Expression of Helios, an Ikaros Transcription Factor Family Member, Differentiates Thymic-Derived from Peripherally Induced Foxp3⁺ T Regulatory Cells

Animals

BALB/c and C57BL/6 mice were obtained from the National Cancer Institute (Frederick, MD). Foxp3-GFP mice (21) and MHC class II-deficient mice (A^bβ^−/−) were obtained by the National Institute of Allergy and Infectious Diseases (NIAID) and were maintained by Taconic Farms (Germantown, NY) under contract by NIAID. The Foxp3-GFP mice were crossed to OT-II TCR transgenic (Tg) RAG^−/− mice obtained from Taconic Farms. Female heterozygous B6.Cg-Foxp3^sf/J (Scurfy) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred to C57BL/6 WT male mice to generate hemizygous male B6 (Cg-Foxp3^sf/Y [Scurfy]) offspring. Armenian hamsters were purchased from Cytogen Research and Development (West Roxbury, MA). Mice bearing loxP-flanked conditional (fl/fl) alleles of Helios on a C57BL/6 background were generated by Ozgene (Bentley Dc, Australia). Ikzf2^fl/fl mice were bred in our laboratory to mice expressing Cre recombinase under the control of the CD4 promoter (CD4-Cre) to produce Ikzf2^fl/fl × CD4-Cre mice, which will be described elsewhere (A.M. Thornton, J. Murphy, and E.M. Shevach, manuscript in preparation). CD4-Cre mice were obtained from Taconic Farms. All animal protocols used in this study were approved by the NIAID Animal Care and Use Committee.

Human cells

Peripheral blood was obtained from healthy adult donors by the Department of Transfusion Medicine at the National Institutes of Health. The acquisition of blood products was approved according to the Institutional Review Board and in accordance with the Declaration of Helsinki.

Media, Abs, and reagents

Mouse cells were grown in RPMI 1640 (Lonza, Walkersville, MD) supplemented with 10% heat-inactivated FCS, penicillin (100 μg/ml), streptomycin (100 μg/ml), 2 mM _L-glutamine, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (all from Lonza), and 50 μM 2-ME (Sigma-Aldrich, St. Louis, MO). Purified anti-CD3 (2C11), PE–anti-CD25 (PC61), and FITC–anti-CD8 were purchased from BD Biosciences (San Jose, CA). Mouse Abs included tricolor–anti-CD4 (Invitrogen, Carlsbad, CA) and Alexa Fluor 647 anti-Foxp3, PE anti-FoxP3, PE-Cy7 anti-CD8, Pacific Blue anti-CD4, Alexa Fluor 750 anti-CD45.1, PE-Cy5.5 anti-CD45.2, and Alexa Fluor 700 anti-CD44 (eBioscience, San Diego, CA). Human Abs included FITC anti-CD4, PE-Cy5.5 anti-CD45RA, PE anti-CD25, allophycocyanin anti–IL-2, and allophycocyanin anti–IFN-γ (Invitrogen); Alexa Fluor 647 anti-CD127 and anti-CD28 (BD Biosciences); Alexa Fluor 647 anti-human FOXP3 (236A/E7) and Alexa Fluor 647 anti–IL-17 (eBioscience); and anti-CD3 (OKT3; Ortho Biotech Products, Bridgewater, NJ). CD8, CD4, and PE microbeads were purchased from Miltenyi Biotec (Auburn, CA). Intracellular staining was performed with the Foxp3 staining buffer kit, according to the manufacturer’s protocol (eBioscience). Flow cytometry analysis was performed using FlowJo software. Human rIL-2 was obtained from the Preclinical Repository of the Biological Resources Branch, National Cancer Institute. TGF-β1 was purchased from PeproTech (Rocky Hill, NJ).

