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

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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 is physiologically required in T cells is unclear. We report herein that inactivation of the Helios 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 (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 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 . 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. Thus, it is important to understand All Abs were purchased from BD Pharmingen or eBioscience, except for the exact role of each protein in target gene activation/repression Abs to CD3 (KT3), CD4 (YTS191.1 or GK1.5), CD8 (YTS169.4), and and T cell development. At the present time, Helios function re- CD44 (IM7), which were produced in-house and conjugated according to standard protocols. For flow cytometry analyses, cells were first incubated mains unknown. with anti-CD16/32 to block Fc receptors. Intracellular staining of Treg cells Downloaded from In this study, we investigated Helios function in T cells by gen- was performed using a Foxp3 staining kit (eBioscience). Cells were ana- erating a null mutation for this gene by homologous recombina- lyzed on a FACSCalibur (BD Biosciences), and data were analyzed with tion. We find that Helios is not essential for T cell differentiation, FlowJo software (Tree Star). Sorting was performed on a FACSVantage SE option DiVa (BD Biosciences). Sort purity was Ͼ95%. homeostasis, and function, and that Helios-deficient mice do not develop T cell malignancies. Proliferation assays http://www.jimmunol.org/ Materials and Methods To induce the proliferation of peripheral CD4ϩ and CD8ϩ T cells, whole Generation of Helios-null mice splenic CD4ϩ and CD8ϩ T cells, as well as splenic CD4ϩCD44ϪCD25Ϫ and CD8ϩCD44Ϫ T cells, were sorted, incubated with CFSE, and then The Helios targeting vector is depicted in Fig. 1A. The C-terminal part of seeded into 96-well plates coated with anti-CD3 (10 ␮g/ml; eBioscience) Helios exon 7 was replaced by a 1.8-kb floxed PGK-neo-poly(A) cassette or anti-CD3 (5 ␮g/ml) plus anti-CD28 (5 ␮g/ml; eBioscience) in complete between the indicated SalI and XbaI sites. The vector was transfected into RPMI medium (RPMI 1640 supplemented with 10% FCS, 25 mM HEPES, P1 129/Sv embryonic stem cells, and homologous recombination events 2mML-glutamine, 1ϫ nonessential amino acids, 1 mM sodium pyruvate, were detected by Southern blot using external probes A and B with BamHI- 50 ␮M 2-ME, 1% penicillin and streptomycin) in triplicate samples for 3 digested genomic DNA. Two embryonic stem cell clones, MBA120 and days. Control cells were cultured without Ab stimulation. Cells were cul- MBA93, were identified as positive and injected into C57BL/6 (B6) blas- tured at 2.5 ϫ 104 cells per well for the anti-CD3 stimulation and unstimu- tocysts to produce chimeric mice. Germline transmission was verified by lated controls, and at 1 ϫ 104 cells per well for anti-CD3 plus anti-CD28. by guest on September 23, 2021 PCR on tail DNA using primers P3 and P4 to detect the wild-type (WT) For Treg cell assays, Treg (CD4ϩCD25ϩCD44Ϫ), Th (CD4ϩCD25Ϫ allele, and P3 and P5 to detect the mutant allele. Mice derived from both CD44Ϫ), and APCs (Thy1.2Ϫ) were sorted from pooled spleen and lymph clones gave similar phenotypes. The experiments described in this paper node cells. Treg or Th cells were incubated with CFSE and cultured in were performed using mice generated from clone MBA120 and back- complete RPMI medium at 5 ϫ 103 cells per well in 96-well plates in crossed once onto the B6 background. triplicate samples with anti-CD3 (1 ␮g/ml) and 105 APCs, in the absence RT-PCR, real-time RT-PCR, and primers or presence of recombinant human IL-2 (100 U/ml). Cells were harvested after 3 days and stained for CD4 and CD25 expression before analysis. For RT-PCR and real-time PCR, RNA was extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed with SuperScript II reverse tran- Th1 and Th2 induction scriptase (Invitrogen). mRNA levels for Aiolos, Helios, Eos, and Ikaros were quantified by real-time RT-PCR using SYBR Green JumpStart Taq Th1 and Th2 polarizing cultures were performed mostly according to Tu et ReadyMix (Sigma-Aldrich). Real-time RT-PCR was performed with the al. (41). Briefly, CD4ϩCD44ϪCD25Ϫ Th cells and Thy1.2Ϫ APCs were LightCycler 480 (Roche) with the following conditions: 95°C for 10 s, sorted from adult spleens. In neutral conditions, 25 ϫ 103 Th cells were 63°C for 15 s, and 72°C for 15 s for 40 cycles. The difference in cDNA cultured with 25 ϫ 103 APCs (mitomycin C treated), recombinant human input was normalized to hypoxanthine phosphoribosyltransferase (HPRT) IL-2 (10 U/ml; PeproTech), soluble anti-CD3 (0.1 ␮g/ml), and anti-CD28 expression levels. The following primers were used: P1, 5Ј-CCAATGG (0.5 ␮g/ml) in 96-well plates in triplicate samples. In Th1 polarizing con- ACAGCACGCCTCG; P2, 5Ј-ATATCTGGGTAGCTGAATCGC; P3, 5Ј- ditions, anti-IL-4 (10 ␮g/ml; eBioscience) and recombinant murine IL-12 TCTATTAGTGTCAGCTTTTTGACAGTTT; P4, 5Ј-GATGAATTCCTT (5 ng/ml; eBioscience) were added to these cultures. In Th2 polarizing ATAGATGTCCTTCAGAGAGCC; P5, 5Ј-ATCTGCACGAGACTAGTG conditions, anti-IL-12 Ab (10 ␮g/ml; eBioscience) and recombinant mu- AGACG; P6, 5Ј-GATGCTAGCCAGAATGTCAGCATGGAGGCTGCC; rine IL-4 (20 ng/ml; PeproTech) were added to the cultures. After 7 days ␤-actin, forward, 5Ј-TGTTACCAACTGGGACGACA, reverse, 5Ј-CCA of culture, cells were restimulated with PMA (50 ng/ml; Sigma-Aldrich) TCACAATGCCTGTGGTA; Helios, forward, 5Ј-ACACCTCAGGACCCA and ionomycin (500 ng/ml; Sigma-Aldrich) for 4 h. GolgiPlug (brefeldin TTCTG, reverse, 5Ј-TCCATGCTGACATTCTGGAG; Aiolos, forward, A; BD Biosciences) was added at 1 ␮l/ml for the last 2 h. Cells were 5Ј-ACAGCAGACCAACCGGTGGGAA, reverse, 5Ј-ACTGGAACGGG harvested and stained to assess surface Thy1.2, CD4, and intracellular IL-4 CGTTCGC; Eos, forward, 5Ј-GAGGAGCACAAGGAGAGGTG, reverse, and IFN-␥. Intracellular staining was performed using the Cytofix/Cyto- 5Ј-CATCTCCAGGTCACGGATTT; Ikaros, forward, 5Ј-CATAAAGAGC perm fixation/permeabilization kit (BD Biosciences). GATGCCACAA, reverse, 5Ј-CAGGACAAGGGACCTCTCTG; HPRT, forward, 5Ј-GTTGGATACAGGCCAGACTTTGTTG, reverse, 5Ј-GATTC AACTTGCGCTCATCTTAGGC. Note that the Aiolos primers were ob- Detection of perforin and granzyme B tained from Thompson et al. (20). Splenic T cells were purified by negative selection using Abs specific for 4 Western blot and Ab production B220, CD11b, CD11c, Ter-119, and DX5. T cells (25 ϫ 10 /well) were then cultured in complete RPMI medium with or without recombinant Nuclear extracts were prepared according to Andrews and Faller (40). Thy- human IL-2 at 100 U/ml in 96-well plates in triplicate samples. After 3 mocyte extracts (5 ␮g) were separated on an 8% SDS-PAGE gel and trans- days, cells were harvested and stained for CD4 and CD8 before intracel- ferred to polyvinylidene difluoride membranes. Extracts (25 ng) from COS lular staining for granzyme B and perforin with a Foxp3 staining buffer cells transfected with a Helios expression vector or from cells (250 ng) system (eBioscience). The Journal of Immunology 2305

