- Email: [email protected]
Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110
Journal of Immunological Methods 297 (2005) 265 – 270 www.elsevier.com/locate/jim
Technical note
Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110 Soo Jung Yanga,b, Sejin Ahna, Chan-Sik Parka,1, Seeyoung Choia, Moon Gyo Kima,T a
Thymic Molecular Development Unit, Laboratory of Cellular and Molecular Immunology, NIAID, NIH, USA b Division of Biological Science, Seoul National University, Seoul, Republic of Korea Received 19 April 2004; received in revised form 6 December 2004; accepted 7 December 2004
Abstract We generated monoclonal antibodies reacting to a mouse thymic epithelial cell specific membrane protein, Thymic Stromal Co-transporter (TSCOT)/Ly110. These antibodies showed specificity to the peptide sequences derived from TSCOT/Ly110 determined by specific peptide inhibition in flow cytometric analyses with cells expressing the protein on the surface. TSCOT/ Ly110 expressing subpopulation can be identified among the CDR1+ or 6C3+ cortical epithelial cells. Furthermore, CDR1 positive cortical thymic epithelial cells can be separated into further distinguishable populations; CDR1+6C3+Ly110+, CDR1+6C3 /lowLy110+, CDR1+Ly110 . Some of TSCOT/Ly110 expressing cells negative for both CDR1 and 6C3 markers were found at the earlier stages of development, while most of the cells are positive for both at 1-week-old stage. After then, downregulation in 6C3 and/or CDR1 expression was noticed until 16 weeks of age. These results suggest that TSCOT/Ly110 is a new marker for the subpopulation of CDR1+ or 6C3+ epithelial cells in the neonatal and adult thymus and is useful for the studies on the epithelial cell differentiation process. Published by Elsevier B.V. Keywords: Flow cytometry; Thymic epithelial cells; TSCOT/Ly110; Specific marker
Among thymic stromal cells present in the microenvironment, epithelial cells are distinctively com-
Abbreviations: MFI, mean fluorescence intensity; TEC, thymic epithelial cell; TSCOT, Thymic Stroma Cotransporter. T Corresponding author. Tel.: +1 301 496 3842; fax: +1 301 402 3184. E-mail address: [email protected] (M.G. Kim). 1 Current address. Laboratory of Cellular Immunology, Ochsner Clinic Foundation, New Orleans, LA, USA. 0022-1759/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.jim.2004.12.021
partmentalized into the cortex and the medulla where thymocytes at different developmental stages reside. It is believed that the different epithelial cells provide critical signals to developing thymocytes. Therefore, for the studies of thymic differentiation, it is important to understand the nature of thymic epithelial cells (TECs) and how they differentiate. However, analysis of specific thymic compartmentalization at the TEC level by flow cytometry using cell surface molecules has been hindered, even though the cell preparation
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protocols with enzyme-mediated dissociation method make investigation on the TEC population possible (Anderson et al., 1993; Kim et al., 1998; Gray et al., 2002). There exist only a small number of reagents that can be used for compartment specific markers, such as CDR1 (Rouse et al., 1988; Derbinski et al., 2001) and 6C3 (Gray et al., 2002) for cortical thymic epithelial cells. From our earlier molecular approaches with PCR-based subtractive library prepared from purified thymic epithelial cells, we identified a new cortical epithelial cell surface protein, Thymic Stroma Co Transporter (TSCOT) (Kim et al., 1998, 2000). This protein is highly TEC specific and was registered as Ly110 (Chen et al., 2000). The message of TSCOT/Ly110 is not detectable in any of the peripheral tissues tested, in contrast to other cortical markers that are expressed in the epithelium of other tissues (Chen et al., 2000; Kim et al., 2000). In order to obtain monoclonal antibodies usable in flow cytometry, two peptide sequences (CLVE: CLVEYQEDQQQKAISN and KESF: KESFKSEAGGSC) near the N-glycosylation sites (Asn 57 and Asn 61) were chosen from the external portion of TSCOT/Ly110 (Fig. 1A). This protein was predicted as putative 12 membrane