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Keywords:

  • T lymphocyte;
  • Chemokine;
  • Integrin;
  • Adhesion;
  • Epithelium

Abstract

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

The integrin CD103 and the chemokine receptor CCR9 are co-expressed on small intestinal CD8+ intraepithelial lymphocytes (IEL), naïve murine CD8+ T cells and by a small population of effector/memory CD8+ T cells, indicating a potential role for CCR9 in regulating CD103 expression and function. Here, we demonstrate that CD103, in contrast to CCR9, is down-regulated on CD8+ T cells following their activation in mesenteric lymph nodes and that effector CD8+ T cells upon initial entry into the small intestinal epithelium are CCR9+CD103. CD103 was rapidly induced on wild-type CD8+ T cells subsequent to their entry into the small intestinal epithelium, however, CCR9–/– CD8+ T cells exhibited a significant delay in CD103 induction at this site. In addition, the CCR9 ligand, CCL25, that is constitutively expressed in the small intestinal epithelium, induced transient, dose-dependent and pertussis toxin-sensitive CD103-mediated adhesion of CD8+ small intestinal IEL to a murine E-cadherin human Fc (mEFc) fusion protein. Together, these results demonstrate a role for CCR9/CCL25 in promoting the induction and function of CD103 on CD8+ IEL and suggest that this chemokine receptor/chemokine pair may function to regulate lymphocyte-epithelial interactions in the small intestinal mucosa.

Abbreviations:
IEL:

Intra epithelial lymphocyte

MLN:

Mesenteric lymph node

PLN:

Peripheral lymph node

mEFc:

Murine-E-cadherin human-Fc fusion protein

WT:

Wild type

1 Introduction

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

The integrin αEβ7 (CD103) is expressed by intraepithelial lymphocytes (IEL) 1, 2, and mediates lymphocyte adhesion to epithelial cells by interacting with its epithelial specific ligand E-cadherin 3. CD103–/– mice exhibit a modest reduction in small intestinal and vaginal IEL number 4, indicating that this interaction is important to maintain T cells within mucosal epithelia. CD103 is also expressed on CD8+ T cells associated with epithelial allografts 5, 6 and is required for CD8+ T cell infiltration and destruction of these grafts 6. In addition, several recent reports have shown CD103 to define a subset of CD4+ cells with potent regulatory function, suggesting a potential role for this integrin also in CD4+ T cell function 7, 8. Despite recent progress in our understanding of the possible functions of CD103, the mechanisms regulating CD103 expression and function remain largely uncharacterized.

In addition to its expression on epithelial associated CD8+ T cells, CD103 is expressed on naïve murine (but not human) CD8+ T cells and a small subset of effector/memory CD8+ T cells in murine lymph nodes and human peripheral blood 9, 10. Remarkably, the majority of these CD103+CD8+ T cells in both human and mouse co-express CCR9 9, 10, a chemokine receptor associated with gut tropic T cells, indicating that CCR9 and CD103 expression may be co-regulated on CD8+ T cells. We have recently demonstrated that CCR9 is selectively maintained on CD8+ T cells following their activation in mesenteric lymph nodes (MLN) but not peripheral lymph nodes (PLN) or spleen 9, and is important for effector CD8+ T cell localization to the small intestinal epithelium 11. In the current study we have examined whether CD103 is similarly regulated during CD8+ T cell activation and the potential role of CCR9 in regulating CD103 expression and function on small intestinal IEL.

2 Results

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

2.1 CD103 and CCR9 are differentially regulated on CD8+ T cells following activation in gut-associated lymphoid tissue

An OVA-specific TCR-transgenic CD8+ (OT-1) adoptive transfer model 9, 11 was used to determine CD103 expression during CD8+ T cell activation in secondary lymphoid tissues. OT-1 cells were adoptively transferred into C57BL/6.Ly5.1 recipients and activated by i.p. injection of OVA+LPS 1 day later. In the absence of activation OT-1 cells in MLN and PLN were CD103+ (Fig. 1A). Three days after i.p. immunization a large proportion of OT-1 cells activated in MLN, but not PLN expressed CCR9 (Fig. 1B), consistent with our previous observations 9, 11. In contrast, CD103 was down-regulated on OT-1 cells activated in both MLN and PLN (Fig. 1C). Thus, CD103 and CCR9 are differentially regulated during CD8+ T cell activation in MLN. In vitro stimulation with anti-CD3 and anti-CD28 antibody also resulted in a loss of CD103 expression on OT-1 cells (data not shown), indicating that CD103 down-regulation in vivo does not require selective signals from antigen-presenting cells.

