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Published online July 20, 2009
The Journal of Immunology, 2009, 183, 2303 -2311
Copyright © 2009 by The American Association of Immunologists, Inc.
doi:10.4049/jimmunol.0901407
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Helios Deficiency Has Minimal Impact on T Cell Development and Function1

Qi Cai*,{dagger},§, Andrée Dierich{dagger},§, Mustapha Oulad-Abdelghani{dagger},§, Susan Chan2,*,{dagger},§ and Philippe Kastner2,*,{dagger},{ddagger},§

* Department of Cancer Biology, {dagger} Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM Unité 964, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7104, Illkirch, France; and {ddagger} Faculté de Médecine, § Université de Strasbourg, Strasbourg, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 {alpha}β 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 {alpha}β and {gamma}{delta} T cells, NKT cells, and regulatory T cells. 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Helios transcription factor (Ikzf2) is a member of the Ikaros family of zinc finger regulators that includes Ikaros (Ikzf1), Aiolos (Ikzf3), Eos (Ikzf4), and Pegasus (Ikzf5). Ikaros family proteins share a similar structure that is characterized by highly conserved zinc finger domains at the N and C termini (1). Four N-terminal zinc fingers are responsible for DNA binding to consensus target sequences, while two C-terminal zinc fingers mediate homo- and heterodimerization between family members (2, 3, 4, 5, 6, 7, 8). Ikaros, Helios, and Aiolos all interact with the nucleosome remodeling and deacetylase (NuRD) histone deacetylase complex (9, 10), suggesting that they play pivotal roles in chromatin remodeling at their target genes.

Ikaros and Aiolos are critical regulators of hematopoiesis. Ikaros is implicated in stem cell renewal, fetal and adult erythropoiesis, and dendritic cell (DC)3 differentiation (11, 12, 13, 14). Ikaros and Aiolos perform distinct functions in B cells. Aiolos is essential for many aspects of B cell differentiation and function, including B cell proliferation, marginal zone vs follicular B cell fate choice, and the development of high-affinity plasma cells (15, 16, 17). Ikaros controls early steps of B cell differentiation, including commitment, as well as Ig class switch recombination in mature B cells (18, 19, 20, 21). Additionally, both Ikaros and Aiolos are required to limit B cell proliferation in response to activation (15, 18). Thus, Ikaros and Aiolos appear to play mostly distinct, but also overlapping, roles in B cells.

In T cells, Ikaros appears to be singularly important. Ikaros deficiency leads to absence of fetal T cell development, while postnatal T cell differentiation is associated with enhanced pre-TCR signaling, leading to increased proliferation of DN4 thymocytes (14, 22). Loss of Ikaros also leads to a decreased {gamma}{delta} T cell pool, as well as altered commitment to the CD4 and CD8 lineages (14, 23, 24). In mature T cells, Ikaros appears to suppress Th1 polarization (25) and to limit proliferation in response to signaling in both CD4+ and CD8+ T cells (26). Finally, Ikaros is involved in silencing Notch signaling during the double-negative to double-positive transition, a function that is likely to contribute to its tumor suppressor function in this lineage (27). Indeed, Ikaros deficiency is strongly associated with development of T cell leukemias that exhibit high levels of Notch activation (28, 29). The multiple abnormalities seen in Ikaros-deficient T cells contrast with the largely normal T cell compartment in Aiolos-deficient mice. However, Aiolos-null T cells also hyperproliferate to activation signals (15), suggesting that, as in B cells, both Ikaros and Aiolos are required to set the threshold for the proliferative response of these cells to activation.

Helios is conspicuous for its high expression from the earliest stages of T cell development (5, 30). Strikingly, Helios is induced >10-fold in CD4+Foxp3+ regulatory T (Treg) cells (31, 32, 33). Its expression in Treg cells does not require Foxp3, a transcriptional regulator essential for Treg cell differentiation (34), suggesting that Helios might function as an upstream regulator of Foxp3, or perhaps define a parallel transcriptional circuit in these cells. Helios is not expressed in mature B cells, DCs, or myeloid cells. At the molecular level, Helios associates with a subset of Ikaros complexes that localize near centromeric heterochromatin in T cells (5), suggesting that it might act as a rate-limiting factor of Ikaros function. Gain-of-function studies, using full-length or dominant-negative (dn) Helios lacking the DNA-binding domain, suggest a key role for this protein in T cell differentiation and function. Overexpression of full-length Helios blocks {alpha}β T cell differentiation at the CD4–CD8– stage in the thymus, and it results in increased frequencies of {gamma}{delta} T cells and NK cells in peripheral lymphoid organs, while overexpression of dn Helios leads to increased T cell proliferation upon TCR stimulation and the development of T lymphomas (35). Furthermore, dn Helios isoforms or allelic loss have been detected in some human T-acute lymphoblastic leukemias or T cell lymphomas (36, 37, 38, 39). These results suggest that Helios is an essential regulator of T cell homeostasis and a tumor suppressor.

While these studies have been informative, they are unclear, as overexpression of either full-length or dn proteins must be interpreted cautiously since they can inhibit the normal function of related endogenous proteins. This is especially true for the Ikaros family, as four of its five members (Ikaros, Aiolos, Helios, and Eos) are coexpressed in T cells. Thus, it is important to understand the exact role of each protein in target gene activation/repression and T cell development. At the present time, Helios function remains unknown.

