Figure 7. Helios is not induced during IL-4-directed in vitro polarization of CD4 T cells. LN cell suspensions from OTII mice were cultured in standard conditions for 6 days to induce IL-4-producing cells in vitro. The cultures included OVA peptide+IL-4+neutralizing anti-IL-12 and anti-IFN-c. Flow cytometry dot plots show cells producing IL-4 versus IFN-c. The graphs show expression of IL-4, GATA-3, Helios, NF- kB1 and c-Maf mRNA relative to b2-microglobulin mRNA by freshly- isolated total LN cell suspensions (open circles), or by cells after Th2 differentiation (black circles). Data show duplicate culture results from 3 independent experiments. doi:10.1371/journal.pone.0020731.g007 any plasma cells or GC, so the appearance of these features after immunization is strong evidence of an induced response. In addition, T cell help is limiting in the primary response to alum- protein and by 7 day after immunization there are only modest numbers of plasma cells induced. By contrast, the transfer of 106 OTII cells i.v. increases the 7 day plasma cell count by some 1000 fold and a high proportion of these plasma cells have switched to IgG1 [59,60]. These considerations allowed us to assess the role of Helios in the induction of help for antibody responses by the transfer of Helios+/+ or Helios2/2OTII cells into wild type congenic mice and observing the popliteal LN response to alumOVA. The 7 day antibody response induced is shown in Fig. 9. The numbers of germinal centre cells (Fas+GL7+B220+) and plasmablasts and plasma cells (collectively CD138+) is comparable in the two sets of chimeras (Fig. 9A). It will be seen that the IL-4, c1 and e transcripts are all strongly induced in these LN (Fig. 9B). There is no difference in the levels of IL-4 and e transcripts induced between the Helios+/+ or Helios2/2OTII cell chimeras. Paradoxically there is a borderline significant increase in the level of c1 germline transcripts in the Helios2/2OTII cell chimeras. This finding was not pursued further as the proportions of plasma cells producing IgG1 were similar in the 2 sets of chimeras (Fig. 9C, D). There was no increase in the low level of switching to IgG2a, in the Helios2/2 chimeras. This indicates that there was no alteration of polarization from Th2 to Th1 as switching to this isotype is typically associated with Th1 responses (Fig. 9C, D). Helios does not regulate the induction of Th2 cytokine expression in CD4 T cells responding to alumOVA Finally, we assessed the role of Helios in transferred OTII cells in the induction of cytokines and transcription factors in response to alumOVA in vivo. Again recipient mice received CFSE-labelled Helios Marks In Vivo Differentiated CD4 Th2 Cells Helios+/+ or Helios2/2OTII LN cells and were immunized the next day with alumOVA in both footpads. In these experiments the responding OTII cells were evaluated 3 days later. As previously shown (Fig. 8A), the Helios2/2OTII cells do not express Helios protein (Fig. 10A). In addition, the Helios+/+ or Helios2/2OTII cells were FACS-sorted and assessed by real-time RT-PCR for Helios mRNA detected with primers recognizing either the junction between exons 4 and 5 or the deleted sequence of exon 7 (Fig. 10B). This shows that Helios mRNA is not induced in Helios2/2OTII cells. OTII cells expressed 1000 fold more IL-4 and 50 fold more IL-13 mRNA than the largely non-OVA-specific and hence non-responding endogenous CD4 T cells (Fig. 10B). Hence, the responses of the Helios+/+OTII cells are comparable to those described in previous sections. Additionally, no obvious difference was found between the responses of Helios+/+ and those of Helios2/2OTII cells in relation to the production and transcriptional control of Th2 or TFh cytokine production. Also, the absence of Helios does not lead to the induction of the Th1 features - IFN-c or T-bet. These results indicate that Helios is dispensable for the regulation of both Th2 and TFh cytokines and transcription factors in response to alumOVA immunization. Discussion In mature hematopoietic cells, Helios expression is restricted to T cells [9,24], suggesting that Helios may control important aspects of T cell differentiation and/or function. Strikingly, the kinetics of Helios expression mirrors that of GATA-3 with about 60% of alumOVA-primed OTII cells expressing Helios after 2–3 divisions. This is shown both at the mRNA and protein levels. Other indirect evidence linking Helios with Th2 responses comes from a genome wide association study. This identified Helios in a sequence variant that correlates with high numbers of blood eosinophils in asthma [61]. Our data also show that Helios expression appears to be regulated by NF-kB1, a transcription factor associated with Th2 responses [40–42,54]. We found that Helios is not induced in OTII cells responding to the Th1 antigen SalOVA, while Ikaros and Aiolos are both expressed to similar levels both in responses to SalOVA as well as alumOVA. This is of interest, for following in vitro polarization of activated CD4 T cells using IL-4, there was increased expression of Il2, Ifnc, and Tnfa and decreased expression of Il4, and Il13 in Ikaros2/2 CD4 T cells compared with wild-type CD4 T cells [20]. Ikaros is also important for IL-10 expression in T cells that have undergone Th2 differentiation, and a decrease in IL-10 expression in Ikaros2/2 CD4 Th2 cells was observed compared with their wild-type counterparts [22]. Ikaros appears to participate in T cell differentiation and maturation in other ways, for it also represses IFN-c [21,22]. At a molecular level, in Th2 cells derived in vitro there is evidence for Ikaros binding sites clustered in the Il5 promoter and within Il4 regulatory regions, including conserved noncoding sequence 1 (CNS-1), the IL-4 intron enhancer (I-E) and the VA enhancer [20,62,63]. The binding of Ikaros to the Ifnc CNS-22 element observed in Th2 cells may reflect its role in silencing Ifnc. By contrast, in Th1 cells Ikaros binds predominantly to CNS-1 and the Il4 promoter, and to a lesser extent to the Ifnc promoter region [20]. Thus, Ikaros appears to exert distinct effects on Il4 and Ifnc in the same cell type (Th1 and Th2 cells) [20]. Ikaros also binds to the Il10 promoter and intronic regulatory regions [22]. A highly conserved Ikaros binding site in the proximal Il10 promoter (2901 bp) is located within 40 bp of a known GATA-3 binding site [22]. In line with this, Ikaros has been found to interact with GATA-1 in erythroid cells [64]. In addition, Ikaros interacts with GATA-1 and GATA-2 in mast cells PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20731