Helios Marks In Vivo Differentiated CD4 Th2 Cells Figure 10. Induction of Th2/TFh-cytokines and transcription factors in CD4 T cells by alumOVA does not require Helios. One day after receiving Helios+/+ or Helios2/2 OTII cells recipient B6 mice were immunized with alumOVA in both footpads. A) Three days later the draining LN were taken for analysis by flow cytometry of CFSE content and the expression of Helios. B) At this stage the Helios+/+ (black circles) or Helios2/2 (open circles) OTII cells were FACS-sorted as CFSE+ CD4 T cells. Largely non-activated endogenous CFSE2 CD4 cells were used as controls (black diamonds). The relative mRNA levels of Th2, TFh and Th1 cytokines (top row) or transcription factors (bottom row) were determined by real time RT-PCR relative to the level of b2-microglobulin mRNA expression. Each symbol represents sorted cells pooled from the two popliteal LN of one mouse. The data are derived from 2 independent experiments with 6 mice in total. Statistical differences between groups are indicated and NS = not significant, ** = p,0.01. doi:10.1371/journal.pone.0020731.g010 Materials and Methods Mice and Chimeras Wild-type C57BL/6J mice were from HO Harlan OLAC Ltd. (Bicester, UK). The Helios mice have been described elsewhere [55]. This strain was maintained on a C57BL6 background by crossing Helios+/2 mice. OTII mice that are transgenic for ab TCR specific for 323–339 OVA-peptide in the context of H-2 I- Ab (Charles River, Wilmington, MA) were crossed to CD45.1+ C57BL/6 congenic mice (The Jackson Laboratory, Bar Harbor, Maine, USA), or NF-kB12/2 C57BL/6 mice [69], or Helios+/2 C57BL/6 mice. NF-kB12/2 mice were a kind gift from Dr. J.H. Caama n˜o (University of Birmingham, UK). All animal procedures were carried out in strict accordance with local ethical approval from the University of Birmingham and the UK Home Office license (Project license 40/2904) as covered by the Animals (Scientific procedures) Act 1986. Fetal liver progenitors were prepared from E14 or E15 mouse embryos derived from pregnant females of the Helios+/2OTII+/2 mouse colony. These cells were frozen in a FCS 10% DMSO solution while each embryo was genotyped by PCR for Helios deficiency as previously described [55] and OTII transgene with primers for mouse OTII TCR a-chain: forward: 59- AAAGGGA- GAAAAAGCTCTCC-39, reverse: 59- CCAGCTGCGTCCCAT- CAC-39. Chimeras were constructed by cell transfer of about 206106 freshly defrosted foetal liver progenitor cells, resuspended in a 150 ml volume of PBS and injected into the tail vein of lethally-irradiated (264.5G) C57BL/6 mice. In studies of the role of Helios in OTII cells, C57BL/6 or CD45.1+ C57BL/6 congenic mice for experiments performed at 3 days or 7 days, respectively, received 106106 CFSE-labelled LN cells from Helios+/+OTII or Helios2/2OTII chimeras. T cell purification, adoptive transfer and immunization CD4 T cells from lymph nodes (LN) of CD45.1+ OTII mice were purified using anti-CD4 MACS microbeads (Miltenyi Biotec, Bisley, UK), CFSE-labelled (Cambridge Bioscience, Cambridge, UK) and 26106 cells were injected i.v. into CD45.2+ C57BL/6 congenic mice. Mice were immunized the following day in both footpads with alum-precipitated ovalbumin as previously described [5,44]. Flow cytometry, T cell analysis and FACS-cell sorting Popliteal LN were prepared as previously described [5,46]. Antibodies against B220- PerCP-Cy5.5 (RA3-6B2), CD4-PerCP- Cy5.5 (RM4-5), CD45.1-PE (A20), CD45.2-PE (104), CD45.2-PE- PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e20731