QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/jcs.00142

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kutlesa, S. Articles by Klein, G. Search for Related Content PubMed PubMed Citation Articles by Kutlesa, S. Articles by Klein, G.

E-cadherin-mediated interactions of thymic epithelial cells with CD103⁺ thymocytes lead to enhanced thymocyte cell proliferation

Snjeana Kutlea¹, Johannes T. Wessels², Angelika Speiser¹, Inge Steiert¹, Claudia A. Müller¹ and Gerd Klein^1,*

¹ University Medical Clinic, Section for Transplantation Immunology and Immunohematology, Tübingen, Germany
² Department of Hematology and Oncology, Children's Hospital, University of Tübingen, Germany

View larger version (76K): [in a new window]   Fig. 1. Expression of cadherins and catenins in the human thymus. The micrographs show immunofluorescence staining of human thymus cryostat sections stained with monoclonal antibodies against E- and P-cadherin and ß- and -catenin. (A) Labelling with anti-E-cadherin antibodies revealed strong staining signals in the medulla (m) and weaker signals in the cortex (c) of the thymic lobules. (B) P-cadherin expression seemed to be restricted to epithelial cells of the cortical area, since medullary epithelial cells did not show any significant staining signals. (C) ß-catenin expression was evenly distributed over the medullary and cortical areas, whereas -catenin expression was found predominantly in the medulla (D). Bar, 100 µm.

View larger version (44K): [in a new window]   Fig. 2. Expression of E-cadherin on medullary epithelial cells. Double-immunofluorescence staining of human thymus cryostat sections with the anti-E-cadherin antibody HECD-1 (A) and the antibody TE4 (B) specific for medullary thymic epithelial cells showed colocalization (C) of both antigens on medullary epithelial cells. Note that not all E-cadherin-expressing cells in the medulla express the TE4 antigen. Bar, 100 µm.

View larger version (104K): [in a new window]   Fig. 3. Expression of cadherins and catenins on isolated thymic epithelial cells. Isolated primary thymic epithelial cells cultured on chamber slides were fixed and stained with antibodies against E-cadherin (A), P-cadherin (B), ß-catenin (C) and -catenin (D) followed by Cy3TM-labelled second antibodies. Control cultures were incubated only with the Cy3TM-labelled anti-mouse (E) or anti-rabbit (F) antibodies. The cultured thymic epithelial cells showed expression of all four analysed antigens. Expression was mainly found on the cell surfaces at sites of cell-cell contacts. Bar, 100 µm.

View larger version (29K): [in a new window]   Fig. 4. (A) Immunoblot analysis of cadherin-catenin expression in the human thymus. Protein extracts of human thymic tissue were separated on 10% polyacrylamide gels and transferred to nitrocellulose filters. After blocking, the filters were divided into five lanes and incubated with antibodies against E-cadherin, P-cadherin, -catenin, ß-catenin and -catenin. After colorimetric development, the 120 kDa bands of E-and P-cadherin (lane 1 and 2), the 102 kDa band of -catenin (lane 3), the 94 kDa band of ß-catenin (lane 4) and the 86 kDa band of -catenin (lane 5) were detected in the thymic extracts. Partial degradation products were observed for E-cadherin and ß-catenin. The positions of 120 and 94 kDa are indicated on the left side. (B) Co-immunoprecipitation of cadherins and catenins isolated from human thymus. Cell lysates of freshly isolated thymic tissue were immunoprecipitated (IP) with monoclonal antibodies against E-cadherin (lanes 1,6) and -catenin (lanes 4,9), with polyclonal antisera against -catenin (lanes 2,7) and ß-catenin (lanes 3,8) or without antibody (lane 5). The precipitated immune complexes were immunoblotted with antibodies against E-cadherin (lanes 1-4) and ß-catenin (lanes 5-9). Co-precipitation of E-cadherin with the different catenins and of ß-catenin with -catenin and E-cadherin, but not with -catenin, revealed functional cadherin-catenin complexes. (C) Immunoblot analysis of cadherin-catenin expression in thymic epithelial cells. Protein extracts of isolated thymic epithelial cells were separated on 10% polyacrylamide gels. After transfer to nitrocellulose, the filters were incubated with antibodies against E-cadherin (lane 1), P-cadherin (2), -catenin (3), ß-catenin (4) and -catenin (5). After colorimetric development, specific bands for all analysed antigens were detected. Partial degradation products were observed for ß-catenin and -catenin (lane 4 and 5). The positions of 120 and 86 kDa are indicated on the left side.

View larger version (14K): [in a new window]   Fig. 5. Flow cytometry analysis of cadherin expression on human thymocytes. Isolated thymocyte suspensions were stained with antibodies to E- and P-cadherin. The staining pattern showed no difference from the isotype control staining (thin line), indicating no expression of both cadherins on human thymocytes.