spanning protein that is highly hydrophobic (Kim et al., 2000) and does not contain any external soluble amino acid stretches long enough to be used as immunogen except for the first external loop with two predicted N-glycosylation sites. The two TSCOT/Ly110 specific monoclonal antibodies, CLVE-1 (rat IgM) and KESF-4 (rat IgM), were successfully generated in Dinona Inc. (Seoul, Korea) by immunizing designed synthetic peptides conjugated with diptheria toxoid into Sprague–Dawley rats. To examine the specificity of the newly generated monoclonal antibodies, we first tested them on the TSCOT/Ly110 expressing A20 cells. Both hybridoma supernatants from CLVE-1 and KESF-4 clone in the single color flow cytometry along with color conjugated anti-rat IgM show the staining patterns specific to the peptide sequences. The specific staining shift disappears after the addition of corresponding peptides but not after the addition of different peptides (Fig. 1B). Although those two peptides are located in the same first external loop,
they do not interfere the specific interaction between the other antibodies and their epitopes. Next we added these antibodies to freshly prepared thymic stromal cells and tested specificity in the CD45 negative gate that includes nonlymphoid thymic stromal cells (Fig. 1C). Approximately 10% of CD45 cells were identified as TSCOT/Ly110 positive with anti-CLVE and anti-KESF. Both antibodies show the identical profiles in the flow cytometric analysis. Those cells also express MHC II molecules on the surface at various levels. MHCII high expressing cells are the majority of TSCOT/ Ly110 positive cells. The presence of CLVE peptide clearly eliminated all the binding of anti-CLVE antibody to TSCOT/Ly110, while KESF peptide did not inhibit binding. Therefore we concluded that these two monoclonal antibodies specifically identify the population expressing TSCOT/Ly110 on the surface of the thymic epithelium. To further define the thymic epithelial cell population, we used compartment specific antibodies CDR1 and 6C3. Both CDR1 and 6C3 were suggested to bind the same molecule aminopeptidase A. When these two monoclonal antibodies were used the flow cytometry with bulk thymic stromal cell preparation, they appeared to bind the same cells (Gray et al., 2002). Among the newborn thymic stromal cells, all CLVE-1 positive cells are CDR1+, indicating that those are cortical TECs (Fig. 2A, top panel). However, when 6C3 is used, only part of CLVE-1 positive cells shows high level of 6C3 binding activity (Fig. 2A, bottom panel). Therefore, if CDR1 and 6C3 bind to the same aminopeptidase A, their epitopes are present at least as two different forms that are distributed differentially among cortical epithelial cells. Therefore, cTEC can be further subdivided into the following populations with our new monoclonal antibodies: CDR1+TSCOT/ Ly110+6C3+ and CDR1+TSCOT/Ly110+6C3 /low and CDR1+TSCOT/Ly110 . In more mature thymus, 6C3 levels appeared to be further decreased in the TSCOT/Ly110+ cells prepared in separate experiments (1 week and 4 weeks samples) as shown in Fig. 2A. To locate gates clearly separating negative and positive populations for CDR1 or 6C3 staining, and to distinguish the differences in the levels of markers, the all-but-one staining controls were included in the
S.J. Yang et al. / Journal of Immunological Methods 297 (2005) 265–270
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Fig. 1. Specificity of CLVE-1 and KESF-4 monoclonal antibodies to the sequences present in the TSCOT/Ly110. (A) Locations of the antigenic peptide sequences on the predicted membrane topology of TSCOT/Ly110. The first putative weak membrane domain is shown as a stippled box and other 11 putative membrane domains are shown in blank boxes with the numbers. Two glycosylation sites (Asn57 and Asn61) are indicated. (B) Competition with specific peptides to the ligand binding activities of anti-TSCOT/Ly110 antibodies. A20 cells stably expressing TSCOT/ Ly110 generated by transfection with pCDNA/TSCOTmycHis plasmid were stained with hybridoma culture supernatant and PE conjugated antirat IgM. Filled area is the histogram of cells