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Blood, 1 March 2007, Vol. 109, No. 5, pp. 2190-2197. Prepublished online as a Blood First Edition Paper on November 16, 2006; DOI 10.1182/blood-2005-01-031930.
NEOPLASIA Expression of a non–DNA-binding isoform of Helios induces T-cell lymphoma in mice1 Departments of Microbiology, 2 Biochemistry, and 3 Pathology, Division of Developmental and Clinical Immunology, University of Alabama at Birmingham
Helios is a zinc-finger protein belonging to the Ikaros family of transcriptional regulators. It is expressed, along with Ikaros, throughout early stages of thymocyte development where it quantitatively associates with Ikaros through C-terminal zinc-finger domains that mediate heterodimerization between Ikaros family members. To understand the role of Helios in T-cell development, we used a retroviral vector to express full-length Helios or a Helios isoform that lacked the N-terminal DNA-binding domain in hematopoietic progenitor cells of reconstituted mice. Constitutive expression of full-length Helios resulted in an inhibition of T-cell development at the double-negative stage within the thymus. Although expression of the DNA-binding mutant of Helios did not contribute to developmental abnormalities at early times after transplantation, 60% of animals that expressed the Helios DNA-binding mutant developed an aggressive and transplantable T-cell lymphoma 4 to 10 months after transplantation. These results demonstrate a vital function for Helios in maintaining normal homeostasis of developing T cells and formally show that non–DNA-binding isoforms of Helios are lymphomagenic if aberrantly expressed within the T-cell lineage.
The Ikaros family of zinc-finger DNA-binding proteins plays a vital role in the generation of all lymphoid-derived hematopoietic cell lineages.1,2 Members of this highly conserved family of proteins that are expressed predominantly in lymphoid cells include Ikaros, Helios, and Aiolos. Each protein is highly homologous throughout an N-terminal DNA-binding domain comprised of 4 Krüppel-like zinc-finger domains of the C2H2 type. Each protein also contains a C-terminal region comprised of 2 zinc-finger domains that mediate dimerization between family members.3–10 Extensive alternative mRNA splicing of exons within the DNA-binding domain of Ikaros, Helios, and Aiolos results in the expression of multiple, functionally distinct isoforms of each protein that are differentially expressed in unique subsets of hematopoietic cells.11–14 All mRNA splice variants characterized to date retain the C-terminal multimerization domain, therefore permitting the association of isoforms that lack the DNA-binding domain with isoforms that retain the ability to bind DNA. Association of non–DNA-binding isoforms of Ikaros with full-length Ikaros proteins has been shown to inhibit DNA-binding and transactivation potential of Ikaros in in vitro assays.5 Inhibition of Ikaros activity by non–DNA-binding Ikaros isoforms is further supported by the phenotype of animals with a targeted deletion of exons encoding the Ikaros DNA-binding domain (Ikaros DN)1 and in animals with a point mutation in one of the zinc-finger exons that disrupts DNA binding without affecting multimerization.15 Heterozygous Ikaros DN animals express similar levels of the dominant-negative and wild-type Ikaros alleles and develop T-cell lymphoma and leukemia with 100% penetrance soon after 3 months of age.16 Prior to the development of lymphoma, thymocytes within the heterozygous animals displayed increased proliferative responses to T-cell receptor (TCR)–mediated signals, which suggests that non–DNA-binding isoforms of Ikaros suppress the antiproliferative activity of full-length Ikaros isoforms during thymocyte development. Lymphoma in the animals was characterized by a loss of heterozygosity at the Ikaros locus, which indicates that the balance of different Ikaros mRNA splice variants is crucial for normal homeostasis of developing thymocytes. The increased proliferative response of B lymphocytes to B-cell receptor stimulation observed in animals with a targeted disruption of the Aiolos DNA-binding domain17 or in transgenic animals that express full-length Helios in the B-cell lineage18 suggests that higher order complexes comprised of Ikaros family members function as key regulators of proliferative responses in both T and B lymphocytes. Consistent with the role of Ikaros family members as tumor suppressor genes in lymphocytes are observations of increased expression of non–DNA-binding isoforms of Ikaros, Aiolos, and Helios in cases of acute lymphocytic leukemia (ALL). An increase in non–DNA-binding isoforms of Ikaros was observed in childhood and adult pre-B–cell ALL and in childhood T-cell ALL.19–23 Expression of non–DNA-binding isoforms of Helios have been noted in adult cases of T-cell ALL.24–26 To date, a loss-of-function mutation in the Helios gene has not been described so it remains unclear as to whether Helios is also functioning as a tumor suppressor in lymphocytes in a manner analogous to Ikaros and Aiolos.
