The 3.1 kb and 9.1 kb TSCOT promoters drive compartment specific expression of EGFP
To study the role of TEC subsets for thymocyte development, we generated an inducible TEC ablation system. For this purpose, we used two different lengths of the TEC specific TSCOT promoter to direct the bicistronic expression of EGFP and NTR for monitering the transgene expression and for inducing cell death, respectively ().
Transgenic mouse lines with TEC targeted expression of NTR-IRES-EGFP
Transgenic mice expressing the 3.1 kb (3.1T-NE) or 9.1 kb fragment (9.1T-NE) containing additional 6 kb upstream sequences were able to express bicistronic NTR-IRES-EGFP in the thymus. As seen in , both transgenic lines showed green fluorescence in the thymus and kidney but not in the spleen or other tissues when tested at newborn stages ( and data not shown). The signals in the newborn kidneys were likely due to a high level of autofluorescence seen as a strong signal in red channels. Nontransgenic thymuses from C57BL/6 mice showed little autofluorescence ( and data not shown).
When adult thymuses were tested for EGFP expression, two founder lines of 3.1T-NE lines (M7 and G8) showed the expression in the medulla and the corticomedullary junction (CMJ) ( and data not shown). In contrast, 9.1T-NE founder lines (B3 and NREP) showed a more limited expression in the cortical area ( and data not shown). No signal was detected in the thymus from C57BL/6 mice. In addition, there was no sign of EGFP expression in any of the thymocytes (data not shown). Based on these results, we concluded that the expression of the transgene is compartment specific due to the difference in the regulatory elements in each length of the promoter.
Specificity of NTR-mediated apoptotic cell death without significant bystander killing
Having established the expression pattern of two transgenes, we tested the inducible cell ablation using NTR. To do this, we chose to test less conventional prodrugs, MTZ and NFT, instead of more commonly used CB1954 (Bailey and Hart, 1997
) since they are more readily available from commercial sources. MTZ and NFT were previously evaluated as useful candidates as NTR prodrugs (Bridgewater et al., 1997
). In addition, CB1954 was reported to show bystander killing through cell permeable metabolites (Bailey et al., 1996
To test the killing effect in vitro first, we introduced NIH3T3 cells with a construct that expresses NTR and EGFP by the CMV promoter, pCMV-NTR-IRES-EGFP. As a control, pEGFP-N1 which does not have the NTR gene, was used. As shown in , EGFP expressing cells showed the NTR activity (red fluorescence) when pCMV-NTR-IRES-EGFP was introduced into the cell. Next, the effect of prodrugs was examined by comparing cell survival after treating cells for 3 days with prodrugs (). The result showed that both MTZ and NFT treatments revealed dose dependent decreases in the number of cells expressing NTR but not the control (pEGFP-N1 transfected) up to 10 µM concentrations of prodrugs. However, a concentration of MTZ and NFT higher than 100 µM showed NTR-independent killing of the cells although the killing effects (NTR+/NTR−) was better (data not shown). At 1,000 µM, nearly all the cells did not survive. From these results, we chose to use 30 µM for the subsequent experiments.
Evaluation of prodrugs, MTZ and NFT for NTR mediated apoptosis in cell culture
Next, we tested whether cell killing in the presence of the prodrug is specific or if the prodrug has a side effect of killing innocent cells. When 293T cells were transfected with pCMV-NTR-IRES-EGFP, the number of NTR expressing cells decreased at a 30 µM concentration (). In fact, EGFP expressing cells were smaller and round but neighboring cells without EGFP expression maintained the intact morphology of cells (arrows in ), suggesting that there was no significant bystander killing at this concentration. Only EGFP+ cell population in the 293T cells transfected with pCMV-NTR-IRES-EGFP showed annexin V staining (, , and data not shown). EGFP− cells showed no binding of annexin V at the ranges of 10–30 µM prodrugs. Neither the DMSO treated pCMV-NTR-IRES-EGFP transfected cells nor cells transfected with pEGFP-N1 showed a sign of non NTR-associated cell death (data not shown). Treatment of MTZ at the same concentration also showed little bystander killing activity (data not shown). These data indicate that bystander killing of the non-NTR-producing cells by the prodrug treatment is insignificant under the condition that we tested. Together, we verified that MTZ and NFT specifically ablated NTR expressing cells in vitro and that two prodrugs are effective in inducing cell ablation.