Cell purification, activation, and RNA isolation of mouse cells

Peripheral lymph nodes (LNs) were harvested from 8-wk-old female BALB/c or C57BL/6 mice. CD4⁺CD25⁻ and CD4⁺CD25⁺ cells were purified as previously described (22). The purity of CD4⁺CD25⁻ and CD4⁺CD25⁺ cells was ~97%. CD8⁺ T cells were purified from mouse LNs using CD8 microbeads. For thymic subpopulations, cells were labeled with PE anti-CD25, tricolor anti-CD4, or FITC anti-CD8 and were purified by cell sorting. For cell activation, purified cells (10⁶/ml) were cultured in complete RPMI 1640 supplemented with 100 U/ml IL-2 at 37°C for 16 h in 24-well plates precoated with 5 μg/ml anti-CD3. RNA was purified using TRIzol reagent (Invitrogen), according to manufacturer’s instructions.

PCR-based cDNA subtraction

Total RNA was subjected to PCR-Select cDNA Subtraction, followed by mirror-orientation selection (BD Clontech, Palo Alto, CA) (23). The CD4⁺ CD25⁺ T cell-specific library resulted in 106 clones. cDNA inserts were sequenced by Lofstrand Labs (Gaithersburg, MD).

Quantitative PCR

RNA from purified or purified and activated cells was isolated using an RNeasy Plus kit (Qiagen, Valencia, CA). RNA was converted to cDNA using SuperScript II reverse transcriptase (Invitrogen) with random primers, according to the manufacturer’s protocol. Primers and FAM-labeled probes were purchased from Integrated DNA Technologies (San Diego, CA). Helios forward: 5′-TCACAACTATCTCCAGAATGTCAGC-3′; reverse: 5′-AGGCGGTACATGGTGACTCAT-3′; probe: 5′-TGGAGGCTGCCG-GGCAGG-3′. Foxp3 forward: 5′-CCCAGGAAAGACAGCAACC-3′TT; reverse: 5′-TTCTCACAACCAGGCCACTTG-3′; probe: 5′-ATCCTACC-CACTGCTGGCAAATGGAGTC-3′. 18S rRNA (Applied Biosystems, Foster City, CA) was used as an internal control for normalization. All PCR reactions were performed in triplicate using TaqMan Universal PCR Master Mix (Applied Biosystems). An ABI prism 7700 sequence detection system (Applied Biosystems) was used for 40 cycles of PCR. Each experiment was performed at least three times, and a representative experiment is shown.

Generation of the 22F6 anti-Helios mAb

A peptide from the Helios protein (aa 51–107) was synthesized by the NIAID Peptide Synthesis Laboratory (Bethesda, MD) and was used to immunize Armenian hamsters. Spleens from immunized animals were fused with SP2/0-Ag14 mouse myeloma cells. Hybridoma supernatants were first screened by ELISA using the peptide and HRP conjugated goat anti-Armenian Hamster IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Supernatants were screened by Western blot analysis on EL-4 whole-cell extracts using HRP-conjugated goat anti-Armenian Hamster IgG or by flow cytometry using intracellular staining methods and FITC-conjugated goat anti-Armenian Hamster IgG as the secondary Ab. The mAb 22F6 was subsequently conjugated to FITC or PE by the NIAID Custom Ab Facility (Bethesda, MD) or to FITC, PE, or Alexa Fluor 647 by BioLegend (San Diego, CA).

Cell purification and activation of human cells

PBMCs were prepared over Ficoll-Paque Plus gradients (GE Healthcare, Piscataway, NJ). The CD4⁺ T cells were enriched by autoMACS positive selection with human CD4 microbeads. The cells were labeled with anti-CD4, anti-CD45RA, anti-CD25, and anti-CD127 and purified by cell sorting with BD Aria. The postsort purity was >95% for CD4⁺CD25⁻ CD127⁺CD45RA⁺ and CD4⁺CD25⁻CD127⁺CD45RA⁻ cells, and the FOXP3 purity was >85% for CD4⁺CD25^hi cells. The cells were activated in vitro with plate-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2.5 μg/ml), with or without 5 ng/ml TGF-β1 in 24-well culture plates (Corning Life Sciences, Acton, MA) at 500,000 cells per well in complete RPMI 1640 supplemented with 100 U/ml IL-2.

