

DC reprogram in vitro-polarized homing receptor expression

The role of DC in the imprinting of T cells for tissue-selective trafficking has been established 4, 2124. However, it is not known if effector T cells are still sensitive to this process or whether they are terminally imprinted. Therefore, we analyzed whether CD8+ effector T cells can switch homing receptor patterns in response to new tissue-specific signals. P14 T cells were primed in vitro with LC or PP-DC as the strongest skin- or gut-polarizing DC, respectively. On day 6, cells were restimulated with DC from the same or the other tissue. Flow cytometry on day 3 after restimulation revealed an adaptation of E-lig and α4β7 expression levels to the new tissue-specific DC signals (Fig. 4). P14 T cells primed with LC down-regulated E-lig and up-regulated α4β7 integrin upon restimulation with PP-DC. Similarly, PP-DC-primed P14 T cells lost α4β7 expression and up-regulated E-lig following restimulation with LC. Similar results were obtained with P-DC and M-DC (data not shown). We conclude that tissue-specific DC can modulate T cell trafficking patterns not only during priming of naive T cells but also on effector T cells during an ongoing immune response.

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Figure 4. Reprogramming of homing receptor profiles by tissue-specific DC in vitro. CD8+ T cells isolated from P14 spleen were primed with LC isolated from skin or with CD11c+ PP-DC. On day 6, T cells were restimulated with either the same or the alternative DC population. On day 3 after restimulation, expression of E-lig and α4β7 integrin was measured by flow cytometry. Numbers give the percentage of the CD8+/homing receptor-positive cells. Data are representative of four independent experiments.

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Reprogramming of in vivo-polarized, skin-homing T cells by M-DC

We were interested in whether the flexibility of homing receptor expression as observed for effector T cells polarized in vitro holds true for in vivo-polarized skin-homing memory T cells. We injected GP33-loaded BM-DC twice i.c. as previously described for the adoptive P14 transfer system 22. On day 10, mice were boosted by another i.c. injection of DC, and P14 T cells were isolated from PLN 3 weeks later. The T cells displayed a strongly polarized skin-homing phenotype and were mainly E-lighigh and α4β7/CCR9low (Fig. 5) by FACS in comparison to naive P14 T cells. Interestingly, a significant T cell population expressed CCR9. After in vitro restimulation of purified CD8+ P14 T cells with LC isolated from skin or CD11c+ DC from PLN, MLN or Peyer's patches (PP), we analyzed homing receptors. The skin-homing phenotype remained stable following restimulation with LC or P-DC (Fig. 5, Table 3). In contrast, restimulation with M-DC efficiently down-regulated E-lig levels and strongly induced α4β7/CCR9 (Fig. 5). Similar results were obtained with PP-DC (data not shown). These results reveal a flexibility of in vivo-polarized skin-tropic memory T cells.

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Figure 5. Reprogramming of in vivo-induced skin-homing receptor profile by M-DC in vitro. Skin-homing CD8+ P14 memory T cells were generated by adoptive transfer and i.c. injection of peptide-pulsed BM-DC on days 1, 3 and 10. On day 30, CD8+ T cells were isolated from PLN by positive magnetic bead separation, and homing receptor expression on ex vivo cells was monitored by flow cytometry in comparison to naive P14 T cells. Restimulation was done with LC or CD11c+ DC isolated from PLN (P-DC) or MLN (M-DC). On day 4, expression of E-lig, α4β7 integrin, and CCR9 on Thy1.1+ T cells was measured by flow cytometry. Expression of CCR9 was measured in a different experiment. Gate was on CD8+ T cells. Numbers give the percentage of total Thy1.1+ cells expressing the indicated homing receptors. Data are representative of four (E-lig and α4β7) and three (CCR9) independent experiments.

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Table 3. Reprogramming of homing receptor patterns on memory T cells by DCa)
E-ligα4β7CCR9
  1. a) Experiments were performed as described in Fig. 5. Numbers indicate the mean percentage of homing receptor-positive, Thy1.1+ cells ± SD from three independent experiments. Differences in the mean values among groups are statistically significant at p<0.001.