stained with plain medium, thick gray line is for specific antibody staining for CLVE-1 in the top panel and for KESF-4 at the bottom panel. The profiles of the competition with 100 ng of HPLC purified CLVE or KESF peptides are shown in dotted lines. Mean fluorescent intensities (MFI) of each experiments are shown on the right. (C) Identification of TSCOT/Ly110 expressing population in the thymic stromal cells. Thymic stromal cells of 4-week-old mice were purified by using 0.25% trypsin and 1 mg/ml DNase I treatment after mincing, and MACS column with CD45 microbead (Miltenyi Biotec). Cells were stained with hybridoma culture supernatant and PE conjugated anti-rat IgM, FITC conjugated MHCII (I-Ab, AF6-120, mouse IgG2a, BD Biosciences), PE and Texas Red tandem conjugated CD45 (30-F11, rat IgG2b, Caltag, Burlingame, CA) and gated for CD45 negative and Propidium Iodide (20 Ag/ml) negative in FL3 channel. Analysis was done using the FlowJo program (Flowjo.com). The panel on the left shows the dot plot of TSCOT/Ly110 and MHCII, the middle two panels show inhibition patterns of the specific peptide and the irrelevant peptide. The right panel shows the dot plot profile with anti-KESF antibody.
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same-day experiments. The all-but-one staining controls contained all the other antibodies except for the one to be set as the negative (CDR1 on the left, 6C3 on the right in Fig. 2B). Three samples at different
ages revealed identical patterns for the negative gates (data not shown). As shown in Fig. 2C, during early development, the percentages of cells expressing CDR1 and 6C3 in
Fig. 2. (A) Analysis of thymic epithelial cells from different ages (newborn, 1 and 4 weeks in separate experiments) with cortical specific markers and TSCOT/Ly110. CDR1 (IgG2a, ATCC) profile is at the top, 6C3 (BP-1/6C3, rat IgG2a, BD Biosciences) at the bottom. All profiles were gated on CD45 negative cells. Zebra plot was used to locate the dip between the separable populations. (B) All-but-one antibody staining to set up gates for TSCOT/Ly110+ population. Antibody mixtures for the negative gate setup are shown on top. The profiles of CDR1 and 6C3 in the CLVE+ gate are shown. CDR1-negative setup on the left, 6C3-negative setup is on the right. For TSCOT/Ly110+ gate setup, antibody mixes including anti-rat IgM without CLVE mAB were used. (C) Developmental profiles of CDR1 and 6C3 in TSCOT/Ly110+ gate in a singleday experiment. CDR1 on top and 6C3 at the bottom. Gates are placed by all-but-one negative control. Percentages of positives and negatives for the two markers are indicated in the profile. Ages of the samples are shown on top. (D) The relationships of CDR1 and 6C3 expression in TSCOT/Ly110-expressing cells. CLVE+ gated cells were display for CDR1 and 6C3 markers. We noticed that newborn samples were variable in terms of relative levels of CDR1 and 6C3 levels, as seen in the difference between panels A and C.
269
Fig. 2 (continued ).
S.J. Yang et al. / Journal of Immunological Methods 297 (2005) 265–270
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TSCOT/Ly110 positive gates are increased until 1 week of age when most of the TSCOT/Ly110 positive cells were positive for both markers. After first week, CDR1 and 6C3 levels in TSCOT/Ly110+ cells appeared to be down regulated continuously until 16 weeks of age and more cells became CDR1 negative in older animal. We displayed TSCOT/Ly110+ population for CDR1 and 6C3 markers in Fig. 2D. In older animals, it becomes especially clear that CDR1+ cells are separable into two populations by the levels of 6C3 (high and low) while CDR1 population expresses low level of 6C3. In conclusion, anti-TSCOT/Ly110 antibodies can serve as a useful new reagent for the studies of the thymic compartments. Using these antibodies, it will be possible to distinguish TEC populations and address roles of specific populations in the thymic differentiation in future experiments. The complexity of cortical epithelial cells may play a role in forming a more functionally stratified thymic microenvironment to support a highly diverse thymocyte population (Petrie, 2003).