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Figure 1. B7 integrin and CCR9 expression during CD8+ T cell activation in LN and entry into the small intestinal epithelium. OT-1 cells were transferred into C57BL/6.Ly5.1 recipient mice and their expression of CD103 or CCR9 in MLN, PLN and small intestinal epithelium determined in the absence of stimulation (A) or at the time points indicated after i.p. administration of OVA + LPS (B and C). OT-1 cells were identified by first gating on Ly5.2+ cells. Results are from one animal and are representative of five to six mice from two separate experiments. (D) α4β7 expression on OT-1 cells in the small intestinal epithelium at the time points indicated after i.p. administration of OVA + LPS. Results are from one representative animal of three.

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2.2 CD103 is induced on CD8+ effector lymphocytes following their entry into the small intestinal epithelium

In the OT-1 transfer model described above, effector OT-1 cells generated in gut-associated lymphoid tissue (GALT) first appear in the small intestinal epithelium 3 days after i.p. administration of OVA+LPS. As expected 11, OT-1 cells localizing to the small intestinal epithelium at day 3 expressed CCR9 and α4β7 (Fig. 1B and D). In contrast, the majority of these early OT-1 immigrants were CD103 (Fig. 1C). CD103 expression increased on intraepithelial OT-1 cells 4 days after administration of OVA + LPS and by day 7 all OT-1 cells in the epithelium expressed CD103 (Fig. 1C). This induction of CD103 on OT-1 IEL was accompanied by a loss of α4β7 expression (Fig. 1D). The large majority of OT-1 cells in the MLN, PLN, spleen, liver and lung remained CD103 during this period (Fig. 1C, data not shown). Together these results demonstrate that CD103 is induced on effector CD8+ T cells after their entry into the small intestinal epithelium.

2.3 CCR9 promotes the induction of CD103 on effector CD8+ T cells entering the small intestinal epithelium

Since effector OT-1 cells arriving in the small intestinal epithelium express CCR9 and the CCR9 ligand, CCL25, is constitutively expressed by small intestinal epithelial cells 12, 13, we determined whether CCR9 was involved in the induction of CD103 on OT-1 cells subsequent to their entry into the intestinal epithelium. For these experiments, CCR9–/– OT-1 (Ly5.2+) and WT OT-1 (Ly5.1+Ly5.2+) cells were co-transferred into recipient (Ly5.1+) mice. A similar percentage of injected CCR9–/– and WT OT-1 cells expressed CD103 (Fig. 2A) and all CD103+ WT OT-1 cells co-expressed CCR9 (Fig. 2A). As with WT OT-1 cells, CCR9–/– OT-1 cells failed to express CD103 in MLN and PLN 3 days after administration of OVA + LPS (Fig. 2C and data not shown), and levels remained low at 4 and 7 days after activation (Fig. 2C, data not shown). A difference in the percentage of CCR9–/– OT-1 cells expressing CD103 compared to WT OT-1 cells was observed in the MLN at day 15, possibly due to recirculation of some cells from the small intestinal mucosa.

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Figure 2. CCR9 is required for early induction of CD103 on effector CD8+ T cells localizing to the small intestinal epithelium. (A) CD103 and CCR9 expression on WT and CCR9–/– OT-1 cells prior to their transfer into C57BL6.Ly5.1 recipients. (B) CD103 expression on WT and CCR9–/– OT-1 cells isolated from the IEL compartment at the times indicated after i.p. administration of OVA + LPS. Results are from one animal and are representative of eight to nine mice from three experiments. (C) CD103 expression on WT and CCR9–/– OT-1 cells isolated from the IEL and MLN compartment at the times indicated after i.p. administration of OVA + LPS [mean (SEM), n=5–6, *p<0.05, **p<0.01]. (D) CD103 expression on CD8+ IEL from CCR9–/– and WT mice. WT OT-1 cells were identified by first gating on Ly5.1+Ly5.2+ cells and CCR9–/– OT-1 cells were identified by gating on Ly5.2Ly5.2+ cells. Results are from three pooled mice from one representative experiment of two performed.

