Figure 4. OTII cells induced to express Helios by alumOVA do not express Foxp3. C57BL/6 mice received CFSE-labelled OTII cells and were then immunized with alumOVA in the footpads. Three days later the draining LN were taken for analysis by flow cytometry of CFSE content and the expression of Helios, and Foxp3 on endogenous CD4 T cells and OTII cells. The top left dot plot shows the expression of CD4 and CFSE and its resolution into the central dot plot with Foxp3 and CFSE content of cells in CD4 T cell gate. The bottom left dot plot shows Foxp3 and Helios by Foxp3+ endogenous CD4 T cells that contain no CFSE. The bottom right dot plot shows Foxp3 and Helios expression by OTII cells captured in the CFSE+ CD4+ gate. The median percentages and SD for the mice studied (2 independent experiments n = 8 in total) are shown in each dot plot. doi:10.1371/journal.pone.0020731.g004 expected, Foxp3 is expressed at much lower levels in the responding OTII cells than in endogenous CD4 T cells. Both TFh and OEf OTII cells have significantly more Helios mRNA than the endogenous CD4 T cells, but the trend for TFh to express more Helios than OEf was not significant. To clarify this, we have compared Helios protein expression in OEf and TFh OTII cells with that in non-responding endogenous CD4 T cells excluding regulatory T cells as they express high level of Helios (Fig. 5C). This shows that TFh OTII cells reproducibly and significantly express higher level of Helios than OEf and these in turn express more Helios than the non-responding CD4 T cells other than regulatory T cells. Helios, like IL-4 and IL-13, requires NF-kB1 for its induction in CD4 T cells by alumOVA NF-kB1-deficient mice are impaired in Th2 responses including experimental allergic airway inflammation and intestinal helminth infection [40–42,52,53]. In addition, we have recently published evidence that the induction of IL-4, IL-13 and CXCR5 by alum- protein vaccine is under NF-kB1 control [54]. On the other hand, NF-kB1-deficient CD4 T cells still become effectors that upregulate IL-2, IL-21 and IFN-c mRNA and provide help for extrafollicular antibody responses. To test if NF-kB1-deficiency in CD4 T cells selectively affects Helios upregulation, chimeras were constructed by transferring either NF-kB1+/+OTII cells or NF- kB12/2OTII cells into wild-type congenic recipients. Three days after footpad immunization with alumOVA NF-kB1+/+OTII cells from the draining node expressed 100 fold more IL-4 mRNA and at least 1000 fold more IL-13 mRNA than NF-kB12/2OTII cells Helios Marks In Vivo Differentiated CD4 Th2 Cells (Fig. 6). NF-kB1+/+OTII cells expressed 10 fold more Helios mRNA than NF-kB12/2OTII cells, while both Ikaros and Aiolos were similarly expressed in the two cell types. Thus, optimal Helios expression, like that of IL-4 and IL-13, requires NF-kB1-signaling. Helios is not expressed during in vitro IL-4 directs CD4 Th2 cell polarization Although, there are key molecules in CD4 T cells that are upregulated and are involved in the induction of Th2 cytokine in vivo, this does not necessarily imply that they are required in the in vitro Th2-conditionned culture system. We carried out further experiments to assess whether Th2-transcription factors are induced when CD4 T cells are induced to produce Th2 cytokines by being activated through their TCR in the presence of IL-4 in vitro. As expected this treatment strongly upregulated GATA-3 and IL-4 in the cultured cells (Fig. 7). By contrast neither Helios nor c- MAF are induced, while NF-kB1 is modestly upregulated. These findings further highlight the differences between in vitro and in vivo responses that induce Th2 cytokines. The lack of Helios upregulation in the IL-4-directed in vitro Th2-polarization may explain why Helios2/2CD4 T cells secrete Th2 cytokines, similarly than WT CD4 T cells, when polarized in IL-4-directed in vitro stimulation [55]. Helios is not required for Th2 or TFh development in responses to alumOVA The data presented so far suggest Helios is a candidate transcriptional regulator of Th2 and TFh differentiation in vivo at least in response to alum-precipitated protein. To test if Helios is required for the differentiation of CD4 T cells we next set up studies to compare Th2 and TFh differentiation and function induced by alumOVA in Helios+/+ or Helios2/2OTII cells. These were transferred into wild type congenic mice and again their response to footpad immunization with alumOVA was followed. Helios2/2 mice have been described elsewhere [55], briefly in this mutant strain Helios lacks most of exon 7, including the sequences that encode the C-terminal zinc finger domain that is essential for dimerization. This deletion results in loss of Helios protein. The homozygous Helios-deficient mice have a low survival rate and the mutants die during the first week. Exceptionally, when mice survive the critical first week, these mice have normal differentiation and homeostasis of ab and cd T cells, NK T cells and regulatory T cells. Because of the low survival of Helios2/2 pups [55], we crossed heterozygous Helios+/2 mice with OTII mice to generate Helios+/2OTII+/2 mice. These hybrids were then interbred and the resulting foetal liver cells prepared from day 14 Helios2/2OTII+ embryos were used to reconstitute lethally-irradiated congenic C57BL/6 mice and generate Helios2/2OTII cells. CD45.2+ LN cells from Helios+/+OTII+ or Helios2/2OTII+ chimeras were then prepared, CFSE-labelled and transferred into CD45.1+ congenic wild-type recipient mice. Seven days after immunization with alumOVA no Helios protein was detected by intracellular flow cytometry in the Helios2/2OTII cells from the draining LN while about 50% of the Helios+/+OTII cells express this transcription regulator (Fig. 8A). The extent of proliferation and survival by Helios+/+ and Helios2/2OTII cells was comparable when assessed 7 days after immunization (Fig. 8A&B). Helios-deficiency in OTII cells did not alter the proportions and total numbers of effector CD62LloCD44+ OTII cells, or theCXCR5+PD-1+ TFh OTII cells generated (Fig. 8B). PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20731