Proliferation and suppression assay

Purified cells were cultured as previously described (22). For human in vitro suppression assays, 50,000 FACS-sorted allogenic CD4⁺CD25⁻ T cells were stimulated with 25,000 nonirradiated autologous CD3-depleted HLA-DR⁺ APCs and 0.25 μg/ml OKT3 alone or with varying numbers of Treg cells. The cells were cultured for 3 d in 96-well flat-bottom plates (Corning Life Sciences), pulsed with [³H]thymidine deoxyribose (1 μCi/well) for the last 6–8 h, and then quantified with a scintillation counter. The APCs were isolated by depleting CD3⁺ cells with CD3 microbeads over the AutoMACS, followed by positive selection with HLA-DR microbeads.

Generation of mouse-induced Treg cells

CD4⁺GFP⁻ cells and CD4⁺GFP⁺ cells from the LN and spleen of Foxp3-GFP reporter mice were purified by cell sorting. For cell activation, purified cells (10⁶/ml) were cultured in complete RPMI 1640 supplemented with 100 U/ml IL-2 at 37°C for 3 d in 24-well plates precoated with 5 μg/ml anti-CD3 in the presence or absence of 5 ng/ml TGF-β.

Oral tolerance induction

CD4⁺GFP^neg T cells from OT-II TCR Tg × Foxp3-GFP mice were purified from the spleen and LN by cell sorting. Two million cells were transferred i.v. into B6.SJL congenic mice. The mice were given 1.5% OVA-supplemented water for 6 d; the water was changed every 2 d.

Thymocyte transfer

CD4⁺CD8⁻GFP⁺ thymocytes were purified from the thymus of 4-wk-old Foxp3-GFP mice. A total of 6 × 10⁵ cells were transferred i.v. Cells were harvested 4 wk later and analyzed by flow cytometry.

Helios is differentially expressed in Treg cells

To elucidate genes specific to CD4⁺CD25⁺ Treg cells, RNA isolated from CD4⁺CD25⁻and CD4⁺CD25⁺ cells was subjected to Clontech PCR Select Differential Screening, followed by mirror-oriented selection (23). Of the 106 clones specific to the CD4⁺CD25⁺ Treg cell library, 54 clones contained a fragment of the Helios gene (Supplemental Table I). Helios expression was analyzed by quantitative PCR (qPCR) to validate the differential screening (Supplemental Fig. 1A). Helios mRNA was detected in freshly isolated CD4⁺CD25⁺ cells, and it increased ~3-fold upon activation of the cells. Helios was not detected in freshly isolated or activated CD4⁺ CD25⁻ cells, and it was barely detectable in CD8⁺ cells. We were unable to detect mRNA for Helios in B cells, dendritic cells, or cells of the monocyte/macrophage lineage (data not shown). To further characterize Helios, we examined Helios mRNA expression in various thymic subpopulations. We observed that Helios was expressed in the thymus at low levels as early as the double negative (DN) stage (Supplemental Fig. 1B). Helios continued to be expressed at low levels in double positive (DP) cells, CD8⁺ cells, and CD4⁺CD25⁻cells. However, there was a striking increase in Helios mRNA in thymic CD4⁺CD25⁺ cells, concurrent with the first appearance of Foxp3 mRNA (Supplemental Fig. 1C). In peripheral LNs, Helios expression correlated with Foxp3 expression and was detected exclusively in Treg cells.