In this study, we investigated Helios function in T cells by generating a null mutation for this gene by homologous recombination. We find that Helios is not essential for T cell differentiation, homeostasis, and function, and that Helios-deficient mice do not develop T cell malignancies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of Helios-null mice

The Helios targeting vector is depicted in Fig. 1A. The C-terminal part of Helios exon 7 was replaced by a 1.8-kb floxed PGK-neo-poly(A) cassette between the indicated SalI and XbaI sites. The vector was transfected into P1 129/Sv embryonic stem cells, and homologous recombination events were detected by Southern blot using external probes A and B with BamHI-digested genomic DNA. Two embryonic stem cell clones, MBA120 and MBA93, were identified as positive and injected into C57BL/6 (B6) blastocysts to produce chimeric mice. Germline transmission was verified by PCR on tail DNA using primers P3 and P4 to detect the wild-type (WT) allele, and P3 and P5 to detect the mutant allele. Mice derived from both clones gave similar phenotypes. The experiments described in this paper were performed using mice generated from clone MBA120 and backcrossed once onto the B6 background.


Figure 1
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  		FIGURE 1. Generation of Helios–/– mice. A, Targeting vector and recombination strategy. In the illustration 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 contained 9.4 kb of the Helios locus containing exon 7, depicted as a thick line. LoxP sites flanking the PGK-neo cassette are depicted as white triangles. Recombination was verified using outside probes A and B, and further confirmed with probe C. B, BamHI; E, EcoRI; H, HindIII; S, SalI; X, XbaI. B, Southern blot of BamHI-digested embryonic stem cell DNA, analyzed with probes A (left) and B (right). C, PCR of genomic DNA from WT, Helios+/–, and Helios–/– mice using primers P3 and P4 (for the WT allele) or P3 and P5 (for the mutant allele). PCR products were confirmed by hybridization with probe C. D, RT-PCR of Helios and β-actin from WT and Helios–/– thymocyte RNA. P1 and P2 amplify transcript sequences upstream of the mutation. P4 and P6 amplify transcripts encompassing the mutation. E, Western blot of WT and Helios–/– thymocyte nuclear extracts using the Abs Ab1 and Ab2. TATA-box binding protein (TBP) was detected as a loading control. Note that the 50-kDa polypeptide detected in both WT and He–/– cells is likely to be nonspecific since it was not detected with Ab2.

 
RT-PCR, real-time RT-PCR, and primers

For RT-PCR and real-time PCR, RNA was extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed with SuperScript II reverse transcriptase (Invitrogen). mRNA levels for Aiolos, Helios, Eos, and Ikaros were quantified by real-time RT-PCR using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Real-time RT-PCR was performed with the LightCycler 480 (Roche) with the following conditions: 95°C for 10 s, 63°C for 15 s, and 72°C for 15 s for 40 cycles. The difference in cDNA input was normalized to hypoxanthine phosphoribosyltransferase (HPRT) expression levels. The following primers were used: P1, 5'-CCAATGGACAGCACGCCTCG; P2, 5'-ATATCTGGGTAGCTGAATCGC; P3, 5'-TCTATTAGTGTCAGCTTTTTGACAGTTT; P4, 5'-GATGAATTCCTTATAGATGTCCTTCAGAGAGCC; P5, 5'-ATCTGCACGAGACTAGTGAGACG; P6, 5'-GATGCTAGCCAGAATGTCAGCATGGAGGCTGCC; β-actin, forward, 5'-TGTTACCAACTGGGACGACA, reverse, 5'-CCATCACAATGCCTGTGGTA; Helios, forward, 5'-ACACCTCAGGACCCATTCTG, reverse, 5'-TCCATGCTGACATTCTGGAG; Aiolos, forward, 5'-ACAGCAGACCAACCGGTGGGAA, reverse, 5'-ACTGGAACGGGCGTTCGC; Eos, forward, 5'-GAGGAGCACAAGGAGAGGTG, reverse, 5'-CATCTCCAGGTCACGGATTT; Ikaros, forward, 5'-CATAAAGAGCGATGCCACAA, reverse, 5'-CAGGACAAGGGACCTCTCTG; HPRT, forward, 5'-GTTGGATACAGGCCAGACTTTGTTG, reverse, 5'-GATTCAACTTGCGCTCATCTTAGGC. Note that the Aiolos primers were obtained from Thompson et al. (20).

Western blot and Ab production

Nuclear extracts were prepared according to Andrews and Faller (40). Thymocyte extracts (5 µg) were separated on an 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. Extracts (25 ng) from COS cells transfected with a Helios expression vector or from cells (250 ng) transfected with an Ikaros expression vector were used as positive controls. The membrane was blocked at room temperature for 1 h with blocking 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), the membrane was incubated with a 1/104 dilution of HRP-conjugated donkey anti-rabbit Abs or goat anti-mouse Abs (Jackson ImmunoResearch Laboratories), washed, and revealed with Immobilon Western (Millipore).

The polyclonal rabbit anti-mouse Helios Abs Ab1 and Ab2 were generated by immunizing rabbits with a bacterially expressed N-terminal Helios fragment corresponding to aa 1–109 (Ab1) or to a peptide corresponding to Helios aa 369–381 (Ab2). Abs were purified with SulfoLink columns coupled to the immunogens. The rabbit anti-mouse Aiolos Ab was generously provided by A. Rebollo (INSERM Unité 543, Paris, France). The rabbit anti-mouse Ikaros (C-terminal) and monoclonal TATA-box binding protein (TBP) Abs were previously generated in our institute.

Abs and flow cytometry

All Abs were purchased from BD Pharmingen or eBioscience, except for Abs to CD3 (KT3), CD4 (YTS191.1 or GK1.5), CD8 (YTS169.4), and CD44 (IM7), which were produced in-house and conjugated according to standard protocols. For flow cytometry analyses, cells were first incubated with anti-CD16/32 to block Fc receptors. Intracellular staining of Treg cells was performed using a Foxp3 staining kit (eBioscience). Cells were analyzed on a FACSCalibur (BD Biosciences), and data were analyzed with FlowJo software (Tree Star). Sorting was performed on a FACSVantage SE option DiVa (BD Biosciences). Sort purity was >95%.