Acknowledgement We would like to thank Drs. Hyung Geun Song and Kyeong Cheon Jung (Dinona) for the production of hybridoma lines, Drs. Kevin Holmes and Larry Lanz in NIAID FACS facility for the large scale
production and conjugation of antibodies, Jenifer Westrup for last minute antibody production and critically reading manuscript and Dr. Ronald H. Schwartz for his support during this work.
References Anderson, G., Jenkinson, E.J., Moore, N.C., Owen, J.J., 1993. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362, 70. Chen, C., Kim, M.G., Soo Lyu, M., Kozak, C.A., Schwartz, R.H., Flomerfelt, F.A., 2000. Characterization of the mouse gene, human promoter and human cDNA of TSCOT reveals strong interspecies homology. Biochim. Biophys. Acta 1493, 159. Derbinski, J., Schulte, A., Kyewski, B., Klein, L., 2001. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2, 1032. Gray, D.H., Chidgey, A.P., Boyd, R.L., 2002. Analysis of thymic stromal cell populations using flow cytometry. J. Immunol. Methods 260, 15. Kim, M.G., Chen, C., Flomerfelt, F.A., Germain, R.N., Schwartz, R.H., 1998. A subtractive PCR-based cDNA library made from fetal thymic stromal cells. J. Immunol. Methods 213, 169. Kim, M.G., Flomerfelt, F.A., Lee, K.N., Chen, C., Schwartz, R.H., 2000. A putative 12 transmembrane domain cotransporter expressed in thymic cortical epithelial cells. J. Immunol. 164, 3185. Petrie, H.T., 2003. Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus. Nat. Rev., Immunol. 3, 859. Rouse, R.V., Bolin, L.M., Bender, J.R., Kyewski, B.A., 1988. Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells. J. Histochem. Cytochem. 36, 1511.
Technical note
Identifying subpopulations of thymic epithelial cells by flow cytometry using a new specific thymic epithelial marker, Ly110 Soo Jung Yanga,b, Sejin Ahna, Chan-Sik Parka,1, Seeyoung Choia, Moon Gyo Kima,T a
Thymic Molecular Development Unit, Laboratory of Cellular and Molecular Immunology, NIAID, NIH, USA b Division of Biological Science, Seoul National University, Seoul, Republic of Korea Received 19 April 2004; received in revised form 6 December 2004; accepted 7 December 2004
Abstract We generated monoclonal antibodies reacting to a mouse thymic epithelial cell specific membrane protein, Thymic Stromal Co-transporter (TSCOT)/Ly110. These antibodies showed specificity to the peptide sequences derived from TSCOT/Ly110 determined by specific peptide inhibition in flow cytometric analyses with cells expressing the protein on the surface. TSCOT/ Ly110 expressing subpopulation can be identified among the CDR1+ or 6C3+ cortical epithelial cells. Furthermore, CDR1 positive cortical thymic epithelial cells can be separated into further distinguishable populations; CDR1+6C3+Ly110+, CDR1+6C3 /lowLy110+, CDR1+Ly110 . Some of TSCOT/Ly110 expressing cells negative for both CDR1 and 6C3 markers were found at the earlier stages of development, while most of the cells are positive for both at 1-week-old stage. After then, downregulation in 6C3 and/or CDR1 expression was noticed until 16 weeks of age. These results suggest that TSCOT/Ly110 is a new marker for the subpopulation of CDR1+ or 6C3+ epithelial cells in the neonatal and adult thymus and is useful for the studies on the epithelial cell differentiation process. Published by Elsevier B.V. Keywords: Flow cytometry; Thymic epithelial cells; TSCOT/Ly110; Specific marker
Among thymic stromal cells present in the microenvironment, epithelial cells are distinctively com-
Abbreviations: MFI, mean fluorescence intensity; TEC, thymic epithelial cell; TSCOT, Thymic Stroma Cotransporter. T Corresponding author. Tel.: +1 301 496 3842; fax: +1 301 402 3184. E-mail address: [email protected] (M.G. Kim). 1 Current address. Laboratory of Cellular Immunology, Ochsner Clinic Foundation, New Orleans, LA, USA. 0022-1759/$ - see front matter. Published by Elsevier B.V. doi:10.1016/j.jim.2004.12.021