Development of an anti-mouse/human Helios mAb

All members of the Ikaros family of transcription factors contain a large region of six highly conserved zinc fingers. However, the N terminus of the members is not conserved. To more thoroughly examine Helios expression and function, we generated a mAb (22F6) to a large peptide within the nonconserved 110-aa N terminus region of Helios. When Helios expression was analyzed by intracellular staining of permeabilized cells with this mAb, 10% of CD4⁺ T cells in the peripheral LN of mice were positive (Fig. 1A). In agreement with the qPCR, mAb 22F6 reacted preferentially with CD4⁺CD25⁺ Treg cells. Notably, when LN cells were gated on CD4⁺ cells and analyzed for Foxp3 and Helios expression, Helios was expressed on only 70% of Foxp3⁺ cells. A low percentage of non-CD4⁺ cells also reacted with the mAb (Fig. 1A): ~50% of these cells were CD8⁺ and 20% were TCRγδ⁺ (data not shown). Lineage-specific markers have not been defined for the remaining 30% of the non-CD4⁺ cells that reacted with the mAb.

Because 22F6 was raised to a peptide derived from the mouse sequence that differed from the human sequence by only two amino acids, we analyzed the reactivity of 22F6 in human cells. 22F6 also reacted with human PBMCs and was again specific to the CD4⁺ CD25^hi Treg cell subset (data not shown). Although much more variable than in mouse CD4⁺ cells, Helios was expressed in a subset of CD4⁺Foxp3⁺ cells in humans that ranged from 50–90% (Fig. 1B). Despite this wide range of variability, analysis of a large number of normal donors demonstrated that CD4⁺Foxp3⁺Helios⁺ cells represent, on average, 70% of Foxp3⁺ cells in man, which is strikingly similar to that observed in mice (Fig. 1C).

Expression of Helios during T cell development

Because our qPCR studies suggested that Helios mRNA was expressed at different stages of thymic development, we used mAb 22F6 to examine the expression of Helios protein in subsets of adult mouse thymocytes. Helios was expressed at the DN stage of development, in agreement with the qPCR data (Fig. 2A). Specifically, Helios was expressed at low levels as early as DN1, but it was expressed in 100% of DN2 and DN3 cells. By DN4, Helios expression had begun to decrease. In contrast, Foxp3 was not expressed at the DN stage. Although Foxp3⁺Helios⁻ T cells could be detected in the adult thymus (Fig. 2A), they were absent at day 3 of life, when Foxp3⁺ cells first appear in the thymus (Fig. 2B). Although Foxp3⁺Helios⁻ cells can be detected in the thymus as the mice mature, the percentage of Foxp3⁺Helios⁻CD4 single positive (SP) cells in an adult remains lower (15%) than that observed in peripheral lymphocytes (30%). Lio and Hsieh (24) recently proposed a two-step process for thymic Treg cell development and demonstrated that Foxp3⁺ Treg cells can develop from the CD4SPFoxp3⁻CD25^hi subset. Helios was expressed in 30% of CD4SPFoxp3⁻CD25^hi thymocytes, but it remains to be determined whether these cells are the precursors of the CD4⁺ Foxp3⁺ cells (data not shown).

We next examined Helios expression in the developing peripheral tissue. Foxp3⁺Helios⁻ cells are absent in the neonate, do not significantly appear in the spleen until between days 10 and 12 of life, and do not reach the percentage observed in an adult until after the pups have been weaned (Fig. 3A). Phenotypic analysis of the Foxp3⁺Helios⁺ cells shows that the vast majority of the cells in the thymus (Fig. 2B) and peripheral lymphoid tissue (Fig. 3B) are CD44^hiCD25⁺ in adults and neonates. In contrast, when the Foxp3⁺ Helios⁻ cells can first be detected in the periphery, the majority are CD44^lowCD25⁻ (Fig. 3C) and do not acquire the CD44^hiCD25⁺ phenotype until after the mice have been weaned. This result is consistent with the possibility that the precursors of the Foxp3⁺ Helios⁻ cells do not arise in the thymus.