In addition to the function of Ikaros family members in the regulation of lymphoproliferation, Ikaros has also been shown to regulate early developmental decisions within the hematolymphoid system. Mice homozygous for the Ikaros DN mutation died soon after birth and completely lacked all B-, T-, and NK-cell lineages.1 In contrast, mice homozygous for an Ikaros null mutation (targeted deletion of the C-terminal dimerization domain) had no B, NK, or fetal T cells and severely reduced numbers of specific
Because Helios is expressed in early double-negative (DN) thymocytes and quantitatively associates with Ikaros in developing T cells,7,8,27 we wished to determine whether Helios might be functioning as a critical regulator of
Construction of Helios retroviral vectors
A cDNA encoding full-length Helios (kindly provided by Dr Stephen Smale, UCLA, Los Angeles, CA) was cloned into the MSCV-IRES-GFP retroviral vector28 and orientation was confirmed by DNA sequencing. A FLAG epitope tag (DYKDDDDK) was cloned at the 3' end of the DNA-binding Helios mutant using polymerase chain reaction (PCR). Primers used to generate the Helios DNA-binding mutant, Helios Retroviral transduction and transplantation Bone marrow cells were isolated from C57BL/6-Ly-5.1 mice injected intraperitoneally 4 days previously with 5-fluorouracil (150 mg/kg body weight). Transductions were done by coculturing bone marrow on transiently transfected BOSC23 retroviral producer cells for 48 hours. Approximately 1 to 2 million transduced cells were then transplanted into lethally irradiated congenic C57BL/6-Ly-5.2 recipients as previously described. 29 Immunoprecipitation
BOSC23 cells were transfected with MSCV plasmids expressing Helios Western analysis of Helios and Ikaros expression in thymocytes Lysates from 2 x 106 fluorescence-activated cell sorting (FACS)–sorted thymocytes or from transiently transfected BOSC23 cells were analyzed by Western blotting using goat polyclonal antibodies against N-terminal (M-20) and C-terminal (G-20) Helios peptides (Santa Cruz Biotechnology), a rabbit polyclonal antibody against Ikaros (gift from Dr Stephen Smale, UCLA), or a mouse monoclonal antibody against ß-actin (Sigma-Aldrich). Donkey anti–goat, goat anti–rabbit, and goat anti–mouse polyclonal IgG (each conjugated to horseradish peroxidase) were used as secondary reagents for enhanced chemiluminescence (ECL) detection (Amersham Biosciences, Piscataway, NJ). For ß-actin, blots originally probed for Ikaros or Helios were stripped and reprobed with the ß-actin antibody. Antibodies and FACS analysis
Bone marrow, spleen, thymus, and Peyer patches were collected, homogenized, and treated to remove red blood cells by hypotonic shock. Monoclonal antibodies used in immunofluorescence staining were reactive against Thy-1.1 (19XE5), c-Kit (2B8), CD3 (KT31.1), CD4 (GK1.5), CD5 (53-7.3), CD8 (53-6.7), Ter119, B220 (6B2), Gr-1 (8C5), Mac-1 (M1/70), CD43 (S7), CD44 (1M7), CD25 (3C7), IL-7R T-cell proliferation assay: SNARF labeling A single-cell suspension was generated from the spleens of chimeric mice. Erythrocytes were lysed by hypotonic shock, and the remaining cells were labeled with 6 µM SNARF-1 (Molecular Probes, Eugene, OR) for 10 minutes at 37°C. The staining reaction was quenched with fetal calf serum, and the cells were washed and plated at a density of 300 000 cells/200 