Helios Marks In Vivo Differentiated CD4 Th2 Cells Figure 5. Helios is induced in both T zone effector and follicular helper OTII cells. A) Popliteal LN cell suspensions were prepared from OTII cell chimeras immunized 7 days previously with alumOVA in both footpads. The left hand FACS plot shows the CD4+CD45.1+ OTII cells (black gate) and the largely non-responding endogenous CD4+CD45.12 T cells (grey gate). The OTII cells were sorted (right hand pair of dot plots) into TFh cells (CXCR5+PD-1+) and other effector cells OEf (CXCR52PD-12). B) The relative amounts of mRNA for IL-4 and a range of transcription factors was determined by real-time RT-PCR. The values for cells from the responding LN from 8 mice in 3 experiments is shown in the graphs: Endogenous CD4 T cells (black diamonds), OEf (grey diamonds), TFh (open diamonds). C) The histogram shows the level of expression of Helios protein in the TFh and OEf from a responding LN, sorted as in (A), compared with that by endogenous FOXP3- CD4 T cells from the non-responding brachial of the same mouse (sorted using gates shown in Fig 4). The graph shows the geometric mean expressions in the same three populations in the LN from each of 8 mice in 3 experiments. The symbols are as in (B). Mann-Whitney 2-tailed statistical probabilities of differences between the TFh and OEf OTII groups are indicated: NS = not significant, * = p,0.05, ** = p,0.01, *** = p,0.001. doi:10.1371/journal.pone.0020731.g005 Helios deficiency in CD4 T cells does not modify their capacity to provide help for follicular or extrafollicular antibody response to alumOVA We next set out to test the effect of Helios deficiency in antigen- specific CD4 T cells on their capacity to support antibody responses to alumOVA. The B cell response induced by this Th2- inducing antigen is characterized by switching to IgG1 [32,56–60]. Alum-protein antigens injected subcutaneously selectively induce processed c1 and e Ig heavy chain gene germline transcripts in the draining LN. Productive switching to IgG1 occurs in these responding nodes although there is very little switching to IgE in these sites [32,57,58]. Non-immunized popliteal LN have few if Figure 6. Helios and Th2 cytokine mRNA induction in CD4 T cells by alumOVA requires NF-kB1. C57BL/6 mice received CFSE- labeled NF-kB1+/+OTII cells or NF-kB12/2OTII cells and were then immunized with alumOVA in both footpads. Three days later in vivo- primed NF-kB1+/+ (black bars) or NF-kB12/2 (open bars) OTII cells were FACS-sorted as CFSE+ CD4 T cells. The relative Th2-cytokine and Ikaros family of transcription factor mRNA levels were determined by real-time RT-PCR relative to b2-microglobulin mRNA expression. The data are derived from 2 independent experiments with a total of 6 mice per group. Mann-Whitney 2-tailed statistical probabilities of differences between groups are indicated: NS = non significant, ** = p,0.01. doi:10.1371/journal.pone.0020731.g006 PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20731