partmentalized into the cortex and the medulla where thymocytes at different developmental stages reside. It is believed that the different epithelial cells provide critical signals to developing thymocytes. Therefore, for the studies of thymic differentiation, it is important to understand the nature of thymic epithelial cells (TECs) and how they differentiate. However, analysis of specific thymic compartmentalization at the TEC level by flow cytometry using cell surface molecules has been hindered, even though the cell preparation
266
S.J. Yang et al. / Journal of Immunological Methods 297 (2005) 265–270
protocols with enzyme-mediated dissociation method make investigation on the TEC population possible (Anderson et al., 1993; Kim et al., 1998; Gray et al., 2002). There exist only a small number of reagents that can be used for compartment specific markers, such as CDR1 (Rouse et al., 1988; Derbinski et al., 2001) and 6C3 (Gray et al., 2002) for cortical thymic epithelial cells. From our earlier molecular approaches with PCR-based subtractive library prepared from purified thymic epithelial cells, we identified a new cortical epithelial cell surface protein, Thymic Stroma Co Transporter (TSCOT) (Kim et al., 1998, 2000). This protein is highly TEC specific and was registered as Ly110 (Chen et al., 2000). The message of TSCOT/Ly110 is not detectable in any of the peripheral tissues tested, in contrast to other cortical markers that are expressed in the epithelium of other tissues (Chen et al., 2000; Kim et al., 2000). In order to obtain monoclonal antibodies usable in flow cytometry, two peptide sequences (CLVE: CLVEYQEDQQQKAISN and KESF: KESFKSEAGGSC) near the N-glycosylation sites (Asn 57 and Asn 61) were chosen from the external portion of TSCOT/Ly110 (Fig. 1A). This protein was predicted as putative 12 membrane spanning protein that is highly hydrophobic (Kim et al., 2000) and does not contain any external soluble amino acid stretches long enough to be used as immunogen except for the first external loop with two predicted N-glycosylation sites. The two TSCOT/Ly110 specific monoclonal antibodies, CLVE-1 (rat IgM) and KESF-4 (rat IgM), were successfully generated in Dinona Inc. (Seoul, Korea) by immunizing designed synthetic peptides conjugated with diptheria toxoid into Sprague–Dawley rats. To examine the specificity of the newly generated monoclonal antibodies, we first tested them on the TSCOT/Ly110 expressing A20 cells. Both hybridoma supernatants from CLVE-1 and KESF-4 clone in the single color flow cytometry along with color conjugated anti-rat IgM show the staining patterns specific to the peptide sequences. The specific staining shift disappears after the addition of corresponding peptides but not after the addition of different peptides (Fig. 1B). Although those two peptides are located in the same first external loop,
they do not interfere the specific interaction between the other antibodies and their epitopes. Next we added these antibodies to freshly prepared thymic stromal cells and tested specificity in the CD45 negative gate that includes nonlymphoid thymic stromal cells (Fig. 1C). Approximately 10% of CD45 cells were identified as TSCOT/Ly110 positive with anti-CLVE and anti-KESF. Both antibodies show the identical profiles in the flow cytometric analysis. Those cells also express MHC II molecules on the surface at various levels. MHCII high expressing cells are the majority of TSCOT/ Ly110 positive cells. The presence of CLVE peptide