Helios (Ikzf2) belongs to the Ikaros transcription factor family that is made up of five DNA-binding proteins; Ikaros, Helios, Aiolos, Eos and Pegasus [6]. These transcription factors contain two sets of highly conserved C2H2 zinc fingers motifs. At the N terminus, four zinc fingers are responsible for sequence-specific DNA binding, while at the C terminus two zinc fingers enable homodimeric or heterodimeric interactions between family members [7,8]. Ikaros, Helios and Aiolos expression is restricted to cells of the hematopoietic system, whereas Eos and Pegasus are more widely expressed [9,10]. Ikaros and Aiolos are involved in many aspects of B cell differentiation and functions [11–17]. Ikaros also appears to be important during T cell development, and its absence leads to increased double-negative, stage 4, thymocyte proliferation [18,19]. In mature T cells, Ikaros has been reported both to regulate Th2 commitment and silence Th1 differentiation [20–22]. In addition, Eos mediates gene silencing in regulatory T cells (Tregs) in a Foxp3-dependent manner [23]. Finally, Helios is highly expressed at early stages of thymocytes development [9,24]. In mature T cells, Helios expression has been strongly associated with Tregs by several groups [25–30]. Helios has also been observed in a very small number of non-characterized splenic germinal centers cells [9]. By dimerizing with Ikaros, Helios potentially controls its epigenetic function by altering the intracellular localization of Ikaros [9,24,31]. This suggests a possible role for Helios in the acquisition of selective functions by peripheral CD4 T cells. Helios expression has also been associated with naturally occurring regulatory T cells [27], but we show that Helios upregulation in the context of the response to alumOVA occurs independently of Foxp3 expression. Finally, we generated Helios- deficient OTII cells and used these to probe the role of Helios in the acquisition of Th2-features in response to alumOVA. Results Helios is selectively expressed in CD4 T cells committing to Th2 differentiation Our previous studies show that CD4 OTII cells primed to SalOVA or alumOVA respectively acquire Th1 and Th2 features as early as 3 days after immunization. These studies also suggest that Helios expression is selectively associated with Th2 differentiation [5]. To confirm these findings, chimeric mice were constructed by transfer of CFSE-labelled CD45.1+ OTII cells into congenic wild- type CD45.2+ recipients. The day after cell transfer chimeras were immunized in both footpads, with either SalOVA or alumOVA and 7 days after immunization OTII cells were FACS-sorted from the draining popliteal lymph node (LN) (Fig. 1A). Real-time RT-PCR shows the sorted OTII CD4 T cells responding to alumOVA selectively express high levels of IL-4, GATA-3 and Helios mRNA (Fig. 1A). By contrast, OTII cells responding to SalOVA acquire Th1 features, expressing high levels of IFN-c and T-bet but not Helios mRNA (Fig. 1A). Importantly, SalOVA- and alumOVA- primed OTII cells express both Ikaros and Aiolos mRNA to similar levels. Thus, Helios mRNA is induced in OTII cells responding to alumOVA but not to SalOVA (Fig. 1A and [5]). We next tested whether the selective association of IL-4 with Helios expression occurs in response to another antigen that induces differentiation of na¨ıve CD4 T cells to Th2 cells. To this end we immunized mice with FliC, a subunit of the Salmonella flagellar protein – flagellin. In addition to being the target for specific immune recognition by T and B cells, FliC is a ligand for TLR-5 and it induces a strong Th2 response [32,33]. WT mice were immunized intraperitoneally with FliC and 7 days later high levels of IL-4 and Helios mRNA were found in FACS-sorted CD4+CD62Llo (primed) splenocytes (Fig. 1B). By contrast these mRNAs were not present in the CD4+CD62L+ (na¨ıve) population. Helios Marks In Vivo Differentiated CD4 Th2 Cells Figure 1. Helios mirrors IL-4 mRNA expression in CD4 T cells responding to Th2-antigens in vivo. A) One day after receiving CD45.1+ OTII cells groups of congenic CD45.2+ B6 mice were immunized either with SalOVA or alumOVA in both footpads. Seven days later popliteal LN cells were taken and OTII cells were sorted on the basis of CD4 and CD45.1 expression. The right hand graphs marked OTII show the purity of the sorted cells, which were used to prepare cDNA. The bar charts show IL-4, GATA-3, IFN-c, T-bet, Helios, Ikaros and Aiolos mRNA levels +SD, assessed by real-time PCR relative to the level of b2- microglobulin mRNA. The data are representative of 2 independent experiments with a total of 4 mice. The Mann-Whitney 2-tailed test was used to estimate the significance of differences between groups: * = p,0.05, NS = not significant. B) C57BL/6 mice were immunized with the flagellin subunit FliC intraperitoneally. Seven days later CD4 T splenocytes were FACS-sorted as na¨ıve (CD62L+) and primed (CD62Llo) cells. The relative amounts of IL-4 and Helios mRNA were determined by real-time RT-PCR relative to the level of b2-microglobulin mRNA. The data are representative of 2 independent experiments with 8 mice in total. doi:10.1371/journal.pone.0020731.g001 Thus, Helios mRNA is upregulated in splenic and LN CD4 T cells responding to at least two types of Th2 antigens. Kinetics of Helios expression in OTII cells responding to alumOVA mirrors GATA-3 expression We next set out to identify the proportion of OTII cells that upregulate Helios at successive stages in the response to alumOVA. Again CFSE-labelled OTII cells were transferred into congenic wild- type mice and these chimeras were immunized in the footpads with alumOVA. Three days after immunization single OTII cells were PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20731