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The ratio of CCR9–/– to WT OT-1 cells in the small intestinal epithelium was markedly reduced compared to the ratio in MLN, 3 days after administration of OVA + LPS (data not shown), consistent with our previous results 11. The majority of CCR9–/– and WT OT-1 cells infiltrating the small intestinal epithelium at day 3 were CD103, although a lower percentage of CCR9–/– OT-1 cells expressed CD103 (Fig. 2B and C). This difference was more dramatic at day 4, with only 25% (±8) of CCR9–/– OT-1 compared to 80% (±15) of WT OT-1 cells expressing CD103 (Fig. 2B and C). While the percentage of CCR9–/– OT-1 cells expressing CD103 increased by day 7 and 15, it failed to reach levels observed on WT OT-1 cells during this period (Fig. 2B and C). Importantly, CCR9–/– and WT OT-1 cells displayed a similar kinetics in their recruitment into the small intestinal epithelium and in their reduction in α4β7 levels (data not shown), indicating that the delayed induction of CD103 on CCR9–/– OT-1 cells was not due to retarded homing of these cells into the epithelium.

Finally, virtually all CCR9–/– and WT IEL expressed CD103 (Fig. 2D), demonstrating that CCR9 is not required for maximal CD103 expression on these cells. Together, these results indicate a nonredundant role for CCR9 in promoting the initial induction of CD103 on CD8+ T cells as they enter the small intestinal epithelium. However, CCR9-independent signals appear sufficient to generate normal CD103 levels on lymphocytes after prolonged residency within this site.

2.4 Freshly isolated murine IEL adhere to a murine E-cadherin human Fc fusion protein

The ability of chemokines to induce integrin mediated lymphocyte adhesion to vascular endothelium by enhancing integrin affinity and avidity for Ig superfamily ligands, is well documented 14. However, whether chemokines can regulate CD103-mediated lymphocyte adhesion to the non-Ig superfamily member E-cadherin, and thus play a potential role in regulating lymphocyte interactions with intestinal epithelia, has not been examined. We therefore determined whether CCL25 could enhance CD103-mediated adhesion of freshly isolated murine IEL to E-cadherin. For this purpose we constructed a murine E-cadherin Fc (mEFc) fusion protein (Fig. 3A, see Sect. 4), and examined the ability of murine CD8+ IEL to adhere to mEFc in in vitro adhesion assays. Murine IEL adhered to mEFc (Fig. 3B) and neutralizing anti-CD103 but not isotype control antibody or a binding anti-MHC-1 control antibody significantly blocked adhesion (**p<0.01, Fig. 3B, and data not shown). Thus, mEFc is functional, and murine IEL adhesion to mEFc is dependent on the integrin CD103.

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Figure 3. Sequence and functionality of recombinant murine E-cadherin-Fc fusion protein. (A) The sequence of the extracellular juxtamembrane region of WT mouse E-cadherin and the alterations resulting from fusion with the human Fc region are shown. Regions corresponding to the Fc portion are shown in bold. (B) Freshly isolated murine IEL adhere to mEFc fusion protein. Murine IEL were incubated with anti-CD103 or isotype control antibody prior to addition to the mEFc coated adhesion plate (see Sect. 4). Murine IEL adhesion was determined in the presence of 1 mM CaCl2, 0.5 mM MgCl2, and 1 mM MnCl2. Results are the mean (SEM) of triplicate wells and are representative of three experiments performed. **p<0.01 compared to isotype control antibody.

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2.5 CCL25 induces CD8+ IEL adhesion to murine E-cadherin Fc fusion protein

To determine whether CCL25 enhances IEL adhesion to mEFc, murine CD8+ IEL were pre-incubated with CCL25 prior to addition to the mEFc coated adhesion plate (see Sect. 4). CCL25 dose dependently enhanced CD8+ IEL adhesion to mEFc fusion protein in the presence of suboptimal doses of MgCl2 (Fig. 4B) and this effect was significant at 500 and 100 nM CCL25 when compared to adhesion in the absence of chemokine. CCL20, whose receptor CCR6 is not expressed by IEL (Fig. 4A and 15) did not enhance IEL adhesion to mEFc across a similar dose range (Fig. 4B). Pre-incubation of IEL with pertussis toxin or anti-CD103 antibody but not isotype control antibody inhibited CCL25-mediated adhesion to mEFc (Fig. 4C and D). Furthermore, CCL25- but not Mn2+-induced IEL adhesion returned to background levels during subsequent plate washes (Fig. 4E). Finally, CCL25 failed to increase the already high levels of CD103 on freshly isolated CD8+ IEL (data not shown), demonstrating that CCL25 induction of CD103-mediated adhesion was not due to enhanced CD103 levels on these cells. Together, these results support a role for CCL25 in regulating CD103-mediated IEL adhesion to intestinal epithelial cells.