clearly eliminated all the binding of anti-CLVE antibody to TSCOT/Ly110, while KESF peptide did not inhibit binding. Therefore we concluded that these two monoclonal antibodies specifically identify the population expressing TSCOT/Ly110 on the surface of the thymic epithelium. To further define the thymic epithelial cell population, we used compartment specific antibodies CDR1 and 6C3. Both CDR1 and 6C3 were suggested to bind the same molecule aminopeptidase A. When these two monoclonal antibodies were used the flow cytometry with bulk thymic stromal cell preparation, they appeared to bind the same cells (Gray et al., 2002). Among the newborn thymic stromal cells, all CLVE-1 positive cells are CDR1+, indicating that those are cortical TECs (Fig. 2A, top panel). However, when 6C3 is used, only part of CLVE-1 positive cells shows high level of 6C3 binding activity (Fig. 2A, bottom panel). Therefore, if CDR1 and 6C3 bind to the same aminopeptidase A, their epitopes are present at least as two different forms that are distributed differentially among cortical epithelial cells. Therefore, cTEC can be further subdivided into the following populations with our new monoclonal antibodies: CDR1+TSCOT/ Ly110+6C3+ and CDR1+TSCOT/Ly110+6C3 /low and CDR1+TSCOT/Ly110 . In more mature thymus, 6C3 levels appeared to be further decreased in the TSCOT/Ly110+ cells prepared in separate experiments (1 week and 4 weeks samples) as shown in Fig. 2A. To locate gates clearly separating negative and positive populations for CDR1 or 6C3 staining, and to distinguish the differences in the levels of markers, the all-but-one staining controls were included in the
S.J. Yang et al. / Journal of Immunological Methods 297 (2005) 265–270
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Fig. 1. Specificity of CLVE-1 and KESF-4 monoclonal antibodies to the sequences present in the TSCOT/Ly110. (A) Locations of the antigenic peptide sequences on the predicted membrane topology of TSCOT/Ly110. The first putative weak membrane domain is shown as a stippled box and other 11 putative membrane domains are shown in blank boxes with the numbers. Two glycosylation sites (Asn57 and Asn61) are indicated. (B) Competition with specific peptides to the ligand binding activities of anti-TSCOT/Ly110 antibodies. A20 cells stably expressing TSCOT/ Ly110 generated by transfection with pCDNA/TSCOTmycHis plasmid were stained with hybridoma culture supernatant and PE conjugated antirat IgM. Filled area is the histogram of cells stained with plain medium, thick gray line is for specific antibody staining for CLVE-1 in the top panel and for KESF-4 at the bottom panel. The profiles of the competition with 100 ng of HPLC purified CLVE or KESF peptides are shown in dotted lines. Mean fluorescent intensities (MFI) of each experiments are shown on the right. (C) Identification of TSCOT/Ly110 expressing population in the thymic stromal cells. Thymic stromal cells of 4-week-old mice were purified by using 0.25% trypsin and 1 mg/ml DNase I treatment after mincing, and MACS column with CD45 microbead (Miltenyi Biotec). Cells were stained with hybridoma culture supernatant and PE conjugated anti-rat IgM, FITC conjugated MHCII (I-Ab, AF6-120, mouse IgG2a, BD Biosciences), PE and Texas Red tandem conjugated CD45 (30-F11, rat IgG2b, Caltag, Burlingame, CA) and gated for CD45 negative and Propidium Iodide (20 Ag/ml) negative in FL3 channel. Analysis was done using the FlowJo program (Flowjo.com). The panel on the left shows the dot plot of TSCOT/Ly110 and MHCII, the middle two panels show inhibition patterns of the specific peptide and the irrelevant peptide. The right panel shows the dot plot profile with anti-KESF antibody.