FIGURE 1. Generation of He- liosϪ/Ϫ mice. A, Targeting vector and recombination strategy. In the illus- tration of the Helios protein, exons are marked as gray boxes and zinc fingers as white rectangles. Ab1 is specific for the N-terminal region; Ab2 is specific for a region within exon 7. Primers are depicted as black triangles. The targeting vector con- tained 9.4 kb of the Helios con- taining exon 7, depicted as a thick line. LoxP sites flanking the PGK-neo cassette are depicted as white trian- gles. Recombination was verified us- ing outside probes A and B, and fur- ther confirmed with probe C. B, BamHI; E, EcoRI; H, HindIII; S, SalI;

X, XbaI. B, Southern blot of BamHI- Downloaded from digested embryonic stem cell DNA, analyzed with probes A (left) and B (right). C, PCR of genomic DNA from WT, Heliosϩ/Ϫ, and HeliosϪ/Ϫ mice using primers P3 and P4 (for the WT allele) or P3 and P5 (for the mu- tant allele). PCR products were con- http://www.jimmunol.org/ firmed by hybridization with probe C. D, RT-PCR of Helios and ␤-actin from WT and HeliosϪ/Ϫ thymocyte RNA. P1 and P2 amplify transcript sequences upstream of the mutation. P4 and P6 amplify transcripts encom- passing the mutation. E, Western blot of WT and HeliosϪ/Ϫ thymocyte nu-

clear extracts using the Abs Ab1 and by guest on September 23, 2021 Ab2. TATA-box binding protein (TBP) was detected as a loading con- trol. Note that the 50-kDa polypeptide detected in both WT and HeϪ/Ϫ cells is likely to be nonspecific since it was not detected with Ab2.