Analysis of apoptosis on the HEK 293T cells transfected with pCMV-NTR-EGFP after treatment with NFT
Ablation of TEC in 3.1T-NE and 9.1T-NE transgenic fetal thymus
In order to identify the type of cells expressing EGFP, thymuses were treated with 2-dGuo for 1 week to deplete hematopoietic cells and cell suspensions were prepared from the culture to examine TEC subsets using flow cytometry (). As shown in , the pan epithelial marker EpCAM separated the population into 4 groups. After analyzing various combinations for the separation of a distinctive TEC population, we decided to include autofluorescence for the first gate along with EpCAM since it gave the best separation for EGFP expression. Then, 4 separate populations (S1–S4) were further divided into 7 separate subsets (S1, S2a, S2b, S3a, S3b, S4a, and S4b) based on the levels of EpCAM, MHCII, and CDR1 as well as EGFP expression levels (). In contrast to the location of specific compartments in adult thymus, both transgenic lines (3.1T-NE and 9.1T-NE) showed no distinctively separable populations; presumably both promoters are active in the overlapping population and at the precursor stages of TEC development. The negative control profiles for the EGFP expression derived from the nontransgenic thymus were variable among the TEC subsets (data not shown). In addition, transgenic EGFP profiles showed significant overlap in the cases of the lower EGFP expression as expected. Nonetheless, we carefully located the positive gates by considering the shapes of the histogram profiles that can be most separable from the negative profiles. Overall 3.1T-NE showed more EGFP+ cells in S1, S2b, S3b, and S4b subsets. A noticeable qualitative difference between 3.1T-NE and 9.1T-NE was located in subset S2b that was Ep-CAMloCDR1medMHCIImed. EGFPhigh expressor was found only in 9.1T-NE TEC population (marked with *). While 3.1T-NE showed no EGFPhigh expression, 9.1T-NE showed 6.17% EGFP expressing cells. The presence of EGFPhigh cells in the S2b subset appeared reproducible only in 9.1T-NE. These results indicate that thymic epithelial cells prepared from 2-dGuo treated fetal thymus are a heterogeneous mixture that can be divided into more distinct subsets using the TSCOT promoter activity along with EpCAM, MHCII, and CDR1. At this point, although it is difficult to assign these fetal TEC subsets to any specific TEC lineages, CDR1high subsets are more likely to be the cTEC committed cells since CDR1 is a cTEC specific marker.
Expression of EGFP in fetal TEC compartment of 3.1T-NE and 9.1T-NE transgenic mice
We next examined TEC ablation in FTOCs by treating the culture with prodrugs in the presence of 2d-Guo (). shows EGFP expression patterns in the resulting thymic rudiments from 3.1T-NE transgenic thymuses. EGFP signals detectable in the E14.5 fetal thymus disappeared after treatment of prodrug MTZ for a week, while nontransgenic C57BL/6 did not show any sign of green fluorescence. The degree of depletion of EGFP expressing cells was further examined by flow cytometric analysis. As seen in , the overall recoveries of total cell numbers from thymic rudiments (n = 14 each group) with 30 µM MTZ were about 60–70%. Despite a poor recovery of the cells, the degree of depletion in each subset of 3.1T-NE TEC population was in agreement with the relative proportion of EGFP+ cells shown in . Among all subsets, S1 and S4b subsets were highly susceptible showing 64.5% and 58% EGFP expressing cells, respectively (). Treatment of 30 µM MTZ or NFT in 9.1T-NE thymuses also yielded reductions in all the subsets, particularly in S2b, S3a and S4b.