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Figure 4. CCL25 promotes IEL adhesion to murine E-cadherin Fc fusion protein. (A) CCR9 and CCR6 expression by murine CD8+ IEL as assessed by flow cytometry analysis (anti-chemokine receptor (filled) or isotype control antibody (blank)). (B) CCL25 dose dependently enhances murine IEL adhesion to mEFc. CD8+ IEL were pre-incubated with CCL25 (gray bars), CCL20 (white bars) or no chemokine (black bar) at the indicated concentration in the presence of 0.5 mM MgCl2 on ice for 10–20 min prior to addition to the mEFc-coated adhesion plate. No MgCl2 (striped bar). Results are the mean (SEM) of triplicate wells from one representative experiment of six performed. (C) CCL25-mediated adhesion is pertussis toxin (PT) sensitive. IEL were pre-incubated with PT (see Sect. 4) prior to incubation with CCL25 (500 nM) and 0.5 mM MgCl2. CCL25 alone (gray bar), CCL25 and PT (white bar) and MgCl2 alone (0.5 mM, black bar). Results are the mean (SEM) of triplicate wells from one representative adhesion plate of four from two separate experiments. (D) CCL25-mediated adhesion is blocked with anti-CD103 antibody. IEL were incubated with anti-CD103 or isotype control antibody prior to addition to the mEFc coated adhesion plate (see Sect. 4). Results are the mean (SEM) of triplicate wells from one representative adhesion plate of two. (E) CCL25-mediated IEL adhesion to mEFc is transient. IEL were pre-incubated with CCL25 (500 nM, gray bar) or MnCl2 (1 mM, white bar) in the presence of 0.5 mM MgCl2, before addition to the adhesion plates. MgCl2 alone (0.5 mM, black bar). After incubation of IEL on the plates for 5 min at 37°C, plates were washed and IEL adhesion quantitated after five and nine washes. Results are the mean (SEM) of triplicate wells from one representative experiment of three performed. *p<0.05 **p<0.01 and ***p<0.0001.

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3 Discussion

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

The majority of human and murine CD103+CD8+ T cells express the chemokine receptor CCR9 9, 11, indicating a potential role for CCR9 in regulating CD103 expression and function on these cells. Here we demonstrate that CD103 is lost during CD8+ T cell activation in PLN and MLN but is rapidly induced on these cells subsequent to their entry into the small intestinal epithelium. This early induction of CD103 was in part dependent on CCR9. Furthermore, the CCR9 ligand CCL25 dose dependently induced CD103-mediated CD8+ IEL adhesion to its epithelial ligand E-cadherin. Together these results implicate a role for CCL25/CCR9 in regulating lymphocyte-epithelial adhesive interactions in the small intestine. Moreover, these results indicate that the CD103+CCR9+ effector/memory CD8+ T cells observed in human peripheral blood and murine secondary lymphoid organs 9, 10, derive from naïve CD8+ T cells that have been activated in GALT and subsequently exposed to an epithelial environment.

Lymphocyte localization to the small intestinal mucosa is dependent on β7 integrins 16, 17. Gut tropic effector T cells express high levels of α4β7 18 whose interactions with MadCAM-1 on intestinal microvascular endothelium help regulate T cell exit from the circulation 19. Whether CD103 is also involved in effector T cell recruitment to the small intestinal mucosa has been the subject of some debate. As with α4β7 and other molecules involved in leukocyte rolling, CD103 is expressed on the microvillous tips of CD103-transfected K562 cells 20. Furthermore, IEL and a CD103-Fc fusion protein adhere to intestinal but not skin microvascular endothelial cells in a CD103-dependent, E-cadherin-independent fashion in vitro21. Together, these observations are consistent with a potential role for CD103 in effector T cell recruitment from the circulation into the intestinal mucosa. However, effector CD103–/–OT-1 cells are equally efficient in localizing to the small intestinal epithelium as WT OT-1 cells 17. Our observation that CD103 is lost during CD8+ T cell activation in MLN, and induced on effector CD8+ T cells subsequent to their entry into the intestinal epithelium, provides further evidence that CD103 is not involved in regulating effector lymphocyte interactions with the intestinal microvascular endothelium or in lymphocyte entry to the small intestinal epithelium in vivo.