Naive P140.7±0.53.6±1.186.0±7.7
Memory P14 ex vivo52.3±27.54.6±2.028.4±4.3
LC71.5±10.08.7±1.414.0±7.8
P-DC54.8±10.715.7±7.616.6±4.6
M-DC17.9±7.152.2±9.365.9±12.0

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Accumulating data provide evidence that DC play a pivotal role in the regulation of T cell homing patterns. Modulation of CD8+ T cell-homing properties has been established in vitro for DC isolated from skin, PLN, MLN and PP 2124. In vitro priming of naive CD8+ T cells with DC isolated from different tissues revealed that the induction of E-lig for skin-homing 22 or the small intestinal-homing receptors α4β7 and CCR9 2124 was crucially dependent on the tissue origin of the DC, as was the ability of CD8+ T cells primed in vitro with PP-DC to migrate to the small intestine 24. However, the signals that control homing receptor expression are largely unknown. We found an unexpected up-regulation of α4β7 on CD4+ as well as CD8+ T cells in late T cell priming cultures beyond day 6, even with P-DC or LC. Similar observations were made with T cells stimulated with anti-CD3 in the presence of P-DC 23. These data suggest an active suppression of α4β7 by skin-associated DC and a release from suppression when DC undergo apoptosis around day 4 24. Persisting lower levels of α4β7 in cultures with LC, which induce the strongest skin-tropic polarization, support this notion. In line with this hypothesis, in vitro-generated, ‘tissue-neutral’ BM-DC do not suppress α4β7 expression 22. Further evidence for the existence of suppressive signals, in addition to inductive signals, for homing receptor polarization has been previously provided 33, 34. Low E-lig levels were observed on human CD4+ T cells stimulated with PHA in the presence of DC in serum-containing medium compared to serum-free medium. In contrast, serum factors were crucial for the induction of β7 integrin 33. Our study demonstrates that tissue-specific DC modulate homing receptors by delivery of soluble factors. Thus, CM from DC/T cell co-cultures are able to induce their corresponding homing receptors and suppress the homing receptors for the alternative tissue on anti-CD3 mAb-stimulated CD8+ P14 T cells. This shows that soluble factors are sufficient in vitro.

Interestingly, tissue-specific DC did not induce their corresponding T cell homing phenotype in a ‘foreign’ microenvironment. α4β7 integrin was up-regulated in MLN upon i.p. DC injection, E-lig in PLN upon i.c. DC injection, regardless of the tissue origin of the DC. We did not observe α4β7 up-regulation following M-DC injection in PLN or spleen. Similarly, E-lig was not significantly up-regulated in MLN or spleen upon injection of P-DC. These data support our previous findings using BM-DC 22 and demonstrate a functional dominance of the secondary lymphoid tissue microenvironment over the origin of the injected DC. Tissue-resident DC and stromal cells may release soluble factors that overrule the direct influences of the transferred antigen-presenting DC population in trans. Alternatively, the injected DC might adapt functional characteristics of the new tissue microenvironment 4. This hypothesis has gained support through the observation that even mature DC, as used in our experiments, are able to differentiate upon interaction with stromal cells 35. We have previously ruled out efficient antigen cross-presentation by host DC to P14 T cells by injection of heat-killed DC 22. Importantly, the in vivo results also gave evidence for inhibitory factors suppressing E-lig in spleen and MLN, which had not been detected in vitro with M-DC.

Our results reveal that DC-driven polarization of T cell homing patterns is mediated, at least in part, by soluble factors. E-lig and α4β7 up-regulation may be a default program induced upon activation as suggested by previous studies 33, 36, 37. Obviously, microenvironmental signals can efficiently polarize towards skin- or gut-homing receptors, supported by the detection of non-overlapping skin- or gut-tropic memory T cell subsets in blood 10, 3843. The soluble factors that are responsible for homing receptor polarization in our study have to be identified in the future. Iwata et al. reported recently that retinoic acid can enhance expression of the small intestine-homing receptors α4β7 and CCR9, while the same factor suppressed skin-homing receptor and fucosyltransferase VII expression under steady-state conditions 44. Interestingly, small intestine-associated but not skin-associated DC expressed the enzymes required to generate retinoic acid from retinol, an observation that is a first hint to explain some of the tissue-specific characteristics of DC isolated from different tissues 2124. It is conceivable that differential expression of genes such as the retinal dehydrogenase genes 44 is the basis for these DC properties.

Furthermore, an inductive role for IL-12, IL-4 and TGF-β1 in E-lig expression by regulation of fucosyltransferase VII 2628 and for TGF-β1 in the regulation of β7 integrins 29 has been demonstrated in vitro. In our hands, addition of recombinant IL-4 to priming cultures with P-DC or priming with BM-DC from IL-12p35/p40 knockout mice strongly reduced E-lig induction on P14 T cells (data not shown). The unexpected in vitro induction of the skin-homing receptor E-lig by gut-associated and S-DC and the corresponding CM may therefore result from accumulation of unphysiologically high DC-derived IL-12 levels in culture. The in vivo relevance of these cytokines remains to be determined. Interestingly, recent studies showed that acquisition of skin- vs. gut-homing phenotypes by human CD4+ T cells occurred independently of the Th1 or Th2 subset polarization 33, 37, 45.