Proliferation assays

To induce the proliferation of peripheral CD4+ and CD8+ T cells, whole splenic CD4+ and CD8+ T cells, as well as splenic CD4+CD44–CD25– and CD8+CD44– T cells, were sorted, incubated with CFSE, and then seeded into 96-well plates coated with anti-CD3 (10 µg/ml; eBioscience) or anti-CD3 (5 µg/ml) plus anti-CD28 (5 µg/ml; eBioscience) in complete RPMI medium (RPMI 1640 supplemented with 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1x nonessential amino acids, 1 mM sodium pyruvate, 50 µM 2-ME, 1% penicillin and streptomycin) in triplicate samples for 3 days. Control cells were cultured without Ab stimulation. Cells were cultured at 2.5 x 104 cells per well for the anti-CD3 stimulation and unstimulated controls, and at 1 x 104 cells per well for anti-CD3 plus anti-CD28. For Treg cell assays, Treg (CD4+CD25+CD44–), Th (CD4+CD25–CD44–), and APCs (Thy1.2–) were sorted from pooled spleen and lymph node cells. Treg or Th cells were incubated with CFSE and cultured in complete RPMI medium at 5 x 103 cells per well in 96-well plates in triplicate samples with anti-CD3 (1 µg/ml) and 105 APCs, in the absence 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.

Th1 and Th2 induction

Th1 and Th2 polarizing cultures were performed mostly according to Tu et al. (41). Briefly, CD4+CD44–CD25– Th cells and Thy1.2– APCs were sorted from adult spleens. In neutral conditions, 25 x 103 Th cells were cultured with 25 x 103 APCs (mitomycin C treated), recombinant human IL-2 (10 U/ml; PeproTech), soluble anti-CD3 (0.1 µg/ml), and anti-CD28 (0.5 µg/ml) in 96-well plates in triplicate samples. In Th1 polarizing conditions, anti-IL-4 (10 µg/ml; eBioscience) and recombinant murine IL-12 (5 ng/ml; eBioscience) were added to these cultures. In Th2 polarizing conditions, anti-IL-12 Ab (10 µg/ml; eBioscience) and recombinant murine IL-4 (20 ng/ml; PeproTech) were added to the cultures. After 7 days of culture, cells were restimulated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) for 4 h. GolgiPlug (brefeldin A; BD Biosciences) was added at 1 µl/ml for the last 2 h. Cells were harvested and stained to assess surface Thy1.2, CD4, and intracellular IL-4 and IFN-{gamma}. Intracellular staining was performed using the Cytofix/Cytoperm fixation/permeabilization kit (BD Biosciences).

Detection of perforin and granzyme B

Splenic T cells were purified by negative selection using Abs specific for B220, CD11b, CD11c, Ter-119, and DX5. T cells (25 x 104/well) were then cultured in complete RPMI medium with or without recombinant human IL-2 at 100 U/ml in 96-well plates in triplicate samples. After 3 days, cells were harvested and stained for CD4 and CD8 before intracellular staining for granzyme B and perforin with a Foxp3 staining buffer system (eBioscience).

Treg cell suppression assay

Pooled cells from spleen and lymph nodes were first selected for lineage-positive (B220, CD11b, CD11c, NK1.1, Ter-119) cells; these cells were treated with mitomycin C and used as APCs. Lineage-negative cells were then sorted for Treg (CD4+CD25+) and responder Th (CD4+CD25–) cells. Th cells were incubated with CFSE, and 25 x 103 Th cells were cultured with equal numbers of APCs and anti-CD3 (5 µg/ml) in complete RPMI medium in 96-well plates in triplicate samples. Treg cells were added to the cultures at the indicated ratios. In the no stimuli controls, Th cells were cultured with APCs in the absence of anti-CD3. Cells were harvested 3 days later and stained for CD4 before analysis. Suppression of Th proliferation was evaluated as loss of CFSE in the CD4+CFSE+-gated populations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We produced a Helios-null mutation by targeting a PGK-neo cassette into exon 7 of the Helios locus by homologous recombination (Fig. 1A). This mutation deletes most of exon 7, including the sequences that encode the C-terminal zinc fingers, as indicated by Southern blot analyses of mutant embryonic stem cells and PCR analyses of tail DNA from heterozygote and homozygote Helios mutants (Fig. 1, B and C). Helios–/– thymocytes still transcribed Helios mRNA (Fig. 1D, left panel), but these transcripts lacked the deleted sequences (Fig. 1D, right panel). Helios proteins were not detected in thymocyte nuclear extracts by Western blot, either in full-length or truncated forms, using two Abs produced against the N-terminal portion of Helios (Ab1; Fig. 1E, top panel) or the C-terminal region upstream of the deletion (Ab2; Fig. 1E, bottom panel). These results indicate that the exon 7 deletion results in loss of synthesis or rapid degradation of Helios polypeptides in T cells. Therefore, we conclude that this mutation is null.

Although homozygote Helios–/– and heterozygote Helios+/– animals were born at Mendelian frequencies and were of normal body size at birth (data not shown), many Helios–/– pups died within the first weeks of postnatal life (Fig. 2A). This early lethality increased as Helios–/– mice were backcrossed onto the B6 background, and no surviving homozygotes could be obtained after 10 generations of backcrossing. We thus studied the phenotype of Helios mutants on a mixed 129/Sv:B6 background. It is unclear why so many Helios–/– mutants die after birth. However, this is probably not associated with defective hematopoiesis, as 14.5 days postcoitus Helios–/– fetal liver cells efficiently reconstituted all hematopoietic lineages after adoptive transfer into lethally irradiated mice (data not shown). Surviving adult Helios–/– mice, particularly females, were markedly smaller than WT or heterozygote littermates in size and weight (Fig. 2, B and C, and data not shown). Helios–/– animals remained smaller as they aged, but these differences were no longer statistically significant in the male population (Fig. 2C). Helios–/– mice lived to at least 22 mo of age and showed no overt signs of ill health (nine mice analyzed between 16 and 22 mo of age; data not shown). Helios–/– mice exhibited smaller eye-openings, a phenotype that might be linked to an abnormal growth of eyelids (data not shown). Both male and female Helios–/– mice were fertile. These results indicate that Helios–/– mice do not spontaneously develop health-threatening illness if they survive the weaning period.