Four days after administration of OVA and LPS, the majority of OT-1 cells in the small intestinal epithelium expressed CD103, and by day 7, CD103 levels were similar to those observed on endogenous IEL. This was not due to delayed CD103 induction following T cell activation in secondary lymphoid organs since the majority of effector OT-1 cells within secondary lymphoid organs, the liver and the lung, remained CD103 during this period. These results provide strong evidence for a role of the intestinal microenvironment in the induction of CD103 on CD8+ IEL in vivo. A key finding of the current study was that the induction of CD103 on effector CCR9–/– OT-1 cells localizing to the small intestinal epithelium is delayed compared to WT OT-1 cells and fails to reach levels observed on WT OT-1 cells even 15 days after immunization. Since all CD8+ IEL from CCR9–/– and WT mice expressed CD103, CCR9 is clearly not required for the induction of CD103 on IEL. Rather, our results indicate that CCR9 plays a role in promoting the initial induction of CD103 on CD8+ T cells subsequent to their entry into the small intestinal epithelium. We are currently unable to isolate sufficient numbers of purified recent IEL immigrants to address the underlying mechanism by which CCR9 promotes CD103 expression, however, several mechanisms may be proposed. Epithelial-derived CCL25 may directly induce CD103 on CCR9+ effector CD8+ T cells as they enter into the small intestinal epithelium, however, we think this unlikely since addition of CCL25 to MLN preparations containing CCR9+ effector OT-1 cells fail to enhance CD103 expression on these cells in vitro (authors unpublished observation). Alternatively, CCL25 may be required indirectly to recruit recent IEL immigrants into a more intimate or rapid contact with CD103 inducing signals, or may synergize with local factors to promote CD103 expression. In this respect, effector CD8+ T cells entering epithelial allografts fail to up-regulate CD103 when expressing a dominant negative TGFβII receptor 22, suggesting an important role for TGFβ signaling in the induction of CD103 in vivo. Indeed, TGF-β induces CD103 expression on T cells in vitro2326, is constitutively produced in the small intestinal mucosa 27, and, in preliminary studies, enhances CD103 expression on CCR9+ effector OT-1 cells in MLN preparations. Irrespective of the mechanism, these results support a role for CCR9 in promoting the induction CD103 on recent IEL immigrants in vivo and suggest a complex interplay between CCR9, TGF-β and CD103 signaling events in the small intestinal epithelium.

A second key finding of the current study was the ability of CCL25 to enhance IEL adhesion to E-cadherin and supports a role for CCL25/CCR9 in regulating CD103 function on IEL. CCL25 induced IEL adhesion to E-cadherin appeared to be transient, as has been demonstrated for chemokine induced integrin adhesion to Ig superfamily members 28, although the possibility that CCL25 induced weaker CD103 mediated adhesion compared to Mn2+ in our assay cannot be excluded. Transient CD103-mediated lymphocyte adhesion is likely to be required to enable lymphocyte retention within the small intestinal epithelium in the face of rapid epithelial turnover. In this respect, CCL25 may function to dynamically modulate IEL adhesive interactions with small intestinal epithelial cells and thereby contribute to IEL positioning within the epithelium. In addition, signaling through CD103 has been implicated in regulating T lymphocyte proliferation 29, 30, and antibodies to CD103 induce redirected lysis of Fc-receptor bearing target cells 31. Furthermore, E-cadherin is itself a signaling molecule 32. Thus, CCL25 regulation of CD103-mediated adhesion may also influence IEL and epithelial cell function within the small intestinal mucosa.

Chemokines enhance integrin-mediated adhesion to Ig superfamily molecules and extracellular matrix ligands by inducing a high-affinity state of the integrin through inside out signaling events, and/or by triggering lateral movement of integrin molecules in the cell membrane, leading to the formation of integrin clusters and increased integrin avidity 33. Since CD8+ IEL initially expressed high levels of CD103 that were not further enhanced by CCL25, it is likely that CCL25 induces CD103-mediated adhesion by increasing CD103 avidity and/or affinity for E-cadherin. The ability of pertussis toxin to block CCL25-induced IEL adhesion demonstrates a critical role for Gai signaling in this process. Interestingly, CXCL12 induced α4β7-dependent adhesion to MadCAM-1 is only partially dependent on pertussis toxin sensitive signaling 34, suggesting that chemokine regulation of β7 integrin adhesion may differ depending on the chemokine, cell type and/or β7 integrin under study. Further studies comparing the intracellular signaling requirements for chemokine-induced regulation of CD103- and α4β7-dependent adhesion of gut tropic and intestinal T cells should provide further insight into these findings.

