IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4 + T Cells

This information is current as Michael L. Ridley, Veerle Fleskens, Ceri A. Roberts, of September 28, 2021. Sylvine Lalnunhlimi, Aldana Alnesf, Aoife M. O'Byrne, Kathryn J. A. Steel, Giovanni A. M. Povoleri, Jonathan Sumner, Paul Lavender and Leonie S. Taams J Immunol published online 22 April 2020

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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 © 2020 The Authors All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 22, 2020, doi:10.4049/jimmunol.1901283 The Journal of Immunology

IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4+ T Cells

Michael L. Ridley,*,1 Veerle Fleskens,*,1 Ceri A. Roberts,* Sylvine Lalnunhlimi,* Aldana Alnesf,* Aoife M. O’Byrne,* Kathryn J. A. Steel,* Giovanni A. M. Povoleri,* Jonathan Sumner,† Paul Lavender,‡ and Leonie S. Taams*

The expression of anti-inflammatory IL-10 by CD4+ T cells is indispensable for immune homeostasis, as it allows T cells to moderate their effector function. We previously showed that TNF-a blockade during T cell stimulation in CD4+ T cell/monocyte cocultures resulted in maintenance of IL-10–producing T cells and identified IKZF3 as a putative regulator of IL-10. In this study, we tested the hypothesis that IKZF3 is a transcriptional regulator of IL-10 using a human CD4+ T cell–only culture system. IL-10+ CD4+ T cells expressed the highest levels of IKZF3 both ex vivo and after activation compared with IL-10–CD4+ T cells. Pharmacological targeting of IKZF3 with the drug lenalidomide showed that IKZF3 is required for anti-CD3/CD28 mAb– Downloaded from mediated induction of IL-10 but is dispensable for ex vivo IL-10 expression. However, overexpression of IKZF3 was unable to upregulate IL-10 at the mRNA or level in CD4+ T cells and did not drive the transcription of the IL10 promoter or putative local enhancer constructs. Collectively, these data indicate that IKZF3 is associated with but not sufficient for IL-10 expression in CD4+ T cells. The Journal of Immunology, 2020, 204: 000–000. http://www.jimmunol.org/ he production of IL-10 by CD4+ T cells is key for the control deletions of Il10 lead to a pronounced inflammation in the colonic of effector function in response to immune challenge (1–3). mucosa in response to commensal gut bacteria (1). T Even in the absence of pathogens, CD4+ T cell–specific IKZF3 (encoding for the protein Aiolos) is a member of the Ikaros Zinc finger family of transcription factors (4). This is expressed by various immune cell types and has been implicated *Centre for Inflammation Biology and Cancer Immunology, Department of Inflam- in the function of multiple Th subsets (5, 6) as well as in con- mation Biology, School of Immunology and Microbial Sciences, King’s College † trolling CD4/CD8 fate decision in the thymus (7). The expression London, London SE1 1UL, United Kingdom; Department of Infectious Diseases, + School of Immunology and Microbial Sciences, King’s College London, Guy’s of IKZF3 in IL-17–producing CD4 T cells (Th17 cells) is as- Hospital, London SE1 9RT, United Kingdom; and ‡MRC and Asthma UK Centre sociated with a “nonpathogenic” signature, which includes in- by guest on September 28, 2021 in Allergic Mechanisms of Asthma, Peter Gorer Department of Immunobiology, creased IL-10 production (6, 8). IKZF3 has also been shown to School of Immunology and Microbial Sciences, King’s College London, Guy’s Hospital, London SE1 9RT, United Kingdom interact with known regulators of IL10 expression, including its 1M.L.R. and V.F. contributed equally. most closely related family member IKZF1 (encoding Ikaros) (4) ORCIDs: 0000-0002-3953-2384 (M.L.R.); 0000-0002-7900-667X (C.A.R.); 0000- whichhasbeenshowninmicetodirectly affect the expression of 0001-8185-2501 (A.M.O.); 0000-0003-0342-651X (G.A.M.P.); 0000-0001-8710- Il10 (9). 