Figure 2
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  		FIGURE 2. Growth and early survival defects in Helios–/– mice. A, Total numbers and frequencies of WT, Helios+/–, and Helios–/– mice that reached the age of 2 wk. Mice were generated from Helios+/– intercrosses. B, Representative WT (top) and Helios–/– (bottom) male littermates at 6 wk of age. C, Body weights of adult WT and Helios–/– male (left) and female (right) littermates. Symbols indicate pairs of age-matched littermates. Values of p were calculated with a two-tailed paired Student’s t test. *, p = 0.013; **, p = 0.083; ***, p = 0.005.

 
To address the role of Helios in {alpha}β T cell development, we analyzed T cell populations in the thymuses and spleens of WT and Helios–/– mice. Immature CD4–CD8–CD3– double-negative thymocytes were analyzed for CD44 and CD25 expression, while the more mature CD4+CD8+ double-positive thymocytes and the CD4+CD8– and CD4–CD8+ single-positive cells were analyzed for CD3, {alpha}βTCR, CD24, and CD69 expression. All double-negative subpopulations (CD44+CD25–, CD44+CD25+, CD44–CD25+, CD44–CD25–) were similar in frequency between WT and mutant thymuses from adult mice (7–9 wk old) and newborn animals (1 day old) (Fig. 3A–D). Double-positive and single-positive thymocyte populations were also similar in frequencies and absolute numbers according to their CD4 and CD8 profiles, and no differences were observed in terms of CD3, {alpha}βTCR, CD24, and CD69 expression for each population (data not shown). Additionally, peripheral CD4+ and CD8+ T cells from the lymph nodes and spleens were comparable in frequency between WT and mutant mice (Fig. 3E), and no differences were observed in CD62L and CD44 expression in these cell types (data not shown), suggesting that memory T cell populations were unaltered. These results indicate that {alpha}β T cell differentiation and homeostasis proceeds normally in the absence of Helios.


Figure 3
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  		FIGURE 3. {alpha}β T cell development or homeostasis in Helios–/– mice. Thymocytes from WT and Helios–/– adult mice (A and B) and neonates (C and D) were analyzed for expression of the indicated markers. Numbers in the contour plots indicate percentages of the gated cells. Bar graphs indicate mean absolute numbers of the different thymocyte populations analyzed ± SD, as calculated from four independent experiments. E, WT and Helios–/– splenocytes and lymph node cells were analyzed for CD4 and CD8 expression. Results are representative of three similar experiments.

 
NKT cells, {gamma}{delta} 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 Helios–/– mice. {gamma}{delta} T cell and NKT cell (CD3+DX5+) frequencies in the mutant organs were similar to those from WT animals (Fig. 4, A and B). Helios–/– animals also showed normal percentages of thymic and splenic CD25+Foxp3+ Treg cells (Fig. 4C). Furthermore, the above cell types differentiated normally when analyzed in competitive mixed bone marrow chimera experiments where WT and Helios–/– bone marrow cells were transferred to lethally irradiated WT recipients (data not shown). These results indicate that {gamma}{delta} T cells, NKT cells, and Treg cells mature normally in Helios-deficient mice.


Figure 4
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  		FIGURE 4. {gamma}{delta} T, NKT, and Treg cell frequencies in adult Helios–/– mice. WT and Helios–/– thymocytes and splenocytes were analyzed for their populations of {gamma}{delta} T cells, gated on CD4–CD8– cells (A), NKT (CD3+DX5+) cells (B), and Treg (CD25+Foxp3+) cells, gated on CD4 single-positive cells in the thymus and CD4+ cells in the spleen (C). Numbers indicate percentages of the gated cells. Results are representative of more than three experiments.

 
To determine whether loss of Helios affects mature T cell function, we first analyzed the capacity of Helios–/– peripheral CD4+ and CD8+ T cells to proliferate in response to TCR stimulation in vitro, as Ikaros and Aiolos have been shown to limit the proliferation threshold of T and B cells upon activation (14, 15, 18, 26, 28). Naive CD4+CD25–CD44– and CD8+CD44– T cells, as well as whole CD4+ and CD8+ T cells, were sorted from WT and Helios–/– spleens, labeled with CFSE, and cultured for 3 days with anti-CD3 Abs alone or in combination with anti-CD28. These experiments show that WT cells and Helios–/– CD4+ and CD8+ T cells proliferate similarly to TCR stimulation in the absence or presence of costimulatory signals (Fig. 5A).


Figure 5
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  		FIGURE 5. {alpha}β T cell function in the absence of Helios. A, WT (gray shaded histogram) and Helios–/– (solid line histogram) whole CD4+ or naive CD4+CD25–CD44– T cells, and whole CD8+ or naive CD8+CD44– T cells, were stimulated for 3 days with the indicated concentrations (µg/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 Helios–/–CD8+ T cells stimulated with anti-CD3 was not reproducible in all experiments. B, WT and Helios–/– splenic CD4+ T cells were cultured in neutral, Th1, or Th2 polarizing conditions. Gated CD4+Thy1.2– cells were analyzed for intracellular expression of IFN-{gamma} 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 Helios–/– 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 ± SD of positive cells in the CD8+ T cell population, as determined from triplicate samples. Results are representative of three independent experiments.

