The Thymic Stromal Cell Line MTSC4 Induced Thymocyte Apoptosis in a Non- MHC-Restricted Manner

Cell research (2004); 14 (2): 125-133 ARTICLECell research, 14(2), April 2004 http://www.cell-research.com Xue Ying HE et al The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non- MHC-restricted manner Xue Ying HE1, Juan LI1, Xiao Ping QIAN1, Wen Xian FU1, Yan LI1, Li WU2, Wei Feng CHEN1 1Department of Immunology, Peking University Health Science Center,38 Xueyuan Road, Beijing 100083, China. 2The Walter and Eliza Hall Institute, PO Royal Melbourne Hospital, Victoria 3050, Australia. ABSTRACT Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4+8+ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC- restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis. Keywords: MHC, thymic dendritic cells, thymic stromal cell line, thymocyte apoptosis. INTRODUCTION (TDCs) as professional antigen presenting cells express The communications between thymic microenvironmental high levels of MHC class II molecules and their role in stromal cells and thymocytes determine the development negative selection has been demonstrated in vivo and in and fate of thymocytes. During maturation, the thymocytes vitro[3, 4]. Apart from TDCs, other types of thymic stro- interact with the stromal cells, including epithelial cells, mal cells may also be involved in negative selection, such fibroblasts and dendritic cells, to undergo T cell receptor as thymic medullary epithelial cells[5-7]. (TCR) gene rearrangement and TCR/CD3 expression, thy- MTSC4 is a mouse thymic dendritic cell-like cell line mic selection and functional maturation. Although the de- established in our laboratory. It was identified by the ex- tailed molecular mechanism of thymic selection remains pression of vimentin and S-100 protein and lacking of char- unclear, it is generally believed that the positive and negative acteristic epithelial cell molecules, such as cytokeratin, selection of immature thymocytes depends on the avidity of tonofilaments and desmosomes[8]. Further characteriza- T cell receptor (TCR) to peptide/MHC complex and requires tion showed that 70% of MTSC4 cells were CD40 positive, secondary signals which are poorly defined. High avidity express low levels of B7.1, CD8α and MHC class I and interaction between TCR and peptide/MHC molecules in- do not express surface MHC class II and B7.2. The duces negative selection resulting in cell apoptosis, whereas proportion of MTSC4 cells expressing DEC-205 varied low avidity interaction between them leads to positive among the MTSC4 sub-clones (5%-54%). However, upon selection for cell survival [1, 2]. Thymic dendritic cells stimulation by a combination of cytokines (IFN-γ, IL-4, GM-CSF and anti-CD40 mAb), the clone of MTSC4-10 *Correspondence: Wei Feng CHEN, Tel: 0086-10-82802593, Fax: 0086-10-82801436, was upregulated on the expression of MHC class I, class E-mail: [email protected] II and B7.1. The up-regulation of MHC class II was IFN-γ Abbreviations: BSS, balanced salt solution; DN, double-negative; DP, dependent, and omission of any one of the cytokines from double-positive; Int, intermediate; MTEC1, mouse thymic epithelial the cocktail reduced the up-regulation of B7.1 expression. cell line 1; MTSC4, mouse thymic stromal cell line 4; NCS, newborn calf serum; SP, single-positive; TDC, thymic dendritic cell; TRC, thy- Our previous studies also showed that application of an- mic reaggregation culture. other cocktail of cytokines, including IL-1, IL-3, SCF and 125 Thymocyte apoptosis in a non-MHC-restricted manner http://www.cell-research.com Flt3L, did not change the cell phenotype. These results containing 5 mM EDTA). The low-density cell fraction was ob- demonstrated that MTSC4 lacked the surface markers tained by centrifugation for 10 min at 1,700 g, 4oC. To deplete non- typical for thymic epithelial cells and dendritic cells (DCs). DC cells, the recovered low-density cells were incubated with a However, upon activation, they up-regulated the expres- cocktail containing the following mAbs: anti-CD3, anti-CD4, anti- erythroid cell antigen, anti-Gr-1, anti-B220 and anti-Thy1.2 (1/3 sion of some surface molecules normally expressed on saturation concentration) for 30 min at 4oC. Cells were then washed DCs. and depleted with anti-Ig-coated magnetic beads (Paesel and Lorei, To determine the relationship between MTSC4 and the GMBH & Co, Frankfurt, Germany) at 10:1 bead-to-cell ratio. The thymic dendritic cells (TDCs) and to investigate the pos- mixture of beads and cells was resuspended in EDTA-BSS-NCS and sible functions of these cells during thymic selection, we incubated for 25 min with continuous slow rotation at 4oC. To obtain compared MTSC4 to freshly isolated TDCs for their ca- purified TDCs, the cell preparation was stained and sorted for + α+ pacity to induce thymocyte apoptosis. MTSC4 cells could CD11c CD8 cells by flow cytometry Vantage (Becton Dic-kinson, + + Mountain View, CA). The purity of separated TDCs was 95% after efficiently augment the apoptosis of CD4 8 thymocytes reanalyzed on FACS. in a Fas/FasL independent manner. In contrast to TDCs, the induction of thymocyte apoptosis by MTSC4 was not Effects of cytokines on MTEC1, MTSC4 and MTSC4-10 MHC-restricted. These results suggest that different mo- Murine rIFN−γ was purchased from PharMingen. Murine rIL-4, lecular pathways may be involved in the induction of thy- murine rGM-CSF and anti-CD40 mAb (FGK45.5) were obtained mocyte apoptosis in MTSC4 and TDCs. from Immunex Corp. (Seattle, WA) and from Dr. A. Rolink (Basel Institute for Immunology). MTEC1, MTSC4 and MTSC4-10 cells MATERIALS AND METHODS (2.5×104 cells/ml) were cultured in media supplemented with a cocktail of cytokines containing mrIL-4 (100U/ml), mrGM-CSF (200 ng/ Mice ml), IFN-γ (4 ng/ml) for 48 h, then added anti-CD40 mAb (1 µg/ml) Six to eight week-old, specific pathogen-free female BALB/c (H- and the cells were cultured for additional 24 h. Cells were then har- 2d) and C57BL/6 (H-2b) mice were obtained from the Department of vested and analyzed by FACS Calibur (Becton Dickinson, Moun- Laboratory Animal Science, Peking University Health Science Center. tain View, CA). Six to eight week-old C3H (H-2k) mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. Preparation of thymic subsets Fresh adult thymuses were homogenized to a single-cell suspen- Cell lines sion in cold RPMI-1640 medium containing 2% newborn calf serum MTEC1 (mouse thymic medullary epithelial cell line 1), MTSC4 (NCS), using steel mesh. The cells were stained with anti-CD8α- (mouse thymic stromal cell line 4) and its clone, MTSC4-10, were Cychrome, anti-CD4-APC, and sorted for CD4+8+ (DP), CD4+CD8- established from the thymus of BALB/c mice in our laboratory[8, (CD4SP) and CD4-CD8+ (CD8SP) cells by flow cytometry on FACS 9]. 3T3 fibroblast cell line was purchased from ATCC (VA, USA). Vantage. The purity of separated thymocyte subsets was 97-98% after reanalyzed on FACS. Monoclonal antibodies Fluorescence-conjugated monoclonal antibodies used for flow Thymic reaggregation cultures (TRC) cytometry analysis including: anti-MHC class II I-A(M5/114)-phy- The TRC was based on the method established by Anderson et al. coerythrin (PE), anti-CD8α(53-6.7)-PerCP, anti-CD8α(53-6.7)- [4]. Freshly prepared thymocytes and appropriate stromal cells were Cychrome, anti-CD4(RM4-5)-APC, and anti-CD3(145-2C11)-FITC mixed together at necessary ratios and pelleted by centrifugation. were purchased from PharMingen (San Diego, CA). Anti-DEC-205 Following removal of the supernatant, the cell pellet was carefully (NLDC-145)-PE was a kind gift given by Dr. Shortman (Melbourne, transferred to the surface of a 0.8 µm Nucleopore filter supported by Australia). Supernatants or ascites of mAbs used for the depletion gelatin foam sponges. by immuno-magnetic beads or for cell cultures were generated in our laboratory, including: anti-erythroid cell antigen (Ter-119), anti- Flow cytometric analysis Thy1.2 (30H12), anti-Gr-1(RB68C5), anti-CD3 (KT3-1.1), anti- MTSC4 and MTSC4-10 cells were dispersed in 0.1% EDTA- CD4 (RL172), anti-B220 (RA36B2), anti-B7.2 (GL1) , anti-CD11c PBS (pH 8.0) at 37oC for 15 min. After blocking with mouse and rat (N418), anti-MHC II I-A (M5/114) and anti-MHC class I (HB-79). sera, the single cell suspensions were stained with a panel of conjugated mAbs (see Monoclonal antibodies) for 30 min at 4oC. Stained Isolation of thymic dendritic cells (TDCs) cells were analyzed on FACS Calibur. Data acquisition and analysis Thymic DCs were isolated according to Vremec et al [10].

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