In conclusion, the results of the current study suggest a novel role for CCL25/CCR9 in promoting the induction and function of CD103 on small intestinal IEL. While CCR9 appears to play a nonredundant role in promoting the initial expression of CD103 on recent IEL immigrants in vivo the paucity of other chemokine receptors on murine small intestinal IEL (Svensson et al, manuscript in preparation) also suggest that CCL25 is the major chemokine capable of regulating CD103-mediated adhesion in the small intestine at least under noninflammatory conditions. Since CD103 is expressed on epithelial-associated lymphocytes at many mucosal sites, and on lymphocytes associated with epithelial allografts 6, 35, 36, it will be of considerable interest to determine whether the findings presented in the current study are unique to CCL25 and small intestinal lymphocytes or represent a broad novel role for chemokines in regulating lymphocyte-epithelial interactions at epithelial surfaces.

4 Materials and methods

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

4.1 Mice

C57BL/6, C57BL/6.Ly5.1, CCR9–/– and OT-1 TCR-transgenic mice were housed and maintained in the BioMedical Center (BMC) animal facility and in the Microbiology, Immunology and Glycobiology (MIG) animal facility, Lund University. CCR9–/– OT-1 (Ly5.2+) and WT OT-1 (Ly5.1+Ly5.2+) mice were generated as previously described 11.

4.2 Antibodies and reagents

The following antibodies and reagents were used during the course of this study: Anti-murine CD8α (YTS169–4, ATCC, Rockville, MD), anti-FcRII/III (2.4G2, ATCC), anti-human Fc (Sigma-Aldrich, St Louis, MO), anti-murine E-cadherin (DECMA-1, kindly provided by Dr. R. Kemler, Max-Plank Institute of Immune Biology, Freiburg, Germany), polyclonal rabbit anti-mouse CCR9 (K629 37), anti-mouse CCR6 (15 ICI2 Millenium Pharmaceuticals, Boston, MA), anti-mouse CD103 (M290. PharMingen, San Diego, CA, anti-mouse α4β7 (DATK32, PharMingen), anti-mouse CD45.1 (A20, PharMingen), anti-mouse CD45.2 (104, PharMingen), and isotype control antibodies (PharMingen). Goat anti-rabbit Ig and goat anti-rat IgG were from Jackson ImmunoResearch Labs (West groove, PA) streptavidin-allophycocyanin from PharMingen, and recombinant murine CCL25 and CCL20 from R&D systems Inc (Abingdon, GB).

4.3 Adoptive transfer

OT-1 T cell adoptive transfers were performed as previously described 9, 11. Briefly OVA-specific CD8+ OT-1 cells (2×106 –5×106 cells/mouse) were injected i.v. into C57BL/6.Ly5.1 recipients and 1 day later mice received PBS or OVA (5 mg) + LPS (100 μg) i.p. Mice were sacrificed at the time points indicated after immunization and the phenotype of OT-1 cells isolated from various organs determined.

4.4 Cell isolation

Lymphocytes were isolated from tissues as previously described 4, 9. For adhesion assays, IEL were further purified by Magnetic Cell Sorting (MACS) using biotin-coupled anti-murine CD8 antibody and streptavidin micro-beads (Miltenyi Biotec GmbH, Bergish Gladbach, Germany), according to manufacturers instructions and were >98% CD8+ as determined by flow cytometry analysis.