 
To test if Helios–/– CD4+ T cells efficiently differentiate into Th1 or Th2 cells, we measured the capacity of these cells to produce IFN-{gamma} and IL-4 under neutral and polarizing conditions. In neutral conditions, CD4+CD25–CD44– splenic T cells were cultured with Thy1.2– APCs in the presence of anti-CD3 and anti-CD28 Abs, as well as IL-2, for 7 days to evaluate the capacity of these cells to produce cytokines upon stimulation. In Th1 polarizing conditions, CD4+CD25–CD44– splenic T cells were cultured as above in the presence of IL-12 and anti-IL-4 Abs. In Th2 polarizing conditions, the same cells were cultured as above in the presence of IL-4 and anti-IL-12 Abs. After 7 days, all cultures were restimulated with PMA and ionomycin for 4 additional hours before analysis by intracytoplasmic staining. As shown in Fig. 5B, Helios–/– CD4+ T cells differentiated into IFN-{gamma}+ Th1 and IL-4+ Th2 cells as efficiently as did WT CD4+ T cells in all conditions, indicating that CD4+ T cell function is unaffected in Helios–/– mice.

To evaluate if Helios–/– CD8+ T cells function as mature effector cells, we tested their capacity to produce perforin and granzyme B, two molecules important for cytotoxic function, upon stimulation. Splenic T cells were enriched by negative depletion and cultured with IL-2 for 3 days. CD8+ T cells were analyzed for the expression of perforin and granzyme B by intracellular staining. Comparable percentages of CD8+ T cells were induced to express perforin and granzyme B in both WT and Helios–/– cultures (Fig. 5C), suggesting that CD8+ T cell function is normal in Helios–/– mice.

Since Helios expression is strongly induced in Treg cells, we asked if Helios deficiency specifically affects Treg cell function. We first tested if Helios contributes to the low proliferative response of Treg cells to TCR-induced signals. As depicted in Fig. 6A, neither WT nor Helios–/– Treg cells (CD4+CD25+CD44–), purified from lymph nodes and spleens, responded to anti-CD3 stimulation in a 3-day coculture assay with APCs. Additionally, both WT and Helios–/– Treg cells proliferated similarly when stimulated with anti-CD3 Abs in the presence of IL-2, although the proportion of Helios–/– Treg cells that responded to this stimulation was reduced by about one-third. Thus, Helios–/– Treg cells do not exhibit an altered pattern of proliferation under established conditions. We next evaluated if Helios–/– Treg cells could suppress the proliferation of responder Th (CD4+CD25–) cells stimulated with anti-CD3 Abs and APCs. Helios–/– Treg cells suppressed Th cell proliferation as efficiently as did WT Treg cells (Fig. 6B). Collectively, these results indicate that mature Treg cells function normally in the absence of Helios.


Figure 6
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  		FIGURE 6. Mature Treg cell function in Helios–/– mice. A, Purified WT and Helios–/– CD4+CD25+CD44– 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 (CD4+CD25–CD44–) were used as positive controls. CD4+CFSE+ cells were analyzed for CD25 expression and CFSE intensity. Numbers indicate percentages of cells in each gate. Results are representative of two independent experiments performed in triplicate. B, CFSE-stained responder Th cells (CD4+CD25–) were cultured for 3 days with anti-CD3 and APCs. WT and Helios–/– Treg cells (CD4+CD25+) were added at the indicated ratios. Th cells cultured with APCs in the absence of anti-CD3 are shown as the no stimuli control. Proliferation of Th cells is shown as CFSE loss in the CD4+CFSE+-gated population. Numbers indicate percentages of cells in each gate. WT and Helios–/– Treg cells suppressed proliferation in a similar manner regardless of whether the Th cells and APCs were of WT or Helios–/– origin. Results are representative of two independent experiments performed in triplicate.

 
Lastly, we investigated the possibility that Helios–/– T cells might compensate for loss of Helios activity by up-regulating the expression of other Ikaros family members. We tested the mRNA levels of Aiolos, Ikaros, Eos, and Helios in WT and Helios–/– thymocytes by real-time RT-PCR (Fig. 7A). Helios–/– thymocytes exhibited a small but statistically significant increase in Aiolos mRNA levels (WT, 0.97 ± 0.03; Helios–/–, 1.26 ± 0.09), while Ikaros and Eos mRNA levels were similar between genotypes. At the protein level, the expression of Aiolos and Ikaros was comparable between WT and mutant thymocytes (Fig. 7B). These data suggest that Helios–/– T cells do not compensate for Helios deficiency by drastically up-regulating Aiolos and Ikaros expression. Furthermore, Helios mRNA levels remained similar between WT and mutant thymocytes despite loss of Helios at the protein level, suggesting that Helios does not autoregulate its own transcription.


Figure 7
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  		FIGURE 7. Expression of Ikaros, Aiolos, and Eos in Helios–/– cells. A, RNA from WT and Helios–/– 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 ± SD are presented, as calculated from three independent experiments. B, Nuclear extracts from WT and Helios–/– 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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report herein the generation of the first Helios-deficient mouse line. Our data show that Helios is essential for the first weeks of life, as most Helios–/– mice (100% on a pure B6 background) die during this period. The physiological functions that depend strictly on Helios remain unknown, but they are unlikely to be related to hematological or immune defects. Helios expression outside of the hematopoietic system is poorly characterized at present. Together with reports showing that Ikaros controls neural and endocrine functions (42, 43, 44), the severe viability defect of Helios–/– mice provides further evidence that Ikaros family members exert important functions beyond the hematopoietic system.