Thymic Foxp3⁺Helios⁺ Treg cells are a stable lineage

Although the differential expression of CD25 and CD44 on Foxp3⁺ Helios⁻ T cells during development was consistent with the development of this population in the periphery, it remained possible that some Foxp3⁺Helios⁻T cells were derived from Foxp3⁺Helios⁺ precursors. We are unable to isolate Foxp3⁺Helios⁺ and Foxp3⁺ Helios⁻ populations from peripheral lymphoid tissues based on the differential expression of any other cell surface Ag. However, the thymus of young mice is almost devoid of Foxp3⁺Helios⁻ cells. To examine the stability of the thymic Foxp3⁺Helios⁺ population, we sorted CD4⁺CD8⁻Foxp3⁻GFP⁺ cells (~3–5% Foxp3⁺Helios⁻) from the thymus of young mice and transferred them to CD45.1⁺ congenic hosts. After 4 wk, the transferred cells were analyzed for

their expression of Foxp3 and Helios. The transferred cells maintained their expression of Helios and Foxp3 1 m after transfer to normal recipients (Fig. 4), indicating that Foxp3⁺Helios⁺ cells are unlikely to be the precursors of the Foxp3⁺Helios⁻ cells.

Foxp3⁺ cells generated in vitro or in vivo are Foxp3⁺Helios⁻

The observation that Foxp3⁺Helios⁻ cells seem to arise in peripheral lymphoid tissue and do not acquire their full phenotypic profile until after weaning led us to examine the expression of Helios in MHC class II-deficient (^−/−) mice. Previous studies showed that Foxp3⁺ T cells are readily detectable in the thymus and the periphery of MHC class II^−/− mice, and the majority express CD8 (25). We reasoned that if Foxp3⁺Helios⁻ cells are induced in the periphery, this induction would require TCR/MHC class II interactions. When LN cells from adult wild-type (WT) and MHC class II^−/− mice were analyzed for the expression of Foxp3 and Helios, the Foxp3⁺Helios⁻ cells were not observed in MHC class II^−/− mice, but they were readily detectable in WT LNs (Fig. 5A).

We next analyzed Helios expression in Foxp3⁺ T cells that had been induced in vitro in the presence of TGF-β. CD4⁺GFP⁻ and CD4⁺GFP⁺ cells were sorted from Foxp3-GFP reporter mice and activated in the presence or absence of TGF-β. Helios was not expressed in Foxp3⁻ T cells activated in the absence of TGF-β (Fig. 5B). Strikingly, Helios was also not induced when the Foxp3⁻ cells were cultured in the presence of TGF-β, despite the strong induction of Foxp3 expression. Expansion of CD4⁺Foxp3⁺ T cells under similar conditions led to a loss of Foxp3 expression in some of the population, but it did not change the ratio of Foxp3⁺Helios^−/Foxp3⁺Helios⁺ cells in the cells that remained Foxp3⁺. Helios expression was not observed in cells that had lost expression of Foxp3. Although human CD4⁺Foxp3⁻ T cells can be induced to express Foxp3 by stimulation in vitro in the presence of TGF-β, they lack regulatory function (26, 27). Nevertheless, similar results were observed when human CD4⁺CD25⁻CD127⁺CD45RA⁺ cells (<1% FOXP3⁺) and CD4⁺CD25^hiCD127⁻ cells (>85% FOXP3⁺) were activated in the presence or absence of TGF-β (Fig. 5C). Helios expression was never observed when human FOXP3⁻ T cells were activated in vitro under conditions in which FOXP3 expression was readily induced.

