Direct Detection of FoxP3 Expression in Thymic Double-Negative CD4−CD8− Cells by Flow Cytometry

Foxp3 expression is a marker of regulatory T cells (Treg), but how early it is expressed in the thymus is still not fully defined. In this study, we examined Foxp3 expression in double-negative (DN) CD4−CD8− T cell precursors in the thymus by flow cytometry. By increasing the number of collected cells from the conventional 104 cells up to more than 106 cells during flow cytometry, we found that DN cells exhibited higher Foxp3 expression than double-positive (DP) CD4+CD8+ and single-positive (SP) CD4+ or CD8+ (SP) T cells. CD44+ expression positively correlated with Foxp3 in thymic DN cells. Furthermore, TCR-β−CD25+ DN cells exhibited the highest frequency of Foxp3-expressing cells. Almost all Foxp3+ cells expressed CD25in DN cells. These results suggest that Foxp3 expression in DN cells can directly be detected by flow cytometry and it was positively corelated with CD25 and CD44 in DN cells.

In this study, we used flow cytometry to examine Foxp3, CD25, and CD44 expression in DN thymocytes. By increasing the normal sample collection number of 10 4 thymocytes up to more than 10 6 thymocytes, we found that CD44 expression positively correlated with TCR-b and Foxp3 expression in DN cells. Furthermore, CD25 expression positively correlated with Foxp3 and negatively correlated with TCR-b in DN cells, thus, our data describe the relationship among Foxp3, CD25, and CD44 cell surface markers on DN thymocytes. The study reported here had two aims. First, this study evaluated the levels of Foxp3 expression in DN cells by the improved detection mode of Flow cytometry. Second, the efficiency and success of the improved detection mode was compared to traditional mode, it may help us to better understand T reg cell development in the thymus.

Methods
Mice. Female C57BL/6 mice (Taconic Farms, Germantown, NY) were maintained under specific pathogen-free conditions and used for experimentation at 4-6 weeks (young) and 6-10 months (adult) of age according to protocols approved by the Institutional Animal Care and Use Committee at Xi'an Jiaotong University.
To conform whether DN cells express higher levels Foxp3, the cells were also stained with FITC-anti-CD4, PerCP5.5-anti-CD8 and PE-anti-Foxp3 antibodies (eBiosciences).  RT-PCR and real-time RT-PCR. Total RNA was extracted from ,5 3 10 5 sortpurified thymocytes of C57BL/6 mice, using TRIzol (Invitrogen). Chloroform (0.2 ml; Sigma-Aldrich) was added for every 1 ml of TRIzol used. Extracted RNA was shaken vigorously for 15 sec and incubated at room temperature for 2 min. After transferring the aqueous phase to a clean tube, isopropanol (Sigma-Aldrich) was added, followed by incubation at room temperature for 5 min. The RNA pellet was washed with 1 ml of 75% ethanol. After air-drying the RNA pellet for 10 min, it was dissolved in 20 ml of water and incubated at 55uC for 10 min. The OD260 nm/ 280 nm ratio was among 1.9 to 2.0. RNA samples were further assessed by electrophoresis on 1.5% agarose gels, and then visualized under UV light after ethidium bromide staining. RNA preparations were treated with DNase I (according to the standard protocol) to remove genomic DNA. cDNA was synthesized by incubating 20 ml of mRNA in a sprint C1000 terminal cycler (Bio-rad). Negative controls contained all the elements of the reaction mixture except for template DNA. For quantification, relative mRNA expression of specific genes was obtained by the 2 2DDCt method, using b-actin for normalization. The following gene-specific primers (59 R 39) were used: b-actin (forward; GAA ATC GTG CGT GAC ATC AAA G, and reverse; TGT AGT TTC ATG GAT GCC ACA G); Foxp3 (forward; GGC CCT TCT CCA GGA CAG A, and reverse; GCT GAT CAT GGC TGG GTT GT). Diluted cDNA (10 ml) was mixed with 2 ml of primer and 10 ml IQ SYBR Green SuperMix, and was then assayed in triplicate on a CFX-96 real-time system (Bio-Rad) under the following conditions: denaturation at 95uC for 3 min, 40 cycles of 95uC for 15 s, and 60uC for 1 min followed by 30 sec of extension at 72uC. Each sample was analyzed in triplicate.
Statistical analysis. Mean and SD values were calculated with Microsoft Excel. At least three independent experiments were performed, the Tukey-Kramer post-test was used to compare 3 or more means or a two-tailed unpaired Student t test to compare 2 groups, Values of P # 0.05 were considered significant. Statistically significant values are denoted in the figures as follows: *P , 0.05; **P , 0.01; ***P , 0.001.

