FACS-sorted from the popliteal LN as a function of CD69 expression and the number of cell division accomplished (Fig. 2A). In this way individual cells at different stages of the response were obtained: (i) cells that had upregulated CD69, but had not yet started to divide (termed 0 division in Fig. 2); (ii) cells that had divided 2–3 times based on CFSE dilution (2–3 divisions); and (iii) cells that had divided 6 times (6 divisions). Na¨ıve single OTII cells from the popliteal LN of chimeras that had not been immunized were also studied. Levels of mRNA for IL-4 and related transcription factors were then determined by real-time RT-PCR in the single cells derived from each of these 4 OTII subsets (Fig. 2A). The transcriptional regulators involved in Th2 development that we analyzed were – GATA-3 [34,35], c-Maf [36–39], Ikaros [20,22], and NF-kB1 [40–42]. Helios mRNA, in contrast to the other transcription factors, is expressed in fewer than 2% of the na¨ıve OTII cells, but is already induced in 30% of CD69+ OTII cells that have not yet divided (0 division) (Fig. 2A). The proportion of cells expressing Helios then increases linearly through to 6 cell divisions. GATA-3 mRNA is present in approximately 20% of na¨ıve OTII cells (Fig. 2A). This proportion rises to 40% in the CD69+ OTII cells that had not divided. Some 80% of primed OTII cells that have completed 2–3 divisions express GATA-3, and this expression is maintained through 6 divisions. The profile of c-Maf expression is similar although this transcription regulator is present in a higher proportion of na¨ıve cells (40%). Ikaros and NF-kB1 are constitutively expressed in 60–70% of na¨ıve OTII cells and this is rapidly up-regulated in the remaining OTII cells following immunization. As previously reported IL-4 Helios Marks In Vivo Differentiated CD4 Th2 Cells mRNA expression is not seen prior to cell division [43], but becomes apparent in some 7% of the OTII cells that have undergone 6 divisions. A similar proportion of IL-4-secreting OTII cells express intracellular IL-4 protein at this stage (see Refs [44–46] and Fig. 3C). Helios and GATA-3 mRNA expression were respectively seen in 88% and 82% of the OTII cells induced to express IL-4 mRNA (Fig. 2B). We were unable to determine if the few IL-4-producing cells that did not contain Helios or GATA-3 had expressed these transcription factors earlier in the response. We went on to assess whether the proportion of OTII cells expressing Helios and GATA-3 mRNA was related to the proportion of cells expressing these transcription factors at the protein level (Fig. 3). The proportion of OTII cells with GATA-3 and Helios proteins is shown in relation to the number of cell divisions induced by alumOVA in Fig. 3A. This shows that kinetics of upregulation of both of these proteins parallels the upregulation of their mRNA, as shown in Fig. 2A. In addition, more than 80% of Helios-expressing OTII cells also express GATA-3 (Fig. 3B). Overall these results show similar timing and frequency of Helios and GATA-3 mRNA and protein expression during this Th2 response. OTII cells induced to express Helios by alumOVA do not express Foxp3 Helios expression has been associated with naturally occurring regulatory T cells [27]. For this reason we went on to assess if the OTII cells that have been induced to express Helios by alumOVA Figure 2. Transcription factor mRNA related to IL-4 induction in single alumOVA-responding OTII cells. C57BL/6 mice received CFSE- labelled OTII cells and were then immunized with alumOVA in both footpads. Three days later flow cytometry was used to analyze and sort single cells from the draining LN. A) The dot plots in the top row are gated on CD4 T cells and show the CD69 expression as a function of OTII cell proliferation. Na¨ıve LN cells from a group of non-immunized chimeras were used as controls. Histograms show the populations of single OTII cells from popliteal LN isolated as follows: 1) na¨ıve OTII cells, 2) OTII cells that had not divided, but were CD69+ (0 division), 3) OTII cells that had completed 2–3 divisions, 4) OTII cells that had completed 6 divisions. Histograms show the purity of the sorted OTII cells (black lines) within the initial OTII population (filled grey). The lower set of graphs show the percent of single cells expressing b2-microglobulin mRNA that also express IL-4 or transcription factor mRNA as quantified using a duplex RT-PCR in the populations of na¨ıve and responding OTII cells defined above. b2-microglobulin expression indicated more than 90% of 154 wells from each sorted population contained a cell. B) Percent of single OTII cells that had divided 6 times that expressed b2-microglobulin mRNA simultaneously with either GATA-3 (left panel) or Helios (right panel) as quantified by a triplex RT-PCR. The results in A & B are shown for 2 to 3 independent experiments respectively represented by open, grey and black symbols. doi:10.1371/journal.pone.0020731.g002 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20731