Several reports suggest the existence of stable, mutually exclusive tissue-selective memory T cell subsets 10, 3843. Memory of the site of antigen encounter may be epigenetically imprinted in these cells by DNA methylation and histone modification 46 as described for cytokine memory of T cell subsets 47 and recently for the regulation of selectin ligands 48. Here we describe for the first time a reprogramming of polarized E-lig or α4β7/CCR9 expression: tissue-specific DC are not only able to imprint naive T cells but also revert the homing profile of already polarized tissue-selective P14 T cells induced in vitro. Even more striking, skin-homing memory T cells polarized in vivo could also be reverted into a gut-homing phenotype. This raises the question of whether imprinting of homing properties is indeed permanently fixed or whether a certain degree of flexibility is preserved in polarized memory T cells. An epigenetic memory and integration of previous homing patterns may be the basis for such flexibility and could be reflected by simultaneous DNA modification of loci involved in the regulation of homing receptors for different tissues such as skin and gut. Flexible trafficking patterns are essential for the immune system to cope efficiently with disseminating infections and metastasizing tumors.

With regard to the proposed dichotomy of central vs. effector memory T cells with different access to secondary lymphoid tissues 3, 49, central memory T cells may be the subset that can adapt to different tissue microenvironments, whereas effector memory T cells may keep a topographical memory of the first site of antigen encounter. Although this model seems to be more complex than initially thought 45, 50, it may explain how the immune system can keep and integrate several tissue-specific T cell homing patterns.

In summary, our results show that DC govern tissue-selective T cell homing not only during priming, but also in the effector and memory response by reprogramming of established homing patterns. Soluble factors play an important role. Furthermore, the tissue microenvironment crucially influences homing receptor polarization. Therapeutic modulation of tissue-selective trafficking patterns of effector and memory T cells will allow tissue targeting in order to generate efficient immune responses or therapeutic re-routing of pathogenic T cells in organ-specific autoimmune and inflammatory diseases.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

C57BL/6 and TCR-transgenic Thy1.1 congenic P14 mice 30, 31 expressing a TCR specific for the lymphocytic choriomeningitis virus (LCMV)-derived peptide GP33 51 were provided by the breeding facility of the University of Freiburg, Germany. OVA-specific TCR-transgenic DO11.10 mice 52 and BALB/c mice were bred in the BfR (Bundesanstalt für Risikobewertung, Berlin, Germany). All of the experimental procedures were in accordance with institutional, state and federal guidelines on animal welfare.

Peptides

Synthetic H-2Db-binding peptide GP33 from the glycoprotein of LCMV 51 and I-Ad-binding chicken ovalbumin peptide OVA323–339 have been described before 52 and were purchased from BioChip Technologies GmbH (Freiburg, Germany) and the Department of Biochemistry (Humboldt-University, Berlin, Germany), respectively.

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 Gibco, Invitrogen Corporation, Karlsruhe, Germany) and 10 µM 2-mercaptoethanol (Sigma, Deisenhofen, Germany).

Antibodies and flow cytometry

Antibodies were from BD Biosciences (Heidelberg, Germany) and used as FITC, PE or biotin conjugates. The latter were revealed with Streptavidin-Cy-Chrome®. mAb were used at 0.1–1 µg/1×106 cells in 100 µl HBSS/0.3% BSA. Staining for α4β7 integrin and E-lig with E-selectin/human IgG-Fc-Chimera (R&D Systems, Wiesbaden, Germany) was performed as described 22. Control staining for E-lig was done with secondary Ab only, while corresponding isotype control Ab was used as a control for α4β7 and CCR9. Ex vivo cells were washed with 5 mM EDTA/PBS before staining. Rat anti-CCR9 Ab has been described elsewhere 53 and was used as B cell hybridoma supernatant at a dilution of 1:4. Secondary FITC-labeled anti-human IgG and biotinylated anti-rat IgG were from DAKO (Hamburg, Germany). Secondary FITC- or PE-labeled anti-rabbit antibodies (Serotec, Eching, Germany) were diluted 1:50–1:100. Data were acquired and analyzed on a FACScan instrument using CellQuest software (BD Biosciences).

Generation of bone marrow-derived DC

Bone marrow cells were cultured at 7×105 cells/ml in the presence of 40 ng/ml GM-CSF (supernatant from producer line X63-Ag8 54) and 10 ng/ml recombinant IL-4 (Promocell, Heidelberg, Germany) in 10 ml medium in 10 cm Petri dishes (Greiner, Nürtingen, Germany). On day 3, 10 ml fresh medium containing 40 ng/ml GM-CSF was added. On days 5 and 7, 10 ml medium was replaced with fresh GM-CSF medium. DC were used on days 7–9. Purity was routinely about 90%.