Results
Foxp3 expression is detected in thymic double-negative CD4 2 CD8 2 cells by flow cytometry. DN cells only comprise 3-5% of the total thymocytes within the thymus 15,16 , and even less of these DN cells express Foxp3; therefore, accurately detecting Foxp3 expression in DN cells by flow cytometry is difficult, and no studies have yet reported direct detection of Foxp3 expression in DN cells by this method. In order to increase the sensitivity of detecting Foxp3 in this small population of cells, we first increased the cell collection number for each sample from 10 4 up to more than 10 6 and examined the developmental profile of thymocytes in mice using multi-color flow cytometry. Thymocytes from naïve mice were stained with the following combination of antibodies: PE-Cy7-anti-CD4, FITC-anti-CD8, APC-Cy7-anti-CD25, PE-anti-CD44, PE-Cy5-anti-Foxp3, and PerCp-anti-TCR-b. As illustrated in Figure 1, we first gated live thymocytes by size (FSC) and granularity (SSC) in the P1 gate Figure 1A, followed by dividing the live cells into four subpopulations based on CD4 and CD8 expression, which are identified as quadrants Q1-Q4 Figure 1B. The P2 population represents Q1 (CD4 2 CD8 1 , or CD8 1 SP), P3 represents Q2 (CD4 1 CD8 1 , or DP), P4 represents Q3 (CD4 2 CD8 2 , or DN), and P5 represents Q4 (CD4 1 CD8 2 , or CD4 1 SP). Since the cells had also been stained with antibodies against CD44 and CD25 Figure 1C.
The expression of many genes, such as Foxp3 are low in CD4 2 CD8 2 (double negative, DN) thymocytes; The cell collection numbers in the traditional detection mode of Flow cytometry are 3 3 10 4 , the cell collection numbers of DN cells will be 1200 (3 3 10 4 3 4%), DN-Foxp3 cells will be 69 (3 3 10 4 3 4% 3 5.8%), therefore, DN-Foxp3 could not be detected by Flow cytometry, because the cell collection numbers of DN cells are too small ( Figure 1D). We increased the cell collection number for each sample from 10 4 up  to more than 10 6 , the cell collection numbers of DN cells will be 40000(10 6 3 4%), the second gated DN-Foxp3 cell number will be 2320 (10 6 3 4% 3 5.8%) ( Figure 1E). The three populations of thymus from the same sample which is the same conditions, the same setting and same dyeing ( Figure 1F). Therefore, using this improved flow cytometry method, lower expression genes in DN cells could be detected high repeatability to reveal previously uncharacterized data on subsets of DN cells. We greatly improved the accuracy of Foxp3 detection in DN cells.
Foxp3 expression during different stages of thymocyte development in adult and young mice. Thymocytes were harvested from young and adult mice, stained with CD4 (PE-Cy7), CD8 (FITC), CD44 (PE), CD25 (APC-AlexaFluor 755), Foxp3 (PE-Cy5) and Isotype IgG2a as negative control (Foxp3) and analyzed by flow cytometry. The frequency of Foxp3-expressing cells was higher in adult mice than in young mice. Among the thymocyte subpopulations, DN cells had a much higher frequency of Foxp3expressing cells in both adult and young mice (adult, 7.2%; young, 5.8%), followed by CD4 1 SP cells (adult, 4.5%; young, 3.6%), CD8 1 SP cells (adult, 0.7%; young, 0.2%), and DP cells (adult, 0.4%; young, 0.2%) (Figures 4A and 4B). Thus, our results reveal that although Foxp3 is a T reg cell marker, it is also a marker that dynamically changes in thymocytes. DP cells contained the lowest frequency of Foxp3-expressing cells in thymocytes. These results further suggest that Foxp3 expression correlates with cell maturation states.
To conform whether DN cells express higher levels Foxp3, we sorted DN, DP and CD4 1 SP cells from the thymocytes by FACS ( Figure 5A). We examined via RT-PCR the levels of Foxp3 mRNA, the levels of Foxp3 were higher in DN and SP cells than DP cells as determined by densitometry scanning of the gels ( Figure 5B) and the data were pooled from three independent experiments and shown in a plot ( Figure 5C and 5D). To further confirm these findings, we sorted DN, DP and CD4 1 SP cells from the thymocytes and measured the levels of Foxp3 mRNA by real-time RT-PCR. As shown in (Figure 5E), DN and CD4 1 SP cells expressed higher levels of Foxp3 than DP cells (more than 20 times). These results further suggest that Foxp3 expression was much higher in DN cells but not in DP cells.

