Helios Marks In Vivo Differentiated CD4 Th2 Cells Figure 3. Transcription factor and IL-4 protein expression in single alumOVA-responding OTII cells. C57BL/6 mice received CFSE- labelled, or not, OTII cells and were then immunized with alumOVA in both footpads. Three days later flow cytometry was used to analyze the expression of transcription factor and IL-4 proteins in OTII cells from the draining LN. A) Dot plots gated on CD4 T cells show GATA-3 and Helios protein expression in the draining LN suspensions as a factor of CFSE dilution. Cells to the left of the vertical quadrant bar are CFSE2 endogenous CD4 T cells. Histograms on the right of each dot plot show the proportions of OTII cells that had completed the indicated number of divisions and express either GATA-3 (top) and Helios (bottom). Each line represents results from one mouse. Data are representative of 2 independent experiments. The graph on the right hand shows the proportion of OTII cells from each of 8 mice expressing GATA-3 or Helios. B) The dot plot shows the co- expression of Helios and GATA-3 proteins by OTII cells. The graph on the right shows that more than 80% of the OTII cells from each of 8 mice that are positive for Helios also express GATA-3. C) The proportion of IL-4-producing cells within the OTII cells after 5 h restimulation in vitro with OVA- peptide was assessed by intracellular flow cytometry as shown in the dot plot. The graph shows the proportions of cells in the responding LN from 8 mice that expressed IL-4. The data in B–C) are representative of 3 independent experiments. doi:10.1371/journal.pone.0020731.g003 also express Foxp3. Again the LN response of CFSE-labelled OTII cells was evaluated. OTII cells were identified by their CFSE content as CD4+ CFSE+ cells (Fig. 4). Three days after chimeras were immunized with alumOVA a mean of 48%613 of the OTII cells in the draining LN contained Helios. Within the OTII population very few (1.7%61.5) co-expressed Helios and Foxp3. Thus, the majority of the Helios+OTII cells are Foxp32. As reported in Ref [27], 84%64 of the 14%61 of endogenous CD4 T cells that expressed Foxp3 are also Helios positive. These data show that almost all of OTII cells responding to alumOVA that are induced to express Helios do so in a different manner from that of naturally occurring regulatory T cells. Helios is induced in both TFh cells and other CD4 effectors responding to alumOVA Upon immunization CD4 T cells first encounter antigen presented on the surface of dendritic cells in the T zone near high endothelial venules (HEV). By 72 h the expression of CXCR5 enables a proportion of the responding CD4 T cells to migrate into the B follicles [44,46]. These migrant cells are TFh required for the selection of B cells in germinal centers (GC) and for inducing the selected B cells to differentiate into plasma cells, memory B cells or centroblasts [47]. CXCR5highCCR7low T cells have been found to have elevated IL-4 and PD-1 transcript expression [48] and GC are the focus of IL-4 production during Th2-responses [49–51]. To test if Helios is expressed in TFh cells, seven days after immunizing a further set of chimeras with alumOVA PD- 1 + CXCR5+ TFh cells and PD-12CXCR52 other effector (OEf) cells were sorted from the draining LN (Fig. 5A). Largely non- responding endogenous CD45.12 CD4 T cells were also sorted as controls. Real-time RT-PCR shows that both TFh and OEf OTII cells significantly upregulate IL-4 at the population level compared to endogenous CD4 T cells. Nevertheless, TFh OTII cells produce significantly more IL-4 mRNA than in the OEf OTII cells (Fig. 5B). Changes in the expression of transcription factors associated with TFh or Th2 differentiation or both of these are also shown in Fig. 5B. As expected, OEf OTII cells were found to contain significantly more GATA-3 mRNA than TFh OTII cells, and TFh OTII cells strongly express BCL6 mRNA whereas OEf OTII cells do not. There are no major differences in c-Maf, NF- kB1, and Ikaros between TFh and OEf OTII cells. Finally, as PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20731