In mature hematopoietic cells, Helios expression is restricted to T cells (5, 30), suggesting that Helios may control important aspects of T cell differentiation and/or function. We show herein that Helios is not essential for the development, homeostasis, and function of thymic-derived T lymphocytes, in contrast to expectations from earlier overexpression studies suggesting a primary role for this transcription factor in {alpha}β T cell development and as a tumor suppressor. In particular, most of the defects associated with Ikaros deficiency are not detected in Helios-null mice (i.e., lack of fetal T cell development, reduced cellularity in the adult thymus, increases in DN4 and CD4 single-positive thymocyte populations, reduced {gamma}{delta} T cells, increased Th1 and impaired Th2 differentiation, T cell hyperproliferation, and T cell transformation; see Refs. 14 , 22 , 25). Thus, Helios is clearly not required for Ikaros-dependent function in T cells and is not sufficient to sustain T cell development alone. Note, however, that Helios may play a unique role in more specialized T cell functions or subsets that were not studied here. The present mouse line thus provides an important tool to further explore the function of Helios in T cells.

The lack of apparent T cell phenotype in Helios-null mice suggests that other Ikaros family members may compensate for Helios in T cells. This is consistent with previous reports suggesting that Ikaros and Helios bind similar target sequences and belong to similar macromolecular complexes (5, 30). If so, then Ikaros appears to be the dominant family member in the {alpha}β T cell lineage, while Helios may function in a redundant manner in these cells. It would therefore be interesting to study T cell development in animals deficient for both Ikaros and Helios. Our efforts to generate double-mutant mice have so far been unsuccessful, due to the extremely high mortality rate of these mice.

Recent studies have shown that Helios is highly expressed at the mRNA level in Treg cells compared with conventional CD4+ T cells (>10-fold) (31, 32, 33). Interestingly, Helios is expressed early in the Treg cell lineage and is not a downstream target of Foxp3 (33). These observations have led to speculation that Helios (and perhaps Eos, which is similarly expressed in Treg cells) may play a decisive role in shaping Treg cell identity by defining the Treg cell transcriptional program, acting in parallel, or even upstream, of Foxp3 (45). Although the answer to this question will require the comparison of gene expression profiles between WT and Helios–/– Treg cells, the normal numbers and biological properties observed in the mutant Treg cells clearly indicate that Helios is not a master regulator of this lineage. However, a full dissection of Helios activity will require a better understanding of the functional redundancies among Ikaros family members, as well as the development of genetic models where Helios can be studied in combination with Ikaros and/or Eos deficiencies.

Interestingly, Helios deficiency does not alter the proliferative response of CD4+ and CD8+ T cells to TCR stimulation. This result was not anticipated, as overexpression of full-length Helios inhibits T cell proliferation in response to anti-CD3 stimulation (28), and the proliferative response of lymphocytes to Ag receptor-derived signals is known to be exquisitely sensitive to Ikaros family members. Indeed, loss of Ikaros or Aiolos leads to hyperproliferation in activated B and T cells (14, 15, 18, 26, 28). Our observations that Helios does not participate in this process may reflect a low relative abundance of Helios proteins compared with Ikaros and Aiolos in T cells, or a specific network of genes controlled by Ikaros or Aiolos, but not Helios.

Finally, several studies have linked the appearance of dominant-negative Helios to T cell transformation in both humans and mice. Our study does not support a prominent role for Helios as a primary tumor suppressor in T cells, although we cannot exclude the possibility that loss of Helios might cooperate with other oncogenic events to promote leukemogenesis. As Ikaros deficiency is consistently associated with T-acute lymphoblastic leukemia development in mouse models, we propose that T cell transformation occurs in animals (and perhaps isolated human cases) expressing dn Helios because these short isoforms bind and inhibit the activity of functional Ikaros proteins, and not functional Helios.


   Acknowledgments
 
We thank A. Rebollo for the Aiolos Ab, S. Duhautbois-Boine and P. Marchal for technical assistance, E. Blondelle for the embryonic stem cell work; the Institut de Génétique et de Biologie Moléculaire et Cellulaire transgenic facility, G. Duval for help with Ab production, M. C. Antal for histology analyses, C. Ebel and J. Barths for help with flow cytometry, and S. Falcone for animal husbandry.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by institute funds from INSERM, Centre National de la Recherche Scientifique, and Hôpital Universitaire de Strasbourg, and a grant to S.C. and P.K. from the Ligue Nationale Française Contre le Cancer (Equipe Labellisée). Q.C. was funded by a predoctoral fellowship from the Association pour le Recherche sur le Cancer. Back

2 Address correspondence and reprint requests to Dr. Susan Chan and Dr. Philippe Kastner, Department of Cancer Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP 10142, 67404 Illkirch Cedex, France. E-mail address: scpk{at}igbmc.fr Back

3 Abbreviations used in this paper: DC, dendritic cell; B6, C57BL/6; dn, dominant negative; HPRT, hypoxanthine phosphoribosyltransferase; Treg cell, regulatory T cell; WT, wild type. Back