Adoptive P14 T cell transfer and injection of tissue-specific DC

P14 spleen cells (3×106 in 200 µl PBS) were injected i.v. into C57BL/6 mice. One day later, DC from mice treated with B16 melanoma cells expressing FLT3-L 32 were incubated for 30 min at 37°C in RP-10 with GP33 peptide (1 µM) and LPS (0.3 μg/ml) (Sigma). After three washes with PBS, cells were used for injection. DC (3×106 in 200 µl PBS) were injected i.c. at three sites on the shaved abdomen. The i.v. and i.p. injections were performed with 1×106 DC in 200 µl PBS. On day 3.5, mice were killed and lymphoid tissue cells prepared for flow cytometry by gentle teasing of the tissue through a steel mesh and filtration of the suspension through a cell strainer (70 µm, BD Falcon, Heidelberg, Germany).

Isolation of tissue-specific DC and in vitro priming

C57BL/6 mice received 1×106 B16 melanoma cells producing FLT3-L 32 s.c. in 200 μl PBS. Mice were killed 14–17 days later, and CD11c+ DC were isolated from skin-draining PLN, MLN, PP and spleen by CD11c MicroBeads using AutoMACS (Miltenyi Biotec, Bergisch-Gladbach, Germany) as described 22. Langerhans cells were isolated from ear sheets as described 22. Ears were split into dorsal and ventral sheets using forceps. Sheets were incubated for approximately 30 min in 1% trypsin/PBS solution (Gibco) at 37°C until the epidermal layer could be removed by rubbing with forceps. Single-cell suspensions were prepared by extensive up-and-down pipetting of the epidermal sheets, followed by cell strainer filtration (70 μm, BD Falcon) and 16% Nycodenz (Sigma) gradient centrifugation. The purity of the isolated DC was routinely about 90%.

DC were incubated with GP33 peptide (1 μM) for 30 min at 37°C and washed three times. DC (5×103/well) and P14 spleen cells (4×104/well) were co-cultured in 96-well round-bottom plates (Corning Life Sciences, Wiesbaden, Germany) in 200 μl RP-10. In some experiments CD8+ P14 T cells were isolated with CD8 MicroBeads (Miltenyi), and similar results were obtained.

OVA-specific naive CD4+ T cells were isolated from pooled lymph nodes and spleens of DO11.10 mice using CD4-FITC and anti-FITC MultiSort MicroBeads (Miltenyi), followed by release of the magnetic label and subsequent isolation of naive CD4+ T cells with CD62L MicroBeads. Naive CD4+ DO11.10 T cells (1×105) were co-cultured with 1×104 DC in the presence of OVA323–339 peptide (1 μg/ml) in 96-well plates in 200 μl RP-10.

Preparation of CM and P14 T cell priming

DC (6×106) isolated from PLN (P-DC), MLN (M-DC) or spleen (S-DC) were pulsed with peptide GP33 as described above and co-cultured with P14 spleen cells (1.2×106) or purified CD8+ spleen cells (4×105) in 12-well plates (Corning Life Sciences) in 2 ml RP-10. Supernatants were harvested 40 h later by centrifugation and stored at –40°C. CD8+ T cells were isolated from P14 spleens using CD8 MicroBeads (Miltenyi), and 1×104 cells were cultured in 100 µl RP-10 plus 150 µl RP-10 or CM from tissue-specific DC/T cell co-cultures (see above) in 96-well round-bottom plates (Corning Life Sciences). Soluble anti-CD3ϵ (145–2C11, BD Biosciences) was added to cultures at 10 μg/ml.

In vivo generation of skin-homing P14 T cells and reprogramming of T cells by DC

In vitro co-cultures of tissue-specific DC with P14 T cells were set up in 96-well plates as described above. Cells were harvested on day 6. After washing in RP-10, cell suspensions were diluted 1:4 and added to fresh 96-well plates together with 1×104 DC (isolated from different tissues) per well. For in vivo generation of skin-homing T cells, P14 T cells were injected i.v., and mice received 3×106 GP33-pulsed and LPS-activated BM-DC i.c. on days 1, 3 and 10. On day 30, CD8+ cells were purified from PLN using CD8 MicroBeads (Miltenyi) and restimulated with tissue-specific DC. T cells (1×104) and GP33-pulsed DC (1×104) were co-cultured in 200 µl RP-10/2% RCAS in 96-well plates.

Statistical analysis

Statistical analysis of the data was performed by comparison of treatment groups using one-way ANOVA.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

We thank Dr. Daniel J. Campbell (Benaroya Research Institute at Virginia Mason, Seattle, WA, USA) for helpful discussions and careful reading of the manuscript, Dr. Hanspeter Pircher (Institute for Medical Microbiology and Hygiene, University of Freiburg) for kindly providing P14 Thy1.1 mice and Dr. Glenn Dranoff (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA) for the B16 FLT3-L cell line. Bettina Ocker, Freiburg, is acknowledged for excellent assistance.

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