µL media in a 96-well round-bottom plate in RPMI 1640 plus 10% fetal calf serum (Hyclone, Logan, UT). Cells were stimulated with 3 µg/mL anti-CD3 (145-2C11) and 3µg/mL anti-CD28 (37.51). Control cells were left unstimulated. Cells were harvested daily for 4 days following stimulation and stained with anti-CD4 (GK1.5) or anti-CD8 (53-6.7) and analyzed for SNARF-1 dilution on a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). T-cell proliferation: tritiated thymidine incorporation Ecotropic packaging cells (Phoenix-E) were transfected with retroviral constructs using Lipofectamine (Life Technologies, Bethesda, MD). Retrovirus-containing cell supernatant was collected 48 hours after transfection and used to transduce C57BL/6 CD4+ splenic T cells in the presence of polybrene (8 mg/mL). CD4+ T cells were purified using CD4 microbeads (Miltenyi Biotec, Auburn, CA; catalog no. 130-049-201), and then were activated by plate-bound anti-CD3 and anti-CD28 stimulation (2 µg/mL each). Retroviral supernatant was added 24 hours after T-cell activation. GFP+CD4+ T cells were sorted after 2 days of transduction. Proliferative responses were measured by culturing 1 x 105 sorted GFP+ T cells in the presence of irradiated antigen-presenting cells (APCs) and varying concentrations of anti-CD3 (1.5 and 2.0 mg/mL) for 48 hours. Each concentration was set up in quadruplicate wells. Cells were then pulsed with 3H-thymidine for 16 hours and then harvested for counting on a beta plate scintillation counter (Wallac, Turku, Finland). Real-time PCR
Each of the DN, DP, and SP thymocyte subsets underwent FACS once and then were resorted to ensure a final sort purity of more than 98% for each subset. DN1 thymocytes were sorted as CD4–CD8–CD25–B220–Mac-1–IL-7R Image acquisition Images were acquired at 160x magnification using a Leica DMRB upright microscope (Wetzlar, Germany), PL FluoroTar 16x objective with 0.5 numerical aperture and a Hamamatsu C5810 CCD camera. Acquisition software was IPLab version 3.6 (Signal Analytics Corp, BD Biosciences, San Jose, CA). Sections were stained with hematoxylin/eosin and visualized using immersion oil (Leica).
Generation of Helios mutants
To assess the function of Helios in early lymphoid-lineage development and leukemia, we generated retroviral constructs that expressed either full-length Helios (amino acids 1-540) or a Helios mutant that lacked the 4 N-terminal zinc finger domains that mediate Helios DNA binding (
To determine whether deletion of the DNA-binding domain in the  49-285 Helios variant would alter its ability to interact with Ikaros through the C-terminal multimerization domain, we performed an immunoprecipitation experiment using FLAG-tagged Helios 49-285 and an HA-tagged Ikaros isoform (IK4).5 Nuclear extracts from BOSC23 cells transiently transfected with the indicated constructs were incubated with an anti-FLAG monoclonal antibody and immunoprecipitates were then used for Western analysis using an anti-Helios (Figure 1B top panel) or an anti-Ikaros polyclonal antibody (Figure 1B bottom panel). IK4 was only immunoprecipitated in the presence of FLAG-Helios 49-285, which indicates that the 49-285 Helios variant was capable of interacting with Ikaros. The reciprocal experiment using an anti-HA monoclonal antibody to immunoprecipitate IK4 and FLAG-Helios gave similar results (data not shown).