Treg cell suppression assay deleted sequences (Fig. 1D, right panel). Helios proteins were not Pooled cells from spleen and lymph nodes were first selected for lineage- detected in thymocyte nuclear extracts by Western blot, either in positive (B220, CD11b, CD11c, NK1.1, Ter-119) cells; these cells were full-length or truncated forms, using two Abs produced against the treated with mitomycin C and used as APCs. Lineage-negative cells were N-terminal portion of Helios (Ab1; Fig. 1E, top panel) or the C- ϩ ϩ ϩ Ϫ then sorted for Treg (CD4 CD25 ) and responder Th (CD4 CD25 ) terminal region upstream of the deletion (Ab2; Fig. 1E, bottom ϫ 3 cells. Th cells were incubated with CFSE, and 25 10 Th cells were panel cultured with equal numbers of APCs and anti-CD3 (5 ␮g/ml) in complete ). These results indicate that the exon 7 deletion results in RPMI medium in 96-well plates in triplicate samples. Treg cells were loss of synthesis or rapid degradation of Helios polypeptides in T added to the cultures at the indicated ratios. In the no stimuli controls, Th cells. Therefore, we conclude that this mutation is null. cells were cultured with APCs in the absence of anti-CD3. Cells were Although homozygote HeliosϪ/Ϫ and heterozygote Heliosϩ/Ϫ harvested 3 days later and stained for CD4 before analysis. Suppression of ϩ ϩ animals were born at Mendelian frequencies and were of normal Th proliferation was evaluated as loss of CFSE in the CD4 CFSE -gated Ϫ/Ϫ populations. body size at birth (data not shown), many Helios pups died within the first weeks of postnatal life (Fig. 2A). This early lethal- Results ity increased as HeliosϪ/Ϫ mice were backcrossed onto the B6 We produced a Helios-null mutation by targeting a PGK-neo cas- background, and no surviving homozygotes could be obtained af- sette into exon 7 of the Helios locus by homologous recombination ter 10 generations of backcrossing. We thus studied the phenotype (Fig. 1A). This mutation deletes most of exon 7, including the of Helios mutants on a mixed 129/Sv:B6 background. It is unclear Ϫ Ϫ sequences that encode the C-terminal zinc fingers, as indicated by why so many Helios / mutants die after birth. However, this is Southern blot analyses of mutant embryonic stem cells and PCR probably not associated with defective hematopoiesis, as 14.5 days analyses of tail DNA from heterozygote and homozygote Helios postcoitus HeliosϪ/Ϫ fetal liver cells efficiently reconstituted all mutants (Fig. 1, B and C). HeliosϪ/Ϫ thymocytes still transcribed hematopoietic lineages after adoptive transfer into lethally irradi- Helios mRNA (Fig. 1D, left panel), but these transcripts lacked the ated mice (data not shown). Surviving adult HeliosϪ/Ϫ mice, 2306 HELIOS AND T CELLS

altered. These results indicate that ␣␤ T cell differentiation and homeostasis proceeds normally in the absence of Helios. NKT cells, ␥␦ T cells, and Treg cells also differentiate in the thymus, and these cells express high levels of Helios mRNA (Refs. 30, 32, 33 and data not shown). We therefore analyzed these cell types in the thymus and spleen of HeliosϪ/Ϫ mice. ␥␦ T cell and NKT cell (CD3ϩDX5ϩ) frequencies in the mutant organs were similar to those from WT animals (Fig. 4, A and B). HeliosϪ/Ϫ animals also showed normal percentages of thymic and splenic CD25ϩFoxp3ϩ Treg cells (Fig. 4C). Furthermore, the above cell types differentiated normally when analyzed in competitive mixed bone marrow chimera experiments where WT and HeliosϪ/Ϫ bone marrow cells were transferred to lethally irradiated WT recipients (data not shown). These results indicate that ␥␦ T cells, NKT cells, and Treg cells mature normally in Helios-deficient mice. To determine whether loss of Helios affects mature T cell func- tion, we first analyzed the capacity of HeliosϪ/Ϫ peripheral CD4ϩ and CD8ϩ T cells to proliferate in response to TCR stimulation in vitro, as Ikaros and Aiolos have been shown to limit the prolifer- Downloaded from ation threshold of T and B cells upon activation (14, 15, 18, 26, 28). Naive CD4ϩCD25ϪCD44Ϫ and CD8ϩCD44Ϫ T cells, as well ϩ ϩ FIGURE 2. Growth and early survival defects in HeliosϪ/Ϫ mice. A, as whole CD4 and CD8 T cells, were sorted from WT and Ϫ/Ϫ Total numbers and frequencies of WT, Heliosϩ/Ϫ, and HeliosϪ/Ϫ mice that Helios spleens, labeled with CFSE, and cultured for 3 days reached the age of 2 wk. Mice were generated from Heliosϩ/Ϫ intercrosses. with anti-CD3 Abs alone or in combination with anti-CD28. These

Ϫ Ϫ ϩ ϩ http://www.jimmunol.org/ B, Representative WT (top) and HeliosϪ/Ϫ (bottom) male littermates at 6 experiments show that WT cells and Helios / CD4 and CD8 wk of age. C, Body weights of adult WT and HeliosϪ/Ϫ male (left) and T cells proliferate similarly to TCR stimulation in the absence or female (right) littermates. Symbols indicate pairs of age-matched litter- presence of costimulatory signals (Fig. 5A). mates. Values of p were calculated with a two-tailed paired Student’s t test. To test if HeliosϪ/Ϫ CD4ϩ T cells efficiently differentiate into ϭ ءءء ϭ ءء ϭ ء , p 0.013; , p 0.083; , p 0.005. Th1 or Th2 cells, we measured the capacity of these cells to pro- duce IFN-␥ and IL-4 under neutral and polarizing conditions. In neutral conditions, CD4ϩCD25ϪCD44Ϫ splenic T cells were cul- tured with Thy1.2Ϫ APCs in the presence of anti-CD3 and anti- particularly females, were markedly smaller than WT or hetero-