Mucida et al. (28) showed that peripheral conversion of CD4⁺ Foxp3⁻ cells to CD4⁺Foxp3⁺ Treg cells can be readily detected in vivo after oral exposure to Ag, whereas Sun et al. (29) demonstrated that this occurs specifically in the gut-associated tissues, such as the lamina propria (LP). Using this model, we adoptively transferred CD4⁺Foxp3⁻ cells from OT-II TCR Tg × Foxp3-GFP mice into CD45.1⁺ congenic hosts and fed the recipients OVA in the drinking water for 6 d. Foxp3⁺Helios⁺ cells made up ~60% of the endogenous Foxp3⁺ cells in the LP and 70% of Foxp3⁺ cells in the mesenteric LN (mLN) (data not shown); oral administration of OVA did not alter these ratios in the host-derived CD4⁺ LP cells and mLN cells (Fig. 5D). As expected following feeding, OT-II⁺Foxp3⁺ T cells could be detected in the LP. However, the converted cells failed to express Helios, thus demonstrating that Foxp3⁺ T cells generated in vivo and in vitro do not express Helios. In the mLN, a small percentage (~2%) of OT-II cells converted to Foxp3-expressing cells. However, in the mLN, a significant percentage of Foxp3⁻cells expressing Helios was detected, raising the possibility that transient expression of Helios occurs in some or all activated cells upon exposure to oral Ag but that expression of Helios is silenced when Foxp3 expression is present.

Human FOXP3⁺Helios⁻ T cells are enriched in cytokine-producing cells

The issue of the stability of Foxp3 expression was recently questioned, and it seems likely that some Foxp3⁺ cells can lose expression of Foxp3 and become effector T cells (30). If Foxp3⁺ Helios⁻ T cells are primarily generated from conventional T cells in the periphery, one might predict that these cells are intrinsically more prone to exhibit plasticity. In particular, a subpopulation of human FOXP3⁺ cells was recently shown to produce IL-17 when stimulated in the presence of proinflammatory cytokines. Therefore, it was of interest to examine the cytokine-expression profile of human FOXP3⁺ cells based on Helios expression. When human PBMCs were stimulated with PMA and ionomycin for 5 h and cytokine production by Foxp3⁺ T cells was analyzed by intracellular staining, low, but significant, numbers of IL-2– and -17–and IFN-γ–producing cells were detected; however, these were present almost exclusively within the Foxp3⁺Helios⁻ population (Fig. 6). Thus, the lack of expression of Helios seems to define a Treg cell population with the potential for plasticity.

Expression of Helios is not required for Treg cell function

Because Helios expression in the thymus precedes Foxp3, we examined the possibility that Helios could regulate the expression of Foxp3. We generated Ikzf2^fl/fl mice (Supplemental Fig. 2) and crossed them to CD4-Cre mice (Ikzf2^fl/fl × CD4-Cre). Because Helios is expressed prior to CD4 in the thymus, Helios expression is only slightly reduced in DN thymocytes, but it is virtually absent by the CD4 single-positive stage (Supplemental Fig. 3). However, despite the deletion of Helios in peripheral CD4⁺ T cells, Foxp3 expression remains unchanged (Fig. 7A). Thus, Helios does not seem to regulate Foxp3 expression, nor does Foxp3 seem to be required for Helios expression. Because Foxp3 was reported to bind to the Helios promoter, and thus, possibly regulate its expression, we examined the expression of Helios in Scurfy mice that possess a mutation in the Foxp3 gene that results in the absence of Foxp3 protein. Because these mice die at an early age, they were examined at 4 or 17 d after birth. Despite the absence of Foxp3, Helios was expressed in the thymus and spleen of Scurfy mice (Supplemental Fig. 4).

To examine the function of Helios in Treg cells, we purified CD4⁺ CD25⁺ T cells from Ikzf2^fl/fl × CD4-Cre mice and assayed the cells for suppressive function in vitro. Treg cells deficient for Helios were nonresponsive to TCR stimulation and failed to respond to IL-2, but they responded to anti-CD3 and IL-2 in a manner similar to Treg cells from WT mice (Fig. 7B). In addition, Treg cells from the Helios^−/− mice were as suppressive as Treg cells from WT mice (Fig. 7C). Helios expression in expanded human CD4⁺CD127⁻ CD25^hi Treg cells was markedly reduced following electroporation of Helios small interfering (siRNA). The loss of Helios expression did not reverse the nonresponsiveness of the Treg cells nor did it impair their suppressive capacity (Fig. 7D).