Helios Deficiency Has Minimal Impact on T Cell Development and Function Qi Cai, Andrée Dierich, Mustapha Oulad-Abdelghani, Susan Chan and Philippe Kastner This information is current as of September 23, 2021. J Immunol 2009; 183:2303-2311; Prepublished online 20 July 2009; doi: 10.4049/jimmunol.0901407 http://www.jimmunol.org/content/183/4/2303 Downloaded from References This article cites 44 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/183/4/2303.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Helios Deficiency Has Minimal Impact on T Cell Development and Function1 Qi Cai,*†§ Andre´e Dierich,†§ Mustapha Oulad-Abdelghani,†§ Susan Chan,2*†§ and Philippe Kastner2*†‡§ Helios is a member of the Ikaros family of zinc finger transcription factors. It is expressed mainly in T cells, where it associates with Ikaros-containing complexes and has been proposed to act as a rate-limiting factor for Ikaros function. Overexpression of wild-type or dominant-negative Helios isoforms profoundly alters ␣␤ T cell differentiation and activation, and endogenous Helios is expressed at strikingly high levels in regulatory T cells. Helios has also been implicated as a tumor suppressor in human T cell acute lymphoblastic leukemias. These studies suggest a central role for Helios in T cell development and homeostasis, but whether this protein is physiologically required in T cells is unclear. We report herein that inactivation of the Helios gene by homologous recombination does not impair the differentiation and effector cell function of ␣␤ and ␥␦ T cells, NKT cells, and regulatory T cells. Downloaded from These results suggest that Helios is not essential for T cells, and that its function can be compensated for by other members of the Ikaros family. The Journal of Immunology, 2009, 183: 2303–2311. he Helios transcription factor (Ikzf2) is a member of the as Ig class switch recombination in mature B cells (18–21). Ad- Ikaros family of zinc finger regulators that includes Ikaros ditionally, both Ikaros and Aiolos are required to limit B cell pro- T (Ikzf1), Aiolos (Ikzf3), Eos (Ikzf4), and Pegasus (Ikzf5). liferation in response to activation (15, 18). Thus, Ikaros and Aio- http://www.jimmunol.org/ Ikaros family proteins share a similar structure that is characterized los appear to play mostly distinct, but also overlapping, roles in B by highly conserved zinc finger domains at the N and C termini cells. (1). Four N-terminal zinc fingers are responsible for DNA binding In T cells, Ikaros appears to be singularly important. Ikaros de- to consensus target sequences, while two C-terminal zinc fingers ficiency leads to absence of fetal T cell development, while post- mediate homo- and heterodimerization between family members natal T cell differentiation is associated with enhanced pre-TCR (2–8). Ikaros, Helios, and Aiolos all interact with the nucleosome signaling, leading to increased proliferation of DN4 thymocytes remodeling and deacetylase (NuRD) histone deacetylase complex (14, 22). Loss of Ikaros also leads to a decreased ␥␦ T cell pool, (9, 10), suggesting that they play pivotal roles in chromatin re- as well as altered commitment to the CD4 and CD8 lineages (14, by guest on September 23, 2021 modeling at their target genes. 23, 24). In mature T cells, Ikaros appears to suppress Th1 polar- Ikaros and Aiolos are critical regulators of hematopoiesis. Ikaros ization (25) and to limit proliferation in response to signaling in is implicated in stem cell renewal, fetal and adult erythropoiesis, both CD4ϩ and CD8ϩ T cells (26). Finally, Ikaros is involved in 3 and dendritic cell (DC) differentiation (11–14). Ikaros and Aiolos silencing Notch signaling during the double-negative to double- perform distinct functions in B cells. Aiolos is essential for many positive transition, a function that is likely to contribute to its tu- aspects of B cell differentiation and function, including B cell pro- mor suppressor function in this lineage (27). Indeed, Ikaros defi- liferation, marginal zone vs follicular B cell fate choice, and the ciency is strongly associated with development of T cell leukemias development of high-affinity plasma cells (15–17). Ikaros controls that exhibit high levels of Notch activation (28, 29). The multiple early steps of B cell differentiation, including commitment, as well abnormalities seen in Ikaros-deficient T cells contrast with the largely normal T cell compartment in Aiolos-deficient mice. How- *Department of Cancer Biology, †Institut de Ge´ne´tique