CD28 Abs, as well as IL-2, for 7 days to evaluate the capacity of by guest on September 23, 2021 zygote littermates in size and weight (Fig. 2, B and C, and data not Ϫ Ϫ these cells to produce cytokines upon stimulation. In Th1 polariz- shown). Helios / animals remained smaller as they aged, but ing conditions, CD4ϩCD25ϪCD44Ϫ splenic T cells were cultured these differences were no longer statistically significant in the male Ϫ Ϫ as above in the presence of IL-12 and anti-IL-4 Abs. In Th2 po- population (Fig. 2C). Helios / mice lived to at least 22 mo of age and showed no overt signs of ill health (nine mice analyzed be- larizing conditions, the same cells were cultured as above in the tween 16 and 22 mo of age; data not shown). HeliosϪ/Ϫ mice presence of IL-4 and anti-IL-12 Abs. After 7 days, all cultures exhibited smaller eye-openings, a phenotype that might be linked were restimulated with PMA and ionomycin for 4 additional hours before analysis by intracytoplasmic staining. As shown in to an abnormal growth of eyelids (data not shown). Both male and Ϫ/Ϫ ϩ ϩ Ϫ/Ϫ Fig. 5B, Helios CD4 T cells differentiated into IFN-␥ female Helios mice were fertile. These results indicate that ϩ ϩ Ϫ/Ϫ Th1 and IL-4 Th2 cells as efficiently as did WT CD4 T cells Helios mice do not spontaneously develop health-threatening ϩ illness if they survive the weaning period. in all conditions, indicating that CD4 T cell function is unaf- Ϫ/Ϫ To address the role of Helios in ␣␤ T cell development, we fected in Helios mice. Ϫ/Ϫ ϩ analyzed T cell populations in the thymuses and spleens of WT To evaluate if Helios CD8 T cells function as mature ef- and HeliosϪ/Ϫ mice. Immature CD4ϪCD8ϪCD3Ϫ double-nega- fector cells, we tested their capacity to produce perforin and gran- tive thymocytes were analyzed for CD44 and CD25 expression, zyme B, two molecules important for cytotoxic function, upon while the more mature CD4ϩCD8ϩ double-positive thymocytes stimulation. Splenic T cells were enriched by negative depletion ϩ and the CD4ϩCD8Ϫ and CD4ϪCD8ϩ single-positive cells were and cultured with IL-2 for 3 days. CD8 T cells were analyzed for analyzed for CD3, ␣␤TCR, CD24, and CD69 expression. All dou- the expression of perforin and granzyme B by intracellular stain- ϩ ble-negative subpopulations (CD44ϩCD25Ϫ, CD44ϩCD25ϩ, ing. Comparable percentages of CD8 T cells were induced to Ϫ/Ϫ CD44ϪCD25ϩ, CD44ϪCD25Ϫ) were similar in frequency be- express perforin and granzyme B in both WT and Helios cul- ϩ tween WT and mutant thymuses from adult mice (7–9 wk old) and tures (Fig. 5C), suggesting that CD8 T cell function is normal in Ϫ Ϫ newborn animals (1 day old) (Fig. 3A–D). Double-positive and Helios / mice. single-positive thymocyte populations were also similar in fre- Since Helios expression is strongly induced in Treg cells, we quencies and absolute numbers according to their CD4 and CD8 asked if Helios deficiency specifically affects Treg cell function. profiles, and no differences were observed in terms of CD3, We first tested if Helios contributes to the low proliferative re- ␣␤TCR, CD24, and CD69 expression for each population (data sponse of Treg cells to TCR-induced signals. As depicted in Fig. Ϫ Ϫ ϩ ϩ Ϫ not shown). Additionally, peripheral CD4ϩ and CD8ϩ T cells 6A, neither WT nor Helios / Treg cells (CD4 CD25 CD44 ), from the lymph nodes and spleens were comparable in frequency purified from lymph nodes and spleens, responded to anti-CD3 between WT and mutant mice (Fig. 3E), and no differences were stimulation in a 3-day coculture assay with APCs. Additionally, observed in CD62L and CD44 expression in these cell types (data both WT and HeliosϪ/Ϫ Treg cells proliferated similarly when not shown), suggesting that memory T cell populations were un- stimulated with anti-CD3 Abs in the presence of IL-2, although the The Journal of Immunology 2307 Downloaded from FIGURE 3. ␣␤ T cell develop- ment or homeostasis in HeliosϪ/Ϫ mice. Thymocytes from WT and He- liosϪ/Ϫ adult mice (A and B) and neonates (C and D) were analyzed for expression of the indicated markers. Numbers in the contour plots indicate http://www.jimmunol.org/ percentages of the gated cells. Bar graphs indicate mean absolute num- bers of the different thymocyte popu- lations analyzed Ϯ SD, as calculated from four independent experiments. E, WT and HeliosϪ/Ϫ splenocytes and lymph node cells were analyzed for CD4 and CD8 expression. Re-

sults are representative of three sim- by guest on September 23, 2021 ilar experiments.

proportion of HeliosϪ/Ϫ Treg cells that responded to this stimula- Lastly, we investigated the possibility that HeliosϪ/Ϫ T cells tion was reduced by about one-third. Thus, HeliosϪ/Ϫ Treg cells might compensate for loss of Helios activity by up-regulating the do not exhibit an altered pattern of proliferation under established expression of other Ikaros family members. We tested the mRNA conditions. We next evaluated if HeliosϪ/Ϫ Treg cells could sup- levels of Aiolos, Ikaros, Eos, and Helios in WT and HeliosϪ/Ϫ press the proliferation of responder Th (CD4ϩCD25Ϫ) cells stim- thymocytes by real-time RT-PCR (Fig. 7A). HeliosϪ/Ϫ thymocytes ulated with anti-CD3 Abs and APCs. HeliosϪ/Ϫ Treg cells sup- exhibited a small but statistically significant increase in Aiolos pressed Th cell proliferation as efficiently as did WT Treg cells mRNA levels (WT, 0.97 Ϯ 0.03; HeliosϪ/Ϫ, 1.26 Ϯ 0.09), while (Fig. 6B). Collectively, these results indicate that mature Treg cells Ikaros and Eos mRNA levels were similar between genotypes. At function normally in the absence of Helios. the protein level, the expression of Aiolos and Ikaros was 2308 HELIOS AND T CELLS