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same-day experiments. The all-but-one staining controls contained all the other antibodies except for the one to be set as the negative (CDR1 on the left, 6C3 on the right in Fig. 2B). Three samples at different
ages revealed identical patterns for the negative gates (data not shown). As shown in Fig. 2C, during early development, the percentages of cells expressing CDR1 and 6C3 in
Fig. 2. (A) Analysis of thymic epithelial cells from different ages (newborn, 1 and 4 weeks in separate experiments) with cortical specific markers and TSCOT/Ly110. CDR1 (IgG2a, ATCC) profile is at the top, 6C3 (BP-1/6C3, rat IgG2a, BD Biosciences) at the bottom. All profiles were gated on CD45 negative cells. Zebra plot was used to locate the dip between the separable populations. (B) All-but-one antibody staining to set up gates for TSCOT/Ly110+ population. Antibody mixtures for the negative gate setup are shown on top. The profiles of CDR1 and 6C3 in the CLVE+ gate are shown. CDR1-negative setup on the left, 6C3-negative setup is on the right. For TSCOT/Ly110+ gate setup, antibody mixes including anti-rat IgM without CLVE mAB were used. (C) Developmental profiles of CDR1 and 6C3 in TSCOT/Ly110+ gate in a singleday experiment. CDR1 on top and 6C3 at the bottom. Gates are placed by all-but-one negative control. Percentages of positives and negatives for the two markers are indicated in the profile. Ages of the samples are shown on top. (D) The relationships of CDR1 and 6C3 expression in TSCOT/Ly110-expressing cells. CLVE+ gated cells were display for CDR1 and 6C3 markers. We noticed that newborn samples were variable in terms of relative levels of CDR1 and 6C3 levels, as seen in the difference between panels A and C.
269
Fig. 2 (continued ).
S.J. Yang et al. / Journal of Immunological Methods 297 (2005) 265–270
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S.J. Yang et al. / Journal of Immunological Methods 297 (2005) 265–270
TSCOT/Ly110 positive gates are increased until 1 week of age when most of the TSCOT/Ly110 positive cells were positive for both markers. After first week, CDR1 and 6C3 levels in TSCOT/Ly110+ cells appeared to be down regulated continuously until 16 weeks of age and more cells became CDR1 negative in older animal. We displayed TSCOT/Ly110+ population for CDR1 and 6C3 markers in Fig. 2D. In older animals, it becomes especially clear that CDR1+ cells are separable into two populations by the levels of 6C3 (high and low) while CDR1 population expresses low level of 6C3. In conclusion, anti-TSCOT/Ly110 antibodies can serve as a useful new reagent for the studies of the thymic compartments. Using these antibodies, it will be possible to distinguish TEC populations and address roles of specific populations in the thymic differentiation in future experiments. The complexity of cortical epithelial cells may play a role in forming a more functionally stratified thymic microenvironment to support a highly diverse thymocyte population (Petrie, 2003).
Acknowledgement We would like to thank Drs. Hyung Geun Song and Kyeong Cheon Jung (Dinona) for the production of hybridoma lines, Drs. Kevin Holmes and Larry Lanz in NIAID FACS facility for the large scale
production and conjugation of antibodies, Jenifer Westrup for last minute antibody production and critically reading manuscript and Dr. Ronald H. Schwartz for his support during this work.
References Anderson, G., Jenkinson, E.J., Moore, N.C., Owen, J.J., 1993. MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus. Nature 362, 70. Chen, C., Kim, M.G., Soo Lyu, M., Kozak, C.A., Schwartz, R.H., Flomerfelt, F.A., 2000. Characterization of the mouse gene, human promoter and human cDNA of TSCOT reveals strong interspecies homology. Biochim. Biophys. Acta 1493, 159. Derbinski, J., Schulte, A., Kyewski, B., Klein, L., 2001. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat. Immunol. 2, 1032. Gray, D.H., Chidgey, A.P., Boyd, R.L., 2002. Analysis of thymic stromal cell populations using flow cytometry. J. Immunol. Methods 260, 15. Kim, M.G., Chen, C., Flomerfelt, F.A., Germain, R.N., Schwartz, R.H., 1998. A subtractive PCR-based cDNA library made from fetal thymic stromal cells. J. Immunol. Methods 213, 169. Kim, M.G., Flomerfelt, F.A., Lee, K.N., Chen, C., Schwartz, R.H., 2000. A putative 12 transmembrane domain cotransporter expressed in thymic cortical epithelial cells. J. Immunol. 164, 3185. Petrie, H.T., 2003. Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus. Nat. Rev., Immunol. 3, 859. Rouse, R.V., Bolin, L.M., Bender, J.R., Kyewski, B.A., 1988. Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells. J. Histochem. Cytochem. 36, 1511.