Full-length Helios expression partially blocks
To investigate the function of Helios on early lymphocyte development, lethally irradiated mice were recipients of hematopoietic progenitor cells that stably expressed each Helios construct. All GFP control and Helios-reconstituted animals showed stable levels of GFP chimerism in different hematopoietic tissues for longer than 10 months, which indicates that primitive hematopoietic stem cells were transduced with the retroviral constructs (Figure 2A). One observation that was immediately apparent was the extremely low levels of GFP chimerism in the thymus of all animals reconstituted for more than 10 weeks with cells that expressed full-length Helios (Figure 2A; mean percentage of GFP+ thymocytes was 1.7% for full-length Helios, n = 16, and 25.8% for GFP control animals, n = 8). Bone marrow chimerism averaged 13.3% and 19.8% for full-length Helios and GFP control-reconstituted animals, respectively. This indicates that the severe reduction in GFP+ thymocytes expressing full-length Helios was not due to loss of stem cell activity in the presence of Helios. Further analysis showed an approximate 5-fold increase in the frequency of GFP+ DN thymocytes in all full-length Helios-expressing animals compared with GFP control and Helios
To characterize the increased frequency of GFP+ DN thymocytes in full-length Helios animals, we analyzed CD44 and CD25 expression, which divided the CD4–CD8–B220–Mac-1– population into 4 distinct subsets. In full-length Helios animals, we noted significant increases in the frequencies of all DN stages compared with GFP control animals (Figure 3A). Even though the frequency of all DN subsets was increased, the absolute numbers of the DN2, DN3, and DN4 subsets were reduced approximately 3- to 5-fold. There was a 2-fold reduction in the absolute numbers of GFP+ DN1 cells. These data suggest that the significant reduction in thymopoiesis in the presence of full-length Helios (as evidenced by the extremely low GFP chimerism; Figure 2A) may partially be explained by reduced expansion within the DN populations that give rise to DP cells.
Endogenous Helios is expressed throughout thymocyte development To account for the reduction in absolute numbers of DN thymocytes and the overall reduction in thymopoiesis in the presence of constitutive Helios expression, we analyzed the expression pattern of endogenous Helios in FACS-sorted thymocyte subsets by real-time PCR using both ß-actin and HPRT amplification to normalize Helios expression levels between samples. In 3 independent cDNA preparations (2 preparations for the DN1 subset) using RNA isolated from cells that were sorted once and then resorted prior to mRNA isolation, there was not a large variation in the relative level of Helios expression between thymocyte subsets (Figure 3B), with the exception that Helios seemed to be down-regulated in later DN stages (most notably, DN4) and again as DP thymocytes differentiated to the CD4 and CD8 SP lineages. These results establish a correlation between Helios down-regulation and thymocyte proliferation that normally occurs at the DN4 stage.31 Full-length Helios represses T-cell proliferation Because Ikaros and Aiolos had previously been shown to function as tumor suppressor genes for T- and B-lineage cells, we tested whether full-length Helios might also function to suppress T-cell proliferation stimulated by anti-CD3 and irradiated APCs in vitro. Incorporation of tritiated thymidine into stimulated CD4+ thymocytes was inhibited 2- to 2.5-fold in the presence of full-length Helios compared with cells that expressed the control GFP vector at either concentration of anti-CD3 (Figure 3C). These results suggest that retroviral expression of full-length Helios in vivo may be inhibiting thymocyte development by partially restricting thymocyte proliferation within early DN subsets.
Full-length Helios expression leads to increased frequencies of
The observations that Ikaros null mice lack NK cells and certain subsets of
Analysis of NK cell frequency in the spleens of reconstituted animals (n = 13 for full-length Helios and GFP control; n = 6 for Helios 49-285) indicated a 3.5-fold increase in the frequency of mature NK cells (NK1.1+CD3–) that expressed full-length Helios as compared with GFP control or Helios mutant animals (Figure 4B-C, P < .01). There was no difference in the mean frequencies of NK1.1+CD3+ NKT cells between full-length Helios, Helios 49-285, or GFP control reconstituted animals (Figure 4B).