0263 (P.L.); 0000-0002-9337-7194 (L.S.T.). Whereas IKZF3 has been suggested to act as a transcriptional Received for publication October 24, 2019. Accepted for publication April 1, 2020. activator in CD4+ T cells (4, 10), this has mainly been ascribed This work was supported by Versus Arthritis (21139), the King’s Bioscience Institute to its cooperation with other factors, such as FOXP3 (11) and and the Guy’s and St. Thomas’ Charity Prize PhD Program in Biomedical and BLIMP1 in CD4+ regulatory T cells (Tregs) (12), and with STAT3 Translational Science, the Innovative Medicines Institute–funded project Be the Cure (115142-2), and the Department of Health via the National Institute for Health in T follicular helper cells (13). Studies in multiple cell lines Research (NIHR) Comprehensive Biomedical Research Centre award to Guy’s and highlight the ability of IKZF3 to repress through St. Thomas’ National Health Service (NHS) Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. The HDAC and PRC2 recruitment (14–16) as well as by altering views expressed are those of the author(s) and not necessarily those of the NHS, the superstructure (17). NIHR, or the Department of Health. Anti–TNF-a mAb therapy is commonly used in the treatment M.L.R. designed and performed experiments, analyzed the data, and wrote the man- of many inflammatory conditions, including rheumatoid arthritis uscript; V.F. and C.A.R. designed and performed experiments and analyzed data; S.L., A.A., A.M.O., K.J.A.S., and G.A.M.P. performed experiments; J.S. and P.L. (18), inflammatory bowel disease (19), and psoriasis (20). Al- provided plasmids and contributed to technical discussions; L.S.T. supervised the though the mechanisms governing its therapeutic effects are still study and contributed to the designing of experiments, data interpretation, and man- not entirely elucidated, multiple effects on the immune system uscript writing. have been reported, including induction of an anti-inflammatory Address correspondence and reprint requests to Dr. Leonie S. Taams, Centre for + Inflammation Biology and Cancer Immunology, Department of Inflammation Biol- CD4 T cell phenotype (21), modulation of innate immune cell ogy, School of Immunology and Microbial Sciences, King’s College London, 1st function (22, 23), and expansion of Tregs (24), in addition to Floor New Hunt’s House, Guy’s Campus, Great Maze Pond, London SE1 1UL, U.K. blocking TNF-a proinflammatory signaling. We previously dem- E-mail address: [email protected] onstrated that patients with rheumatoid arthritis or ankylosing The online version of this article contains supplemental material. spondylitis treated with anti–TNF-a drugs have increased fre- Abbreviations used in this article: ADA, adalimumab; qPCR, quantitative PCR; Treg, quencies of IL-10+ CD4+ T cells in peripheral blood (10). Fur- regulatory T cell. thermore, CD4+ T cells from the peripheral blood of healthy This article is distributed under the terms of the CC BY 4.0 Unported license. volunteers activated in the presence of anti–TNF-a therapeutics had + Copyright Ó 2020 The Authors increased frequencies of IL-10 cells (10, 25). Gene expression