Depletion of NTR and EGFP expressing TEC from FTOC with prodrugs MTZ and NFT
We further verified the depletion of TEC in 9.1T-NE using UEA-1 binding together with CDR-1 and MHCII. Previously, UEA-1 has been considered as a mTEC specific marker but the appearance of UEA-1 binding has not been clearly established in the fetal thymus. Analysis of UEA-1 and CDR1 revealed two small but distinct subsets, U1 (UEA-1med, MHCIIlo, CDR1lo) and U2 (UEA-1high, MHCIImed, CDR1med) (). It is possible that these two populations may be precursor cells since they do not express high levels of MHCII. Alternatively, they may be the cells committed to the mTEC lineage. The profiles for EpCAM, MHCII, and CDR1 of the same cells revealed that the U2 subset overlaps with the S2b subset, MHCIImed, and CDR1med (). Major CDR1 expressing population was divided into 2 populations by the different levels of MHCII and EGFP expression (U3 and U4). In addition, the U5 subset was identified by a unique pattern of MHCII and high EGFP levels. Using the subset gates, depletion of each subset with MTZ and NFT prodrugs was shown in . One of the major populations, CDR1 expressing U4 subset without EGFP expression did not show any sign of depletion, indicating little non-specific killing. Interestingly, the U2 subset was depleted most severely in 9.1T-NE stroma. Together with results from , subsets S2b and U2, both of which contain the same types of cells, (EpCAMloCDR1medMHCIImedUEA1hi) were depleted most. Although the absolute numbers were small in each subset, a similar pattern of EGFP expression and the degree of depletion with prodrug treatments indicate that we can achieve specific depletion of TEC using the inducible system.
Analysis of 9.1T-NE TEC with UEA-1 marker after prodrug induced depletion
Effect of TEC ablation on thymocyte development
After establishing the efficacy of the inducible cell ablation system, we investigated the effect of TEC depletion on thymocyte development. To do this, we set up a FTOC from two founder lines of 3.1T-NE (M7 and G8) and treated them with increasing doses of MTZ up to 30 µM. Results showed the dose dependent reduction in the recovery of total thymocyte numbers in the culture of 3.1T-NE not C57BL/6, suggesting that NTR independent nonspecific toxicity was minimum (). The results from two founder lines of 3.1T-NE were indistinguishable (data not shown) and therefore we focused on the G8 founder line for 3.1T-NE and NREP for 9.1T-NE for subsequent experiments. The average of total thymocyte recovery of MTZ prodrug treated 9.1T-NE FTOC was 39.1+/−3.67% of DMSO treated 9.1T-NE FTOC ( on the right).
Representative analysis of thymocyte development in the presence of prodrugs during FTOC
Next, we set up a large scale time pregnancy using normal and two transgenic lines (40–60 females per group) for a single experiment. Total thymocyte recoveries from the FTOC of C57BL/6, transgenic 3.1T-NE and 9.1T-NE were compared after treating cultures with MTZ or NFT at 30 µM. Neither prodrug treatments showed a toxic effect in C57BL/6, whereas the treatments clearly induced NTR-dependent killing in the FTOCs of 3.1T-NE and 9.1T-NE (). There were noticeable variations in the absolute numbers (per lobe) recovered from untreated cultures among the strains (). However, a comparison of the percentages of total thymocyte recovery with and without treatments showed a clear effect only in transgenic FTOCs.
As shown in , the relative recoveries of γδ T cells, DN1, DN4, DP, and CD4 cells from C57BL/6 showed little interference by the presence of prodrugs in FTOC. Neither MTZ nor NFT seemed to have the nonspecific toxicity in those cell populations. The only deviation with an unknown reason resided in the CD8 population. Inclusion of prodrugs in the FTOC showed a severe reduction in DN4, DP, and CD4 population in both transgenic 3.1T-NE and 9.1T-NE. Although the general patterns of two transgenic lines were similar, the recovery of γδ T cells and DN3 was different between the two transgenic lines. As summarized in , the relative recovery of γδ T cells was clearly diminished in the 9.1T-NE FTOC, whereas DN3 cells were reduced in the 3.1T-NE line. The recoveries of absolute numbers of DP cells showed significant and comparable reductions in both lines.