Animals expressing the
As noted previously (Figures 2 and 4), there was little difference in the phenotypes of T-lineage subsets between control and Helios
The accumulation of abnormal T cells in the 49-285 animals originated in the thymus, which was suggested by the substantial increase in GFP+ thymocytes prior to GFP+ thymocyte expansion at other hematopoietic sites (Figure 5A). Among moribund 49-285 animals, abnormal DP thymocytes could be detected in both the bone marrow and spleen (Figure 5B). Characterization of lymphoma cells in the thymus and spleen of Helios mutant animals
The
Helios
To test whether expression of Helios
Lymphoma is transplantable into secondary hosts
To determine whether thymocytes or splenocytes from moribund
Loss of Ikaros is not required for lymphoma in moribund
In the context of Ikaros heterozygous animals, loss of the wild-type Ikaros allele was associated with progression to lymphoma.16 To determine if loss of Ikaros was required for disease progression in the sick, Helios
The contrasting phenotypes between Ikaros DN mutant animals,1 which displayed a complete absence of  ß T cells, and Ikaros null animals,2 which retain the ability to differentiate into mature ß T cells, suggests that the dominant-negative allele of Ikaros may be interfering with the function of another factor that is essential for the earliest stages of ß T-cell development. One possible candidate factor could be Helios, which is expressed at the earliest stages of thymocyte development (Figure 3B). If Helios were the factor being inhibited by the DN allele of Ikaros to block T-cell development, then we would have anticipated that expression of full-length Helios would promote ß T-cell development and Helios 49-285 expression would inhibit thymocyte development. In contrast, full-length Helios suppressed thymocyte development by a mechanism that is perhaps best explained by the ability of Helios to inhibit thymocyte proliferation (Figures 2 and 3C). Loss-of-function mutations in other Ikaros family members, including Ikaros and Aiolos, have shown that both factors function as tumor suppressors for lymphocytes. Data showing that expression of the 49-285 Helios mutation in thymocytes results in T-cell lymphoma and enhanced SP T-cell proliferation (Figures 5–6) also support an antiproliferative function for the full-length DNA-binding isoforms of Helios.
Expression of a non–DNA-binding isoform of Helios resulted in no detectable abnormalities in development of T, B, or NK cells in the bone marrow, thymus, or peripheral lymphoid organs before the onset of T-cell lymphoma (Figures 2
Animals that expressed the The normal development of lymphoid cells and the control of lymphoproliferative responses is regulated by a complex interaction between multiple members of the Ikaros gene family and proteins that mediate changes in chromatin structure. The experiments described in this study establish Helios as an important regulator of thymocyte development and indicate that aberrant mRNA splicing or deletion of the DNA-binding domain of Helios results in significantly increased risk for development of T-cell lymphoma and lymphoproliferative disease.
Contribution: Z.Z. performed research, designed experiments, and collected and analyzed data; C.S.S. performed research, designed experiments, and collected and analyzed data; J.T.B. performed research and analyzed data; R.K. performed research and analyzed data; C.V.C. performed research and analyzed data; and C.A.K. designed research, analyzed data, and wrote the paper. Conflict-of-interest disclosure: The authors declare no competing financial interests. Z.Z. and C.S.S. contributed equally to this manuscript. Correspondence: Christopher A. Klug, Departments of Microbiology, Biochemistry, and Pathology, Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham, AL; e-mail: chrisk{at}uab.edu.
This work was supported by NIH grants RO1DK55650 and RO1AI055667. J.T.B. and C.S.S. were supported by the Immunologic Diseases and Basic Immunology training grant (T32AI07051). We wish to thank Dr John F. Kearney for his support; Dr Stephen Smale and Dr Bradley Cobb for the Helios cDNA and insightful comments related to the work; and Drs Richard Lopez, Colleen Witt, and Sheetal Purohit for valuable discussions. Thanks to Weifan Jia for technical support.
Submitted January 21, 2006; accepted September 22, 2006.
Prepublished online as Blood First Edition Paper, November 16, 2006
DOI: 10.1182/blood-2005-01-031930
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Abstract
Introduction
T-cell subsets and thymic dendritic cells.