FIGURE 4. ␥␦ T, NKT, and Treg cell frequencies in adult HeliosϪ/Ϫ mice. WT and HeliosϪ/Ϫ thymocytes and splenocytes were analyzed for their populations of ␥␦ T cells, gated on CD4ϪCD8Ϫ cells (A), NKT (CD3ϩDX5ϩ) cells (B), and Treg (CD25ϩFoxp3ϩ) cells, gated on CD4 single-positive cells in the thymus and CD4ϩ cells in the spleen (C). Numbers indicate percentages of the gated cells. Results are representative of more than three experiments. comparable between WT and mutant thymocytes (Fig. 7B). during this period. The physiological functions that depend strictly These data suggest that HeliosϪ/Ϫ T cells do not compensate for on Helios remain unknown, but they are unlikely to be related to

Helios deficiency by drastically up-regulating Aiolos and Ikaros hematological or immune defects. Helios expression outside of the Downloaded from expression. Furthermore, Helios mRNA levels remained similar hematopoietic system is poorly characterized at present. Together between WT and mutant thymocytes despite loss of Helios at with reports showing that Ikaros controls neural and endocrine the protein level, suggesting that Helios does not autoregulate functions (42–44), the severe viability defect of HeliosϪ/Ϫ mice its own transcription. provides further evidence that Ikaros family members exert im- portant functions beyond the hematopoietic system.

Discussion In mature hematopoietic cells, Helios expression is restricted to http://www.jimmunol.org/ We report herein the generation of the first Helios-deficient mouse T cells (5, 30), suggesting that Helios may control important as- line. Our data show that Helios is essential for the first weeks of pects of T cell differentiation and/or function. We show herein that life, as most HeliosϪ/Ϫ mice (100% on a pure B6 background) die Helios is not essential for the development, homeostasis, and by guest on September 23, 2021

FIGURE 5. ␣␤ T cell function in the absence of Helios. A, WT (gray shaded histogram) and HeliosϪ/Ϫ (solid line histogram) whole CD4ϩ or naive CD4ϩCD25ϪCD44Ϫ T cells, and whole CD8ϩ or naive CD8ϩCD44Ϫ T cells, were stimulated for 3 days with the indicated concentrations (␮g/ml) of anti-CD3 and anti-CD28 Abs. Proliferation was assayed by loss of CFSE intensity. Results are representative of three similar experiments, each performed in triplicate samples. Note that the small difference seen between WT and HeliosϪ/ϪCD8ϩ T cells stimulated with anti-CD3 was not reproducible in all experiments. B, WT and HeliosϪ/Ϫ splenic CD4ϩ T cells were cultured in neutral, Th1, or Th2 polarizing conditions. Gated CD4ϩThy1.2Ϫ cells were analyzed for intracellular expression of IFN-␥ and IL-4. Numbers indicate percentages of each quadrant. Results are representative of three similar experiments conducted in triplicate wells. The small differences observed between WT and HeliosϪ/Ϫ cells were not reproduced in other experiments. C, Splenic T cells were stimulated with IL-2 for 3 days and analyzed for intracellular expression of granzyme B and perforin. Bar graph shows the mean percentage Ϯ SD of positive cells in the CD8ϩ T cell population, as determined from triplicate samples. Results are representative of three independent experiments. The Journal of Immunology 2309

FIGURE 6. Mature Treg cell func- tion in HeliosϪ/Ϫ mice. A, Purified WT and HeliosϪ/Ϫ CD4ϩCD25ϩ CD44Ϫ Treg cells from spleen and lymph nodes were stimulated for 3 days with anti-CD3 and APCs, in the absence or presence of IL-2. Th cells (CD4ϩCD25ϪCD44Ϫ) were used as positive controls. CD4ϩCFSEϩ cells were analyzed for CD25 expression and CFSE intensity. Numbers indi- cate percentages of cells in each gate. Results are representative of two in- dependent experiments performed in triplicate. B, CFSE-stained responder Th cells (CD4ϩCD25Ϫ) were cul- tured for 3 days with anti-CD3 and APCs. WT and HeliosϪ/Ϫ Treg cells (CD4ϩCD25ϩ) were added at the in-

dicated ratios. Th cells cultured with Downloaded from APCs in the absence of anti-CD3 are shown as the no stimuli control. Pro- liferation of Th cells is shown as CFSE loss in the CD4ϩCFSEϩ-gated population. Numbers indicate per- centages of cells in each gate. WT Ϫ/Ϫ and Helios Treg cells suppressed http://www.jimmunol.org/ proliferation in a similar manner re- gardless of whether the Th cells and APCs were of WT or HeliosϪ/Ϫ ori- gin. Results are representative of two independent experiments performed in triplicate.