The relative percentage of thymocyte subsets recovered from prodrug treatment
In conclusion, TEC depletion of the transgenic thymus with prodrugs resulted in a reduction of thymocyte recovery for the mainstream αβ T cell lineages in both transgenic lines, but the effects during the earlier developmental stages were different. Interference by NTR-mediated TEC depletion for DN3 was restricted in 3.1T-NE, while that for γδ T cells was noticeable only in 9.1T-NE.
Developmental stage and lineage specific roles of TEC subsets revealed by RTOC
When we set up a FTOC using 14.5 day old fetal thymuses, the culture contained thymocytes at DN2 and DN3 stages that already committed to αβ and γδ T cell lineages. Therefore, the thymocyte population in the FTOC is a mixture of cells at various developmental stages to begin with. To have a better assessment of the role of the TEC subset for thymocyte development, we employed a RTOC with E14.5 fetal liver progenitor cells. We prepared the TEC in the presence of 2d-Guo, aggregated it with E14.5 fetal liver progenitor cells, and analyzed cells 7 days later (). Thymocytes from the RTOC consisted mostly of DN cells (50.9%). The γδ T cells were more abundant in RTOC (4.86%) compared to that in adults (0.96%). DN subpopulations in the RTOC were clearly separated into DN1–DN3 using CD44 and CD25 profiles. A small fraction of the DN population in the RTOC was of Sca1+cKit+ cells.
Analysis of thymocytes recovered from RTOC of fetal liver progenitor cells and TEC preparation depleted with prodrugs
Next, we examined the effect of TEC ablation using the same strategy for the FTOC. When the RTOC was analyzed at day 9, non-transgenic C57BL/6 stroma showed no significant difference for the DN profiles upon the prodrug treatment ( and data not shown). However, the DN profiles changed in 3.1T-NE and 9.1T-NE transgenic RTOCs. We calculated the ratio of DN3/DN1 without including DN2 since the cell numbers in the DN2 population were too low to obtain an accurate measurement. The ratio of DN3/DN1 of 3.1T-NE was greatly diminished. In contrast, the DN3/DN1 ratio of 9.1T-NE RTOC was comparable with and without prodrug treatment ().
The levels were also similar to that of the FTOC with non-transgenic C57BL/6 thymus, indicating that transition from DN1 to DN3 was not affected in 9.1T-NE. In regards to the recovery however, γδ T cells were greatly reduced in 9.1T-NE but not in the 3.1T-NE RTOC () consistent with the earlier results from the FTOC ( and ).
We also examined the cell populations after 14 days of the RTOC and found a very poor recovery of the thymocytes in the RTOC with 9.1T-NE TEC preparation (). The total numbers and relative recovery of DN and γδ T cells were also severely reduced in the 9.1T-NE RTOC ( and data not shown). Interestingly, there was an opposing effect of TEC depletion on the DN3/DN1 ratio and γδ T cells between two lines of mice. The γδ T cell development was dramatically reduced in 9.1T-NE, whereas 3.1T-NE showed a reduction in the DN3/DN1 ratio but not γδ T cells (). However, flow cytometric profiles showed a relatively good DN3/DN1 ratio with or without the prodrug treatment in TEC preparation of 9.1T-NE. The RTOC with depleted 3.1T-NE showed consistently low DN3/DN1 ratios upon prodrug treatment, and 9.1T-NE showed abrogation of γδ T cells. These results are consistent with the results from the FTOC and the RTOC at day 9, supporting the idea of lineage and stage specific roles of TEC.
Analysis of RTOC at day 14 of fetal liver and depleted TEC treated with prodrugs