View larger version (59K): [in a new window]   Fig. 6. Detection of integrin E receptor (CD103) on human thymocyte subpopulations. (A) DN, DP and SP thymocyte subpopulations were isolated by magnetic cell sorting and subsequently labelled with the antibody against CD103. No expression of CD103 was observed in the DP thymocyte cell population, and only a marginal expression was seen in the CD4+ SP population. 12% of the DN subpopulation expressed CD103. The highest expression, however, was observed in the CD8+ SP population, in which more than one third of this subpopulation expressed CD103. (B) MACS-isolated CD4- CD8- DN thymocytes were triple-stained with antibodies against CD103 (FITC), CD25 (APC), and TCR /ß (PE) or TCR / (PE), respectively. The CD103+ DN thymocytes did not show any significant expression of TCR /ß or CD25. A subpopulation of CD103+ DN thymocytes expressed the TCR /, the majority of the CD103+ DN cells, however, did not express TCR /. (C) Isolated CD8+ SP thymocytes were analysed for TCR expression. By one round of magnetic cell sorting, a purity of >92% of CD8+ cells was achieved. 4.3% of the CD8+ cells expressed the / TCR, whereas 89% of these cells expressed the /ß TCR. By double staining of CD8+ cells it was shown that CD103+ CD8+ cells mostly expressed the /ß TCR, but not the / TCR. (D) Four-colour flow cytometric analysis was performed with MACS-sorted CD8+ SP thymocytes, which were labelled with antibodies against CD103 (FITC), CD24 (PE), CD62L (Cy5) and CD69 (APC). All CD103+ CD8+ SP thymocytes expressed CD69, but not CD24. When the CD103+ CD8+ cells were gated it could be demonstrated that all of these cells express L-selectin (CD62L) and CD69 simultaneously.

View larger version (124K): [in a new window]   Fig. 7. Localisation of integrin E (CD103) in the human thymus. (A) Staining of thymus cryostat sections with the antibody against CD103 revealed that the cell surfaces of individual thymocytes (arrowheads) within the medulla (m) are strongly labelled. Note that only a few thymocytes within the cortical region (c) were stained with the CD103 antibody. (B) Control staining was performed with the antibody W6/32. HK, an inactive variant of the anti-MHC class I antibody W6/32.HL. No background staining was observed with this antibody. Bar, 100 µm.

View larger version (52K): [in a new window]   Fig. 8. Inhibition of thymocyte adhesion to thymic epithelial cells by function-blocking antibodies to integrin E and E-cadherin. (A,B) The micrographs show adhesive interactions of MACS-isolated CD8+ thymocytes with primary thymic epithelial cells in the absence (A) or presence (B) of anti-CD103 antibodies. Without antibody treatment, the small, round and darkly stained thymocytes adhered strongly to the adherent epithelial cells. By contrast, anti-CD103 treatment strongly diminished thymocyte adhesion. Bar, 100 µm. (C) Thymocyte cell adhesion to thymic epithelial cells was quantified after BCECF-labelling of CD8+ thymocytes. Fluorescence intensity of the labelled cells used for the cell adhesion assay was set to 100% (lane 1). Around 40% of the CD8+ thymocytes attached to the adherent thymic epithelial cell layer (lane 2). The adhesion process was not influenced by addition of the W6/32. HL control antibody (lane 3). (D) Adhesive interactions of CD8+ thymocytes with thymic epithelial cells were inhibited by antibodies against E-cadherin and CD103. Fluorescence intensity of cell binding in the absence of the antibodies was set to 100% (lane 1). Both anti-E-cadherin (lane 2) and anti-CD103 (lane 3) antibodies diminished cell binding to 30-40%. Combined addition of both antibodies did not show an enhanced inhibitory effect (lane 4).

View larger version (22K): [in a new window]   Fig. 9. Inhibition of cell proliferation by anti-CD103 antibodies. (A) Isolated CD8+ SP thymocytes were co-cultured with primary thymic epithelial cells (Ep/CD8+), which induces a net increase in cell proliferation after 48 hours of culture. Antibodies against HLA-A,B,C (Ep/CD8+/HL) used as control antibodies did not significantly interfere with the proliferation process, whereas antibodies against E-cadherin (Ep/CD8+/a.E-cad) slightly blocked cell proliferation. Anti-CD103 antibodies (Ep/CD8+/a.CD103), however, drastically reduced cell proliferation. Results represent the mean of triplicate experiments. (B) CD103- CD8+ SP thymocytes, when co-cultured with primary thymic epithelial cells for 48 hours, did not show a net increase in cell proliferation (Ep/CD8+/CD103-) whereas CD8+-SP-containing CD103+ CD8+ SP thymocytes did (Ep/CD8+). Antibodies against CD103 inhibited cell proliferation drastically (Ep/CD8+/a.CD103).