Helios Is Associated with CD4 T Cells Differentiating to T Helper 2 and Follicular Helper T Cells In Vivo Independently of Foxp3 Expression Karine Serre1*., Ce´ cile Be´ ne´ zech1, Guillaume Desanti1, Saeeda Bobat1, Kai-Michael Toellner1, Roger Bird1, Susan Chan2, Philippe Kastner2, Adam F. Cunningham1, Ian C. M. MacLennan 1 * . , Elodie Mohr1*.¤ 1 School of Immunity and Infection, MRC Centre for Immune Regulation, Institute for Biomedical Research, University of Birmingham, Birmingham, England, United Kingdom, 2 Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, INSERM Unite´ 964, Centre National de la Recherche Scientifique, Unite´ Mixte de Recherche 7104, Universite´ de Strasbourg, Strasbourg, France Abstract Background: Although in vitro IL-4 directs CD4 T cells to produce T helper 2 (Th2)-cytokines, these cytokines can be induced in vivo in the absence of IL-4-signalling. Thus, mechanism(s), different from the in vitro pathway for Th2-induction, contribute to in vivo Th2-differentiation. The pathway for in vivo IL-4-independent Th2-differentiation has yet to be characterized. Findings: Helios (ikzf2), a member of the Ikaros transcription regulator family, is expressed in thymocytes and some antigen- matured T cells as well as in regulatory T cells. It has been proposed that Helios is a specific marker for thymus-derived regulatory T cells. Here, we show that mouse ovalbumin-specific CD4 (OTII) cells responding to alum-precipitated ovalbumin (alumOVA) upregulate Th2 features - GATA-3 and IL-4 - as well as Helios mRNA and protein. Helios is also upregulated in follicular helper T (TFh) cells in this response. By contrast, OTII cells responding to the Th1 antigen - live attenuated ovalbumin-expressing Salmonella - upregulate Th1 features - T-bet and IFN-c - but not Helios. In addition, CD4 T cells induced to produce Th2 cytokines in vitro do not express Helios. The kinetics of Helios mRNA and protein induction mirrors that of GATA-3. The induction of IL-4, IL-13 and CXCR5 by alumOVA requires NF-kB1 and this is also needed for Helios upregulation. Importantly, Helios is induced in Th2 and TFh cells without parallel upregulation of Foxp3. These findings suggested a key role for Helios in Th2 and TFh development in response to alum-protein vaccines. We tested this possibility using Helios-deficient OTII cells and found this deficiency had no discernable impact on Th2 and TFh differentiation in response to alumOVA. Conclusions: Helios is selectively upregulated in CD4 T cells during Th2 and TFh responses to alum-protein vaccines in vivo, but the functional significance of this upregulation remains uncertain. Citation: Serre K, Be´ne´zech C, Desanti G, Bobat S, Toellner K-M, et al. (2011) Helios Is Associated with CD4 T Cells Differentiating to T Helper 2 and Follicular Helper T Cells In Vivo Independently of Foxp3 Expression. PLoS ONE 6(6): e20731. doi:10.1371/journal.pone.0020731 Editor: Jacques Zimmer, Centre de Recherche Public de la Sante´ (CRP-Sante´), Luxembourg Received December 23, 2010; Accepted May 10, 2011; Published June 3, 2011 Copyright: 2011 Serre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a program grant from the British Medical Research Council (ICMM), MAMOCELL, a NEST project from the European Union (K- MT), and from a scientific project committee grant from the University of Birmingham (KS), and from a Wellcome Trust award (KS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: karine@kserre.net (KS); I.C.M.MacLennan@bham.ac.uk (ICMM); emohr@igc.gulbenkian.pt (EM) . These authors contributed equally to this work. ¤ Current address: Instituto Gulbenkian de Cieˆncia, Oeiras, Portugal Introduction CD4 T helper 2 (Th2) cells are protective when they control immunity to extracellular parasites but their involvement in allergic inflammatory responses shows they can also be pathogenic. The canonical Th2-cytokine is IL-4 which is often produced with IL-5 and IL-13 and the genes encoding these 3 cytokines are located in a contiguous gene cluster [1–3]. There are still gaps in our understanding of how CD4 Th2 cells are induced in vivo [4]. To get insight into the specific features and the signalling pathway(s) that operate(s) in vivo we studied Th2- differentiation of na¨ıve ovalbumin (OVA)-specific transgenic CD4 T (OTII) cells that had been transferred into wild-type congenic mice. Alum-precipitated ovalbumin (alumOVA) was used to induce the Th2-associated transcription factor GATA-3 and IL-4, while live attenuated ovalbumin-expressing Salmonella (SalOVA) was used to induce Th1-associated T-bet and IFN-c. a previous report from our laboratory extensive analysis of the diversity of these different CD4 T cell responses was made by using low-density, real-time RT-PCR microfluidic cards [5]. One of the novel outcomes of this study was that, by 3 days after immunization, the transcription regulator Helios was induced in OTII cells in response to alumOVA but, not to SalOVA. The objective of the current report is to assess whether this selective expression of Helios is strictly associated with Th2 cells differentiated in vivo. PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20731