Discussion
In this study, we directly detected the expression of Foxp3 in thymic DN cells by multi-color flow cytometry and observed that DN and CD4 1 SP cells contained a higher frequency of Foxp3-expressing cells than DP cells. We also found that CD44 expression positively correlated with Foxp3 that CD25 1 DN cells expressed higher Foxp3 levels, and that CD25 2 DN cells do not expressed Foxp3.
We used multi-color flow cytometry to analyze the thymic DN cell population. We determined surface marker expression on thymocytes and found that Foxp3 expression changed in different stages of T cell development. One previous study did not find expression in DN cells by the RT-PCR method 17 ; other used MACS to isolate DN cells from thymocytes and remove any contaminating NK, B, and macrophage, but this group also did not observe any Foxp3 expression in DN cells 18 . By increasing the cell collection from the normally used 10 4 cells up to more than 10 6 cells during the acquisition phase of flow cytometry, we were able to identify a Foxp3-expressing population within DN cells and were further able to analyze its expression in each of the DN cell subpopulations (Figure 1). CD25 1 DN cells expressed higher Foxp3 levels; whereas CD25 2 DN cells expressed www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 5781 | DOI: 10.1038/srep05781 much lower Foxp3 levels ( Figure 1D). Furthermore, Foxp3 also positively correlated with CD44 ( Figure 2). The frequency of Foxp3expressing cells was much higher in DN cells than in DP or SP cells. Among the CD25 1 cells specifically, CD4 1 SP cells had the highest frequency of Foxp3-expressing cells compared to the DN and DP cells, which were similar to each other ( Figure 3C). These results tell us that Foxp3 expression positively correlates with CD25 expression and the state of cell maturation. Almost 100% of Foxp3 1 cells were CD25 1 in DN cells, while only 4.3% of Foxp3 1 cells were CD25 1 in DP cells; meanwhile, 34% of Foxp3 1 cells were CD25 1 cells in SP cells ( Figure 3E). Our experiment testing Foxp3 expression in thymocytes from young and adult mice also suggested that Foxp3 expression correlated with cell maturation status (Figure 4). To conform whether DN cells express higher levels Foxp3, we sorted DN, DP and CD4 1 SP cells from the thymocytes by FACS. We examined the levels of Foxp3 via RT-PCR and real time RT-PCR ( Figure 5). Our data also showed that CD25 expression negatively correlated with TCR-b expression. This raises the question of whether some function of CD25 may interrupt TCR signaling transformation through Foxp3 expression. Perhaps TCR signaling controls Foxp3 expression 19 , while Foxp3 or CD25 expression can also interfere with TCR signaling? Also, how do CD25 1 DN cells express Foxp3 to seed development of T reg cells in thymus? This is a very interesting question. T reg cells may originate from DN cells, where they do not express TCR; once they move into the CD4 1 SP stage, they express TCR, but this TCR is non-functional. Consistent with this, neither anti-CD3 nor anti-TCR can induce T reg cell proliferation 20 , but anti-CD25 mAb treatment can not only block T cell activation but also prevent activation-induced cell death 21 . The precise functional role of CD25 in the TCR signaling pathway and its relationship with Foxp3 expression is currently unclear and requires further study. In order to enhance the link between the Foxp3-expressing DN cells, perhaps consider also including some functional tests. We could sort these Foxp3-expressing DN cells and evaluate their capability in vitro or in vivo to give rise to functional T reg cells that can suppress the proliferation of activated CD81 cells.
In this study, we determined a reliable method for analyzing the relationship among Foxp3, CD25, and CD44 expression in DN cells by increasing the cell collection number from 10 4 up to more than 10 6 thymocytes, the approach is standards method of flow cytometry, we apply the new protocol to detect T cell base on BD software which is modern FACS data analysis software has introduced easily accessible compensation utilities that simply make fluorescence compensation the first part of the analysis procedure with any primary FACS data set. Since primary data can be collected with any FACS instrument just by avoiding the compensation step 22 . Today's FACS technology readily supports the collection of primary data and frees us to do better and more accurate analyses. In facts, it can directly clear detect to the expression of Foxp3 in DN cells. In order to improve the detection efficiency, we should pay attention to: (i) in our experiment, the data of capacity in computer very large, a specimen is equivalent to 30-100 times normal, it was very slowly when we did compensation correction, so we had prepare one sample to collect 10 4 thymocyte s, specially for compensation correction; (ii) Multiple antibody can not be together staining, especially the lower expression of gene should be used to separate; (iii) This study used the off-line compensation technology, it needs very careful for repeated verification accuracy of compensation.
In summary, characterization of DN cells is technically difficult, because the very small number of DN in mice thymus. In the present study, we have developed a reliable and efficient multiple -color flow cytometry method. We reveal that DN cells have a higher frequency of Foxp3-expressing cells than DP or CD4/CD8 (SP) cells; CD44 1 DN cells express Foxp3; CD25 1 DN cells express Foxp3; and CD25 2 DN cells do not expressed Foxp3. Taken together, our results suggest Foxp3 expression in DN cells can directly be detected by flow cytometry and Foxp3 expression was positively corelated with CD25 and CD44 in DN cells.