Helios Marks In Vivo Differentiated CD4 Th2 Cells Figure 8. Helios is not required for Th2 or TFh cell induction in response to alumOVA in vivo. One day after receiving Helios+/+ or Helios2/2CD45.2+OTII cells congenic CD45.1+ B6 mice were immunized with alumOVA in both footpads. A) Seven days later the draining LN were taken for analysis by flow cytometry of CFSE content and the expression of Helios. B) The proportion of OTII cells in the CD4 T cells was assessed as CD4+ CD45.2+ cells (left hand dot plots). The middle and right hand dot plots are gated on OTII cells and show the proportions of effector cells, identified as CD4+CD45.2+CD44+CD62Llo and TFh cells, identified as CD4+CD45.2+CXCR5+PD-1+ cells. The graphs show the percentages (top) or total numbers (bottom) of cells in LN. Left graphs compare all Helios+/+ OTII cells (black diamonds) with all Helios2/2 OTII cells (open diamonds). The centre graphs compare those cells in these populations that are CD44+CD62Llo OTII effectors. The right graphs compare the CXCR5+PD-1+ TFh cells (right). The data are derived from 2 independent experiments with 8–9 mice in total. Statistical differences between groups are indicated and NS = not significant, ** = p,0.01. doi:10.1371/journal.pone.0020731.g008 but in this case negatively regulates IL-4 expression [62]. It is plausible that Ikaros interacts with GATA-3 in a similar manner in CD4 Th2 cells. As Helios dimerizes with Ikaros, it has been proposed that it acts as a rate limiting factor for Ikaros functions, thereby controlling its intracellular, cytoplasmic or nucleic, localization [9,24,31]. Ikaros and Helios share the similar binding DNA sequence (GGGAAT) [24]. Thus, it was tempting to speculate that Helios, Ikaros and GATA-3 may be part of a macromolecular complex that regulates accessibility at the Il4 locus in CD4 Th2 cells. Finally, to add to the complexity both Ikaros and Helios have been shown to be able to potentiate as well as to inhibit gene expression [9,21,24]. So why were Helios2/2 CD4 T cells not impaired in Th2 differentiation? Perhaps this is because its function is compensated for by other members of the Ikaros family. Consistent with this, it is striking that a more severe phenotype is manifested by the Ikaros dominant negative homozygotes as compared with the Ikaros mice [65]. This suggests that Ikaros dominant negative dimerizes and interferes with the activity of other Ikaros-like factors in the hematopoietic system. In addition, even though Ikaros has been shown to be required in vitro in IL-4-directed Th2 responses [20,22] this does not necessarily imply that it is critical for the induction of early Th2-features in CD4 T cells in Th2 responses in vivo. We and others have shown that molecules important in vitro, including IL-4, may not be required in vivo for induction of primary Th2-features in CD4 T cells [57]. Thus, given the diversity in homodimerization and heterodimerization of the Ikaros family members, a full dissection of Helios activity during differentiation of Th2 cells in vivo may be required to get a clear understanding of the functional redundancies among Ikaros family members. This may require the development of genetic models where Helios can be studied in combination with Ikaros deficiency. Are alum-precipitated antigens appropriate to explore the role of Helios? Undoubtedly Helios expression is associated with CD4 T cells responding to this form of antigen. Nevertheless, it is plausible that other types of Th2 responses may be more suited to assess the role of Helios in vivo. For instance, expression of the IL-4 receptor alpha, which is required for both IL-4 and IL-13 signalling is not required on smooth muscle cells for the development of experimental allergic asthma [66]. On the other hand, signalling through IL-4Ra contributes to initiation of Th2 immunity and pulmonary pathology during Nippostrongylus brasi- liensis infections [67]. It also confers resistance during acute Schistosomiasis [68]. Thus, studies of the response of Helios-deficient mice to Th2-dependent parasites might reveal a role for this transcription factor during infections. Finally, it has been proposed that Helios is a specific marker that distinguishes thymus-derived Foxp3 Tregs from those that are peripherally induced [27]. Our results show that Helios is also PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20731