Received for publication May 5, 2009. Accepted for publication June 11, 2009.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Cobb, B. S., S. T. Smale. 2005. Ikaros-family proteins: in search of molecular functions during lymphocyte development. Curr. Top. Microbiol. Immunol. 290: 29-47. [CrossRef][Medline]
  2. Molnar, A., K. Georgopoulos. 1994. The Ikaros gene encodes a family of functionally diverse zinc finger DNA-binding proteins. Mol. Cell Biol. 14: 8292-8303. [Abstract/Free Full Text]
  3. Hahm, K., P. Ernst, K. Lo, G. S. Kim, C. Turck, S. T. Smale. 1994. The lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced mRNAs derived from the Ikaros gene. Mol. Cell. Biol. 14: 7111-7123. [Abstract/Free Full Text]
  4. Sun, L., A. Liu, K. Georgopoulos. 1996. Zinc finger-mediated protein interactions modulate Ikaros activity, a molecular control of lymphocyte development. EMBO J. 15: 5358-5369. [Medline]
  5. Hahm, K., B. S. Cobb, A. S. McCarty, K. E. Brown, C. A. Klug, R. Lee, K. Akashi, I. L. Weissman, A. G. Fisher, S. T. Smale. 1998. Helios, a T cell-restricted Ikaros family member that quantitatively associates with Ikaros at centromeric heterochromatin. Genes Dev. 12: 782-796. [Abstract/Free Full Text]
  6. Morgan, B., L. Sun, N. Avitahl, K. Andrikopoulos, T. Ikeda, E. Gonzales, P. Wu, S. Neben, K. Georgopoulos. 1997. Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation. EMBO J. 16: 2004-2013. [CrossRef][Medline]
  7. Perdomo, J., M. Holmes, B. Chong, M. Crossley. 2000. Eos and pegasus, two members of the Ikaros family of proteins with distinct DNA binding activities. J. Biol. Chem. 275: 38347-38354. [Abstract/Free Full Text]
  8. McCarty, A. S., G. Kleiger, D. Eisenberg, S. T. Smale. 2003. Selective dimerization of a C2H2 zinc finger subfamily. Mol. Cell 11: 459-470. [CrossRef][Medline]
  9. Kim, J., S. Sif, B. Jones, A. Jackson, J. Koipally, E. Heller, S. Winandy, A. Viel, A. Sawyer, T. Ikeda, et al 1999. Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes. Immunity 10: 345-355. [CrossRef][Medline]
  10. Sridharan, R., S. T. Smale. 2007. Predominant Interaction of both Ikaros and Helios with the NuRD complex in immature thymocytes. J. Biol. Chem. 282: 30227-30238. [Abstract/Free Full Text]
  11. Nichogiannopoulou, A., M. Trevisan, S. Neben, C. Friedrich, K. Georgopoulos. 1999. Defects in hemopoietic stem cell activity in Ikaros mutant mice. J. Exp. Med. 190: 1201-1214. [Abstract/Free Full Text]
  12. Lopez, R. A., S. Schoetz, K. DeAngelis, D. O'Neill, A. Bank. 2002. Multiple hematopoietic defects and delayed globin switching in Ikaros null mice. Proc. Natl. Acad. Sci. USA 99: 602-607. [Abstract/Free Full Text]
  13. Allman, D., M. Dalod, C. Asselin-Paturel, T. Delale, S. H. Robbins, G. Trinchieri, C. A. Biron, P. Kastner, S. Chan. 2006. Ikaros is required for plasmacytoid dendritic cell differentiation. Blood 108: 4025-4034. [Abstract/Free Full Text]
  14. Wang, J. H., A. Nichogiannopoulou, L. Wu, L. Sun, A. H. Sharpe, M. Bigby, K. Georgopoulos. 1996. Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation. Immunity 5: 537-549. [CrossRef][Medline]
  15. Wang, J. H., N. Avitahl, A. Cariappa, C. Friedrich, T. Ikeda, A. Renold, K. Andrikopoulos, L. Liang, S. Pillai, B. A. Morgan, K. Georgopoulos. 1998. Aiolos regulates B cell activation and maturation to effector state. Immunity 9: 543-553. [CrossRef][Medline]
  16. Cariappa, A., M. Tang, C. Parng, E. Nebelitskiy, M. Carroll, K. Georgopoulos, S. Pillai. 2001. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 14: 603-615. [CrossRef][Medline]
  17. Cortes, M., K. Georgopoulos. 2004. Aiolos is required for the generation of high affinity bone marrow plasma cells responsible for long-term immunity. J. Exp. Med. 199: 209-219. [Abstract/Free Full Text]
  18. Kirstetter, P., M. Thomas, A. Dierich, P. Kastner, S. Chan. 2002. Ikaros is critical for B cell differentiation and function. Eur. J. Immunol. 32: 720-730. [CrossRef][Medline]
  19. Reynaud, D., I. A. Demarco, K. L. Reddy, H. Schjerven, E. Bertolino, Z. Chen, S. Smale, S. Winandy, H. Singh. 2008. Regulation of B cell fate commitment and immunoglobulin heavy-chain gene rearrangements by Ikaros. Nat. Immunol. 9: 927-936. [CrossRef][Medline]
  20. Thompson, E. C., B. S. Cobb, P. Sabbattini, S. Meixlsperger, V. Parelho, D. Liberg, B. Taylor, N. Dillon, K. Georgopoulos, H. Jumaa, et al 2007. Ikaros DNA-binding proteins as integral components of B cell developmental-stage-specific regulatory circuits. Immunity 26: 335-344. [CrossRef][Medline]
  21. Sellars, M., B. Reina-San-Martin, P. Kastner, S. Chan. 2009. Ikaros controls isotype selection during immunoglobulin class switch recombination. J. Exp. Med. 206: 1073-1087. [Abstract/Free Full Text]
  22. Winandy, S., L. Wu, J. H. Wang, 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. [Abstract/Free Full Text]