4.5 Construction and production of murine E-cadherin-Fc (mEFc)

Full-length murine E-cadherin cDNA in pUC19 under the control of CMV-IE promoter and the neo resistance marker under the control of the polyoma enhancer/tk promoter was kindly provided by Dr. R. Kemler (Freiburg, Germany 38). A phosphorylated HindIII linker [d(pCCCAAGCTTGGG)] was ligated to the KpnI cleaved plasmid DNA, immediately upstream of the 5′- end of mouse E-cadherin cDNA. A 3′ XhoI cohesive site immediately upstream of the transmembrane region of mouse E-cadherin cDNA was introduced by PCR from E-cadherin cDNA in pUC19. PCR with the primers 5′-GCGCGCTGAGATGGACAGAGAAGAC-3′ and 5′-GCAATCCTGCTGCCACGATCTCGAGCTTCATGCAGTTG-3′ using cloned plaque-forming unit (PFU) polymerase (Stratagene, La Jolla, CA) amplified a product that was subsequently cleaved with NdeI and XhoI to generate a 413-bp fragment from the end of the E-cadherin extracellular region. A fragment encoding the rest of the extracellular region of E-cadherin was derived after digestion of plasmid pUC 19 with HindIII and NdeI and ligated to the NdeI-XhoI fragment. The HindIII-XhoI fragment was ligated upstream of the coding sequence for the hinge and Fc region of human IgG1 in a derivative of pCDM8 39, and the integrity of the whole construct confirmed by double-stranded sequencing using the Sequenase kit (United States Biochemical Corp). Finally, the murine E-cadherin-Fc (mEFc) cDNA was excised from pCDM8 using HindIII and NotI and ligated into the expression vector pCEP4 (Invitrogen Corp., Carlsbad, CA) cleaved with the same enzymes. Production and purification of mEFc was performed as previously described for human E-cadherin-Fc 40. SDS-PAGE and Western blot analysis of mEFc with anti-human Fc or anti-mouse E-cadherin antibody under reducing conditions demonstrated the presence of a doublet of ∼125 and 140 kDa (data not shown). Since E-cadherin is synthesized as a pro-protein 41 these bands presumably correspond to the mature and immature form of E-cadherin. The fusion protein migrated at ∼240 kDa under non-reducing conditions, as expected for dimeric fusion protein linked through disulfide bonds in the hinge of the Fc region.

4.6 Cell adhesion assays

Ninety-six-well plates (Linbro, ICN Flow laboratories, Horsham, MA) were coated with mEFc or human IgG1 (Calbiochem-Novabiochem Corp, 0.3126 μg/well) in TBS supplemented with 1 mM CaCl2 at 4oC for 18 h. Unspecific binding was blocked with TBS containing 1% BSA (Sigma-Aldrich) and 1 mM CaCl2 for 2 h at room temperature. Purified CD8α+ murine IEL were labeled with BCECF-AM [2′, 7′-bis(2-carboxyethyl)-5-(and 6)-carboxyflourescein acetomethylester, Molecular Probes, Leiden, The Netherlands] according to the manufacturer's instructions and 100,000 labeled IEL were added to each well. The adhesion plate was centrifuged for 3 min at 60×g and input fluorescence determined using a fluorescence plate reader (SpectraMAX GEMINI XS, Molecular Devices, Sunnyvale, CA). Cells were then incubated at 37oC for 3–5 min by floating the adhesion plate on preheated water. Nonadherent cells were removed by washing plates with pre-warmed HBSS with 1 mM CaCl2, and the remaining fluorescence in each well determined as above. For antibody blocking experiments, IEL were incubated on ice for 15 min with the indicated antibody (20 μg/ml) prior to addition to the adhesion plate. For pertussis toxin experiments IEL were pre-incubated with pertussis toxin (200 ng/ml) for 30 min at 37oC. This treatment had no effect on IEL viability as assessed by trypan blue exclusion.

4.7 Statistical analysis

Statistical analysis was performed using the Mann-Whitney U test or the Student's t-test where appropriate.

Acknowledgements

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

We would like to thank Dr. C. Parker (Dana Farber Cancer Institute, Boston, MA) for invaluable advice in the construction of the mEFc fusion protein, Drs. B. Malisson and M-A.Wurbel (Centre d'Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Medicale-Centre National de la Recherche Scientifique-Université de la Méditerranée, Marseille, France) for providing CCR9–/– mice, Dr. G. Marquez Marquez (National Center for Biotechnology/CSIC, Madrid, Spain) for providing anti-CCR9 antibody and Dr. D. Soler (Millenium Pharmaceuticals) for providing the anti-CCR6 antibody. This work was supported by grants from the Swedish Research Council (VR-Medicine), the Crafoordska, Österlund, Åke Wiberg, Richard and Ruth Julins, Nanna Svartz and Kocks foundations, the Lund Family American Cancer, the Royal Physiographic and the Swedish Medical Society and a Crohns and Colitis Foundation of America (CCFA) project grant to W. A.

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