Helios Marks In Vivo Differentiated CD4 Th2 Cells Figure 9. Helios2/2 OTII cells responding to alumOVA induce IgG class switched plasma cells at comparable levels to those of Helios+/+ OTII cells. Chimeras were constructed by transfer of Helios+/+ or Helios2/2CD45.2+OTII cells into congenic CD45.1+ B6 mice. One day later they were immunized with alumOVA in both footpads and the B cell response in the draining LN at 7 days was assessed. A) Representative flow cytometry plots show gating of antibody-producing cells (B220intCD138+) and GC B cells (B220+Fas+GL7+). The graphs show the percentages (top) or the numbers (bottom) of GC B cells (left) or antibody-producing cells (right) in the two groups of chimeras. The data are derived from 2 independent experiments with 8–9 mice in each group. B) The graphs compare the levels of IL-4 mRNA and Cc1, Ce and Cc2a germline transcripts (GT) per LN section in each of the 8 chimeras in both groups. C) Dot plots show the gating of IgG1 and IgG2a, B220+CD138+ antibody-producing cells. The graphs show the % B220+CD138+ cells that were IgG1 expressing or IgG2a expressing in the draining LN of each of 5 chimeras in the two groups. D) Photomicrographs of sections of a draining LN from a representative Helios+/+OTII cell chimera (left) and Helios2/2OTII cell chimera (right). IgG1+ cells are blue, IgG2a+ are brown. Black arrow heads highlight rare IgG2a-producing cells. All results are from 2 independent experiments. The Mann- Whitney 2 tailed statistical differences between groups shown are indicated and NS = not significant, * = p,0.05, ** = p,0.01. doi:10.1371/journal.pone.0020731.g009 induced in peripheral mature CD4 T cells that respond to Th2 antigens without upregulation of Foxp3. Even though Helios is expressed at high levels in naturally occurring Tregs, two groups failed to detect a role for this transcription factor in the development and function of Tregs in their respective Helios knock-out mice [27,55]. Thus, although Helios is a molecule expressed in thymocytes, Tregs and Th2 cytokine-producing cells its role in the biology of these cells remains obscure. PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e20731