  23. Urban, J. A., S. Winandy. 2004. Ikaros null mice display defects in T cell selection and CD4 versus CD8 lineage decisions. J. Immunol. 173: 4470-4478. [Abstract/Free Full Text]
  24. Urban, J. A., W. Brugmann, S. Winandy. 2009. Ikaros null thymocytes mature into the CD4 lineage with reduced TCR signal: a study using CD3{zeta} immunoreceptor tyrosine-based activation motif transgenic mice. J. Immunol. 182: 3955-3959. [Abstract/Free Full Text]
  25. Quirion, M. R., G. D. Gregory, S. E. Umetsu, S. Winandy, M. A. Brown. 2009. Cutting edge: Ikaros is a regulator of Th2 cell differentiation. J. Immunol. 182: 741-745. [Abstract/Free Full Text]
  26. Avitahl, N., S. Winandy, C. Friedrich, B. Jones, Y. Ge, K. Georgopoulos. 1999. Ikaros sets thresholds for T cell activation and regulates chromosome propagation. Immunity : 333-343.
  27. Kleinmann, E., A. S. Geimer Le Lay, M. Sellars, P. Kastner, S. Chan. 2009. Ikaros represses the transcriptional response to Notch signaling in T-cell development. Mol. Cell. Biol. 28: 7465-7475. [CrossRef]
  28. Winandy, S., P. Wu, K. Georgopoulos. 1995. A dominant mutation in the Ikaros gene leads to rapid development of leukemia and lymphoma. Cell 83: 289-299. [CrossRef][Medline]
  29. Dumortier, A., R. Jeannet, P. Kirstetter, E. Kleinmann, M. Sellars, N. R. Dos Santos, C. Thibault, J. Barths, J. Ghysdael, J. A. Punt, et al 2006. Notch activation is an early and critical event during T-cell leukemogenesis in Ikaros-deficient mice. Mol. Cell. Biol. 26: 209-220. [Abstract/Free Full Text]
  30. Kelley, C. M., T. Ikeda, J. Koipally, N. Avitahl, L. Wu, K. Georgopoulos, B. A. Morgan. 1998. Helios, a novel dimerization partner of Ikaros expressed in the earliest hematopoietic progenitors. Curr. Biol. 8: 508-515. [CrossRef][Medline]
  31. Fontenot, J. D., J. P. Rasmussen, M. A. Gavin, A. Y. Rudensky. 2005. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat. Immunol. 6: 1142-1151. [CrossRef][Medline]
  32. Sugimoto, N., T. Oida, K. Hirota, K. Nakamura, T. Nomura, T. Uchiyama, S. Sakaguchi. 2006. Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis. Int. Immunol. 18: 1197-1209. [Abstract/Free Full Text]
  33. Hill, J. A., M. Feuerer, K. Tash, S. Haxhinasto, J. Perez, R. Melamed, D. Mathis, C. Benoist. 2007. Foxp3 transcription-factor-dependent and -independent regulation of the regulatory T cell transcriptional signature. Immunity 27: 786-800. [CrossRef][Medline]
  34. Zheng, Y., A. Y. Rudensky. 2007. Foxp3 in control of the regulatory T cell lineage. Nat. Immunol. 8: 457-462. [CrossRef][Medline]
  35. Zhang, Z., C. S. Swindle, J. T. Bates, R. Ko, C. V. Cotta, C. A. Klug. 2007. Expression of a non-DNA-binding isoform of Helios induces T-cell lymphoma in mice. Blood 109: 2190-2197. [Abstract/Free Full Text]
  36. Nakase, K., F. Ishimaru, K. Fujii, T. Tabayashi, T. Kozuka, N. Sezaki, Y. Matsuo, M. Harada. 2002. Overexpression of novel short isoforms of Helios in a patient with T-cell acute lymphoblastic leukemia. Exp. Hematol. 30: 313-317. [CrossRef][Medline]
  37. Fujii, K., F. Ishimaru, K. Nakase, T. Tabayashi, T. Kozuka, K. Naoki, M. Miyahara, H. Toki, K. Kitajima, M. Harada, M. Tanimoto. 2003. Over-expression of short isoforms of Helios in patients with adult T-cell leukaemia/lymphoma. Br. J. Haematol. 120: 986-989. [CrossRef][Medline]
  38. Tabayashi, T., F. Ishimaru, M. Takata, I. Kataoka, K. Nakase, T. Kozuka, M. Tanimoto. 2007. Characterization of the short isoform of Helios overexpressed in patients with T-cell malignancies. Cancer Sci. 98: 182-188. [CrossRef][Medline]
  39. Fujiwara, S. I., Y. Yamashita, N. Nakamura, Y. L. Choi, T. Ueno, H. Watanabe, K. Kurashina, M. Soda, M. Enomoto, H. Hatanaka, et al 2008. High-resolution analysis of chromosome copy number alterations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified, with single nucleotide polymorphism-typing microarrays. Leukemia 22: 1891-1898. [CrossRef][Medline]
  40. Andrews, N. C., D. V. Faller. 1991. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 19: 2499[Free Full Text]
  41. Tu, L., T. C. Fang, D. Artis, O. Shestova, S. E. Pross, I. Maillard, W. S. Pear. 2005. Notch signaling is an important regulator of type 2 immunity. J. Exp. Med. 202: 1037-1042. [Abstract/Free Full Text]
  42. Elliott, J., C. Jolicoeur, V. Ramamurthy, M. Cayouette. 2008. Ikaros confers early temporal competence to mouse retinal progenitor cells. Neuron 60: 26-39. [CrossRef][Medline]
  43. Ezzat, S., S. L. Asa. 2008. The emerging role of the Ikaros stem cell factor in the neuroendocrine system. J. Mol. Endocrinol. 41: 45-51. [Abstract/Free Full Text]
  44. Kiehl, T. R., S. E. Fischer, S. Ezzat, S. L. Asa. 2008. Mice lacking the transcription factor Ikaros display behavioral alterations of an anti-depressive phenotype. Exp. Neurol. 211: 107-114. [CrossRef][Medline]
  45. Chatila, T.. 2007. The regulatory T cell transcriptosome: E pluribus unum. Immunity 27: 693-695. [CrossRef][Medline]



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