function of thymic-derived T lymphocytes, in contrast to expecta- role in more specialized T cell functions or subsets that were not by guest on September 23, 2021 tions from earlier overexpression studies suggesting a primary role studied here. The present mouse line thus provides an important for this transcription factor in ␣␤ T cell development and as a tool to further explore the function of Helios in T cells. tumor suppressor. In particular, most of the defects associated with The lack of apparent T cell phenotype in Helios-null mice sug- Ikaros deficiency are not detected in Helios-null mice (i.e., lack of gests that other Ikaros family members may compensate for Helios fetal T cell development, reduced cellularity in the adult thymus, in T cells. This is consistent with previous reports suggesting that increases in DN4 and CD4 single-positive thymocyte populations, Ikaros and Helios bind similar target sequences and belong to sim- reduced ␥␦ T cells, increased Th1 and impaired Th2 differentia- ilar macromolecular complexes (5, 30). If so, then Ikaros appears tion, T cell hyperproliferation, and T cell transformation; see Refs. to be the dominant family member in the ␣␤ T cell lineage, while 14, 22, 25). Thus, Helios is clearly not required for Ikaros-depen- Helios may function in a redundant manner in these cells. It would dent function in T cells and is not sufficient to sustain T cell de- therefore be interesting to study T cell development in animals velopment alone. Note, however, that Helios may play a unique deficient for both Ikaros and Helios. Our efforts to generate

FIGURE 7. Expression of Ikaros, Aiolos, and Eos in HeliosϪ/Ϫ cells. A, RNA from WT and HeliosϪ/Ϫ thymocytes was analyzed for Aiolos, Ikaros, Eos, and truncated Helios transcripts by real-time RT-PCR. Expression levels were normalized to HPRT. Mean expression levels Ϯ SD are presented, as calculated from three independent experiments. B, Nuclear extracts from WT and HeliosϪ/Ϫ thymocytes were analyzed by Western blot using Helios, Aiolos, and Ikaros-specific Abs. TATA-box binding protein (TBP) was analyzed as a loading control. Results are representative of three independent experiments. 2310 HELIOS AND T CELLS double-mutant mice have so far been unsuccessful, due to the ex- 5. Hahm, K., B. S. Cobb, A. S. McCarty, K. E. Brown, C. A. Klug, R. Lee, tremely high mortality rate of these mice. K. Akashi, I. L. Weissman, A. G. Fisher, and S. T. Smale. 1998. Helios, a T cell-restricted Ikaros family member that quantitatively associates with Ikaros at Recent studies have shown that Helios is highly expressed at the centromeric heterochromatin. Genes Dev. 12: 782–796. mRNA level in Treg cells compared with conventional CD4ϩ T 6. Morgan, B., L. Sun, N. Avitahl, K. Andrikopoulos, T. Ikeda, E. Gonzales, P. Wu, Ͼ S. Neben, and K. Georgopoulos. 1997. Aiolos, a lymphoid restricted transcription cells ( 10-fold) (31–33). Interestingly, Helios is expressed early factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J. in the Treg cell lineage and is not a downstream target of Foxp3 16: 2004–2013. (33). These observations have led to speculation that Helios (and 7. Perdomo, J., M. Holmes, B. Chong, and M. Crossley. 2000. Eos and pegasus, two members of the Ikaros family of proteins with distinct DNA binding activities. perhaps Eos, which is similarly expressed in Treg cells) may play J. Biol. Chem. 275: 38347–38354. a decisive role in shaping Treg cell identity by defining the Treg 8. McCarty, A. S., G. Kleiger, D. Eisenberg, and S. T. Smale. 2003. Selective cell transcriptional program, acting in parallel, or even upstream, dimerization of a C2H2 zinc finger subfamily. Mol. Cell 11: 459–470. 9. Kim, J., S. Sif, B. Jones, A. Jackson, J. Koipally, E. Heller, S. Winandy, A. Viel, of Foxp3 (45). Although the answer to this question will require A. Sawyer, T. Ikeda, et al. 1999. Ikaros DNA-binding proteins direct formation the comparison of profiles between WT and He- of chromatin remodeling complexes in lymphocytes. Immunity 10: 345–355. liosϪ/Ϫ Treg cells, the normal numbers and biological properties 10. Sridharan, R., and S. T. Smale. 2007. Predominant Interaction of both Ikaros and Helios with the NuRD complex in immature thymocytes. J. Biol. Chem. 282: observed in the mutant Treg cells clearly indicate that Helios is not 30227–30238. a master regulator of this lineage. However, a full dissection of 11. Nichogiannopoulou, A., M. Trevisan, S. Neben, C. Friedrich, and Helios activity will require a better understanding of the functional K. Georgopoulos. 1999. Defects in hemopoietic stem cell activity in Ikaros mu- tant mice. J. Exp. Med. 190: 1201–1214. redundancies among Ikaros family members, as well as the devel- 12. Lopez, R. A., S. Schoetz, K. DeAngelis, D. O’Neill, and A. Bank. 2002. Multiple opment of genetic models where Helios can be studied in combi- hematopoietic defects and delayed globin switching in Ikaros null mice. Proc. nation with Ikaros and/or Eos deficiencies. Natl. Acad. Sci. USA 99: 602–607. Downloaded from 13. Allman, D., M. Dalod, C. Asselin-Paturel, T. Delale, S. H. Robbins, Interestingly, Helios deficiency does not alter the proliferative G. Trinchieri, C. A. Biron, P. Kastner, and S. Chan. 2006. Ikaros is required for ϩ ϩ response of CD4 and CD8 T cells to TCR stimulation. This plasmacytoid dendritic cell differentiation. Blood 108: 4025–4034. result was not anticipated, as overexpression of full-length Helios 14. Wang, J. H., A. Nichogiannopoulou, L. Wu, L. Sun, A. H. Sharpe, M. Bigby, and K. Georgopoulos. 1996. Selective defects in the development of the fetal and inhibits T cell proliferation in response to anti-CD3 stimulation adult lymphoid system in mice with an Ikaros null mutation. Immunity 5: (28), and the proliferative response of lymphocytes to Ag - 537–549. 15. Wang, J. H., N. Avitahl, A. Cariappa, C. Friedrich, T. Ikeda, A. Renold, derived signals is known to be exquisitely sensitive to Ikaros fam- http://www.jimmunol.org/ K. Andrikopoulos, L. Liang, S. Pillai, B. A. Morgan, and K. Georgopoulos. 1998. ily members. Indeed, loss of Ikaros or Aiolos leads to hyperpro- Aiolos regulates B cell activation and maturation to effector state. Immunity 9: liferation in activated B and T cells (14, 15, 18, 26, 28). Our 543–553. 16. Cariappa, A., M. Tang, C. Parng, E. Nebelitskiy, M. Carroll, K. Georgopoulos, observations that Helios does not participate in this process may and S. Pillai. 2001. The follicular versus marginal zone B lymphocyte cell fate reflect a low relative abundance of Helios proteins compared with decision is regulated by Aiolos, Btk, and CD21. Immunity 14: 603–615. Ikaros and Aiolos in T cells, or a specific network of genes con- 17. Cortes, M., and K. Georgopoulos. 2004. Aiolos is required for the generation of high affinity bone marrow plasma cells responsible for long-term immunity. trolled by Ikaros or Aiolos, but not Helios. J. Exp. Med. 199: 209–219. Finally, several studies have linked the appearance of dominant- 18. Kirstetter, P., M. Thomas, A. Dierich, P. Kastner, and S. Chan. 2002. Ikaros is negative Helios to T cell transformation in both humans and mice. critical for B cell differentiation and function. Eur. J. Immunol. 32: 720–730.