et de Biologie Mole´culaire et ever, Aiolos-null T cells also hyperproliferate to activation signals Cellulaire, INSERM Unite´964, Centre National de la Recherche Scientifique, Unite´ (15), suggesting that, as in B cells, both Ikaros and Aiolos are ‡ § Mixte de Recherche 7104, Illkirch, France; and Faculte´deMe´decine, Universite´de required to set the threshold for the proliferative response of these Strasbourg, Strasbourg, France cells to activation. Received for publication May 5, 2009. Accepted for publication June 11, 2009. Helios is conspicuous for its high expression from the earliest The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance stages of T cell development (5, 30). Strikingly, Helios is induced ϩ ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. Ͼ10-fold in CD4 Foxp3 regulatory T (Treg) cells (31–33). Its 1 This work was supported by institute funds from INSERM, Centre National de la expression in Treg cells does not require Foxp3, a transcriptional Recherche Scientifique, and Hoˆpital Universitaire de Strasbourg, and a grant to S.C. regulator essential for Treg cell differentiation (34), suggesting that and P.K. from the Ligue Nationale Franc¸aise Contre le Cancer (Equipe Labellise´e). Q.C. was funded by a predoctoral fellowship from the Association pour le Recherche Helios might function as an upstream regulator of Foxp3, or per- sur le Cancer. haps define a parallel transcriptional circuit in these cells. Helios is 2 Address correspondence and reprint requests to Dr. Susan Chan and Dr. Philippe not expressed in mature B cells, DCs, or myeloid cells. At the Kastner, Department of Cancer Biology, Institut de Ge´ne´tique et de Biologie Mo- le´culaire et Cellulaire, BP 10142, 67404 Illkirch Cedex, France. E-mail address: molecular level, Helios associates with a subset of Ikaros com- [email protected] plexes that localize near centromeric heterochromatin in T cells 3 Abbreviations used in this paper: DC, dendritic cell; B6, C57BL/6; dn, dominant (5), suggesting that it might act as a rate-limiting factor of Ikaros negative; HPRT, hypoxanthine phosphoribosyltransferase; Treg cell, regulatory T function. Gain-of-function studies, using full-length or dominant- cell; WT, wild type. negative (dn) Helios lacking the DNA-binding domain, suggest a Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 key role for this protein in T cell differentiation and function. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901407 2304 HELIOS AND T CELLS Overexpression of full-length Helios blocks ␣␤ T cell differenti- transfected with an Ikaros expression vector were used as positive controls. ation at the CD4ϪCD8Ϫ stage in the thymus, and it results in The membrane was blocked at room temperature for 1 h with blocking increased frequencies of ␥␦ T cells and NK cells in peripheral buffer (5% fat-free milk, 0.1% Tween 20 in PBS) and then incubated with the primary Ab at 4°C overnight. After washing (0.1% Tween 20 in PBS), lymphoid organs, while overexpression of dn Helios leads to in- the membrane was incubated with a 1/104 dilution of HRP-conjugated creased T cell proliferation upon TCR stimulation and the devel- donkey anti-rabbit Abs or goat anti-mouse Abs (Jackson ImmunoResearch opment of T lymphomas (35). Furthermore, dn Helios isoforms or Laboratories), washed, and revealed with Immobilon Western (Millipore). allelic loss have been detected in some human T-acute lympho- The polyclonal rabbit anti-mouse Helios Abs Ab1 and Ab2 were gen- erated by immunizing rabbits with a bacterially expressed N-terminal He- blastic leukemias or T cell lymphomas (36–39). These results sug- lios fragment corresponding to aa 1–109 (Ab1) or to a peptide correspond- gest that Helios is an essential regulator of T cell homeostasis and ing to Helios aa 369–381 (Ab2). Abs were purified with SulfoLink a tumor suppressor. columns coupled to the immunogens. The rabbit anti-mouse Aiolos Ab was While these studies have been informative, they are unclear, as generously provided by A. Rebollo (INSERM Unite´543, Paris, France). overexpression of either full-length or dn proteins must be inter- The rabbit anti-mouse Ikaros (C-terminal) and monoclonal TATA-box binding protein (TBP) Abs were previously generated in our institute. preted cautiously since they can inhibit the normal function of related endogenous proteins. This is especially true for the Ikaros Abs and flow cytometry family, as four of its five members (Ikaros, Aiolos, Helios, and Eos) are coexpressed in T cells.