19. Reynaud, D., I. A. Demarco, K. L. Reddy, H. Schjerven, E. Bertolino, Z. Chen, by guest on September 23, 2021 Our study does not support a prominent role for Helios as a pri- S. Smale, S. Winandy, and H. Singh. 2008. Regulation of B cell fate commitment mary tumor suppressor in T cells, although we cannot exclude the and immunoglobulin heavy-chain gene rearrangements by Ikaros. Nat. Immunol. possibility that loss of Helios might cooperate with other onco- 9: 927–936. 20. Thompson, E. C., B. S. Cobb, P. Sabbattini, S. Meixlsperger, V. Parelho, genic events to promote leukemogenesis. As Ikaros deficiency is D. Liberg, B. Taylor, N. Dillon, K. Georgopoulos, H. Jumaa, et al. 2007. Ikaros consistently associated with T-acute lymphoblastic leukemia de- DNA-binding proteins as integral components of B cell developmental-stage- velopment in mouse models, we propose that T cell transformation specific regulatory circuits. Immunity 26: 335–344. 21. Sellars, M., B. Reina-San-Martin, P. Kastner, and S. Chan. 2009. Ikaros controls occurs in animals (and perhaps isolated human cases) expressing isotype selection during immunoglobulin class switch recombination. J. Exp. dn Helios because these short isoforms bind and inhibit the activity Med. 206: 1073–1087. of functional Ikaros proteins, and not functional Helios. 22. Winandy, S., L. Wu, J. H. Wang, and K. Georgopoulos. 1999. Pre-T cell receptor (TCR) and TCR-controlled checkpoints in T cell differentiation are set by Ikaros. J. Exp. Med. 190: 1039–1048. Acknowledgments 23. Urban, J. A., and S. Winandy. 2004. Ikaros null mice display defects in T cell selection and CD4 versus CD8 lineage decisions. J. Immunol. 173: 4470–4478. We thank A. Rebollo for the Aiolos Ab, S. Duhautbois-Boine and P. 24. Urban, J. A., W. Brugmann, and S. Winandy. 2009. Ikaros null thymocytes ma- Marchal for technical assistance, E. Blondelle for the embryonic stem ture into the CD4 lineage with reduced TCR signal: a study using CD3␨ immu- cell work; the Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellu- noreceptor tyrosine-based activation motif transgenic mice. J. Immunol. 182: laire transgenic facility, G. Duval for help with Ab production, 3955–3959. 25. Quirion, M. R., G. D. Gregory, S. E. Umetsu, S. Winandy, and M. A. Brown. M. C. Antal for histology analyses, C. Ebel and J. Barths for help with flow 2009. Cutting edge: Ikaros is a regulator of Th2 cell differentiation. J. Immunol. cytometry, and S. Falcone for animal husbandry. 182: 741–745. 26. Avitahl, N., S. Winandy, C. Friedrich, B. Jones, Y. Ge, and K. Georgopoulos. 1999. Ikaros sets thresholds for T cell activation and regulates prop- Disclosures agation. Immunity 333–343. The authors have no financial conflicts of interest. 27. Kleinmann, E., A. S. Geimer Le Lay, M. Sellars, P. Kastner, and S. Chan. 2009. Ikaros represses the transcriptional response to Notch signaling in T-cell devel- opment. Mol. Cell. Biol. 28: 7465–7475. References 28. Winandy, S., P. Wu, and K. Georgopoulos. 1995. A dominant mutation in the 1. Cobb, B. S., and S. T. Smale. 2005. Ikaros-family proteins: in search of molecular Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83: functions during lymphocyte development. Curr. Top. Microbiol. Immunol. 290: 289–299. 29–47. 29. Dumortier, A., R. Jeannet, P. Kirstetter, E. Kleinmann, M. Sellars, 2. Molnar, A., and K. Georgopoulos. 1994. The Ikaros gene encodes a family of N. R. Dos Santos, C. Thibault, J. Barths, J. Ghysdael, J. A. Punt, et al. 2006. functionally diverse zinc finger DNA-binding proteins. Mol. Cell Biol. 14: Notch activation is an early and critical event during T-cell leukemogenesis in 8292–8303. Ikaros-deficient mice. Mol. Cell. Biol. 26: 209–220. 3. Hahm, K., P. Ernst, K. Lo, G. S. Kim, C. Turck, and S. T. Smale. 1994. The 30. Kelley, C. M., T. Ikeda, J. Koipally, N. Avitahl, L. Wu, K. Georgopoulos, and lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced B. A. Morgan. 1998. Helios, a novel dimerization partner of Ikaros expressed in mRNAs derived from the Ikaros gene. Mol. Cell. Biol. 14: 7111–7123. the earliest hematopoietic progenitors. Curr. Biol. 8: 508–515. 4. Sun, L., A. Liu, and K. Georgopoulos. 1996. Zinc finger-mediated protein inter- 31. Fontenot, J. D., J. P. Rasmussen, M. A. Gavin, and A. Y. Rudensky. 2005. A actions modulate Ikaros activity, a molecular control of lymphocyte development. function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. EMBO J. 15: 5358–5369. 6: 1142–1151. The Journal of Immunology 2311

32. Sugimoto, N., T. Oida, K. Hirota, K. Nakamura, T. Nomura, T. Uchiyama, and 38. Tabayashi, T., F. Ishimaru, M. Takata, I. Kataoka, K. Nakase, T. Kozuka, and S. Sakaguchi. 2006. Foxp3-dependent and -independent molecules specific for M. Tanimoto. 2007. Characterization of the short isoform of Helios overex- CD25ϩCD4ϩ natural regulatory T cells revealed by DNA microarray analysis. pressed in patients with T-cell malignancies. Cancer Sci. 98: 182–188. Int. Immunol. 18: 1197–1209. 39. Fujiwara, S. I., Y. Yamashita, N. Nakamura, Y. L. Choi, T. Ueno, H. Watanabe, 33. Hill, J. A., M. Feuerer, K. Tash, S. Haxhinasto, J. Perez, R. Melamed, D. Mathis, K. Kurashina, M. Soda, M. Enomoto, H. Hatanaka, et al. 2008. High-resolution analysis of chromosome copy number alterations in angioimmunoblastic T-cell and C. Benoist. 2007. Foxp3 transcription-factor-dependent and -independent lymphoma and peripheral T-cell lymphoma, unspecified, with single nucleotide regulation of the regulatory T cell transcriptional signature. Immunity 27: polymorphism-typing microarrays. Leukemia 22: 1891–1898. 786–800. 40. Andrews, N. C., and D. V. Faller. 1991. A rapid micropreparation technique for 34. Zheng, Y., and A. Y. Rudensky. 2007. Foxp3 in control of the regulatory T cell extraction of DNA-binding proteins from limiting numbers of mammalian cells. lineage. Nat. Immunol. 8: 457–462. Nucleic Acids Res. 19: 2499. 35. Zhang, Z., C. S. Swindle, J. T. Bates, R. Ko, C. V. Cotta, and C. A. Klug. 2007. 41. Tu, L., T. C. Fang, D. Artis, O. Shestova, S. E. Pross, I. Maillard, and W. S. Pear. Expression of a non-DNA-binding isoform of Helios induces T-cell lymphoma in 2005. Notch signaling is an important regulator of type 2 immunity. J. Exp. Med. mice. Blood 109: 2190–2197. 202: 1037–1042. 42. Elliott, J., C. Jolicoeur, V. Ramamurthy, and M. Cayouette. 2008. Ikaros confers 36. Nakase, K., F. Ishimaru, K. Fujii, T. Tabayashi, T. Kozuka, N. Sezaki, early temporal competence to mouse retinal progenitor cells. Neuron 60: 26–39. Y. Matsuo, and M. Harada. 2002. Overexpression of novel short isoforms of 43. Ezzat, S., and S. L. Asa. 2008. The emerging role of the Ikaros stem cell factor Exp. Hematol. Helios in a patient with T-cell acute lymphoblastic leukemia. 30: in the neuroendocrine system. J. Mol. Endocrinol. 41: 45–51. 313–317. 44. Kiehl, T. R., S. E. Fischer, S. Ezzat, and S. L. Asa. 2008. Mice lacking the 37. Fujii, K., F. Ishimaru, K. Nakase, T. Tabayashi, T. Kozuka, K. Naoki, transcription factor Ikaros display behavioral alterations of an anti-depressive M. Miyahara, H. Toki, K. Kitajima, M. Harada, and M. Tanimoto. 2003. Over- phenotype. Exp. Neurol. 211: 107–114. expression of short isoforms of Helios in patients with adult T-cell leukaemia/ 45. Chatila, T. 2007. The regulatory T cell transcriptosome: E pluribus unum. Im- lymphoma. Br. J. Haematol. 120: 986–989. munity 27: 693–695. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021