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901283 2 IKZF3 IS ASSOCIATED WITH IL-10+ CD4+ T CELLS analysis from one of these studies highlighted IKZF3 as a potential (PeproTech) for 24 h at a density of 1 3 106 cells/ml. Viral supernatants regulator of IL-10 expression, at least in Th17 cells (10). were mixed with TransDux MAX (Cambridge Bioscience), added to the In this study we sought to address the hypothesis that IKZF3 is a cells, and cultured. After 3 d, the cells were supplemented with fresh 10% + FCS RPMI 1640 and 20 U/ml recombinant human IL-2 and rested from transcriptional regulator of IL-10 production in CD4 T cells. stimulation for 3 d. These cells were subsequently sorted on live CD3+ GFP+/2 cells (Supplemental Fig. 1D). Cells were rested overnight at a 5 Materials and Methods density of 1 3 10 cells/ml, then stained for IL-10, IL-17A, IFN-g, and IKZF3. Cells and cell culture Plasmids and cloning Peripheral blood was obtained from healthy adult volunteers with written informed consent (Bromley Research Ethics Committee ref- The selected regions of the human IL10 locus (indicated in Table II) were erence 06/Q0705/20). PBMCs were isolated using density gradient amplified by PCR using the BAC RP11-262N9 (Thermo Fisher Scientific) + + centrifugation. CD4 T cells and CD14 monocytes were isolated as a template, and TOPO-cloned into pCR-Blunt II-TOPO (Invitrogen). + by MACS using the manufacturer’s protocol. CD14 monocytes These were then sequenced to confirm 100% conformity to the reference + were isolated using anti-CD14 microbeads to ∼98% purity (Miltenyi sequence. These regions of interest were subcloned into a pGL4.26 vector + Biotec), and CD4 T cells were isolated using negative selection to (Promega). ∼95% purity (Miltenyi Biotec). FLAG-cMAF-pCMV was a gift from P. Lavender (King’s College Cells were cultured in RPMI 1640 (Life Technologies) supplemented London), and HA-IKZF3 was PCR-cloned from a pCMV sport vector with 10% FCS and 1% penicillin–streptomycin and 10 mg/ml L-glutamine purchased from Source BioScience. (culture medium). CD4+ T cell cultures were stimulated with anti-CD3/CD28 mAb stimulation by coating tissue culture plate wells with 1.25 mg/ml a-CD3 Luciferase assay mAb OKT3 (Janssen-Cilag) in PBS for 3 h at 37˚C. Wells were washed with sterile PBS before adding the cells (1 3 106 cells/ml) together with 1 mg/ml HEK293T cells were seeded at a density of 200,000 cells/ml in 96-well Downloaded from anti-CD28 mAb (clone CD8.2; BD Biosciences). For cocultures, 0.5 3 plates. On the next day, each well was transfected with 1 mg of poly- 106 CD14+ peripheral blood monocytes were cultured with 0.5 3 106 ethylenimine (Sigma-Aldrich) mixed with 0.2 mg of experimental pGL4.26, autologous CD4+ T cells in 1 ml of culture medium in the presence of 0.01 mg of control pRL4, and 0.2 mg of -pCSIG or empty 100 ng/ml anti-CD3 mAb (OKT3). HEK293T cells (gifted from the vector. After 18 h of transfection, the cell culture media was replaced and left Stuart Neil laboratory, King’s College London, London, U.K.) were for a further 48 h before harvesting the cells. cultured in DMEM supplemented with 10% FCS, 1% penicillin–strep- Luciferase assays were performed using the Dual-Glo Luciferase Kit (Promega) according to the manufacturer’s instructions, and data were tomycin,and10mg/mlL-glutamine. Where indicated, adalimumab http://www.jimmunol.org/ (ADA; obtained from Guy’s Hospital Pharmacy) was added at 1 mg/ml. collected on a Tecan Spark 10M. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample to control for transfection Flow cytometry efficiency and further normalized to the empty vector control. For intracellular cytokine staining, CD4+ T cells or CD4+ T cell/monocyte Statistical analysis cocultures were stimulated for 3 h in the presence of PMA (50 ng/ml; Sigma-Aldrich), ionomycin (750 ng/ml; Sigma-Aldrich), and GolgiStop Statistical analysis was performed using GraphPad Prism version 8. A (as per the manufacturer’s instructions; BD Biosciences). Cells were Wilcoxon test was used for comparisons between two groups, unless oth- erwise stated. Significant p values are reported as *p , 0.05, **p , 0.01, washed and stained with CD3-PE Cy7 (UCHT1; BioLegend) and LIVE/ , , DEAD efluor 780 (Thermo Fisher Scientific). Cells were then fixed in 2% ***p 0.001, and ****p 0.0001. PFA and permeabilized with 0.5% saponin (Thermo Fisher Scientific). by guest on September 28, 2021 Cells were subsequently stained for the following cytokines: IL-10–Alexa Results Fluor 488 (JES3-9D7; BioLegend), IL-17A–PE (BL168; BioLegend), IFN- a + g–Pacific blue (4S.B3; BioLegend), and TNF–allophycocyanin (MAb11; TNF- blockade maintains IL10 transcription in CD4 T cells BioLegend). We previously observed a transient increase in the frequency of For intranuclear staining of IKZF3, cells were fixed and permeabilized + + with FOXP3 staining buffer (BioLegend) for 15 min at room temperature IL-10 CD4 T cells when PBMCs were stimulated with anti-CD3 before being stained for CD3-PE Cy7, IL-10–Alexa Fluor 488, IL-17A–PE, mAb, which was maintained in the presence of TNF-a blockade IFN-g–Pacific blue, TNF-BV605 (MAb11; BioLegend), and either IKZF3- (10, 25). Because we aimed to use a reductionist CD4+ T cell Alexa Fluor 647 (EPR9342[B]; Abcam) or isotype control (EPR25A; culture in our experiments, we first sought to determine the ki- Abcam) for 30 min. Standard gating strategy for intracellular cytokine netics of IL-10 expression in cultures of anti-CD3/CD28 mAb– staining is shown in Supplemental Fig. 1A–C. stimulated CD4+ T cells rather than PBMC cultures. CD4+ T cells RNA isolation and quantitative PCR were purified and stimulated with plate-bound anti-CD3 and sol- mRNA was isolated using an RNeasy Mini Kit (QIAGEN). cDNA was uble anti-CD28 mAb for 1–3 d with or without the anti–TNF-a Ab transcribed using a High-Capacity cDNA Reverse Transcription Kit ADA before being restimulated with PMA and ionomycin for (Applied Biosystems) according to the manufacturer’s protocol. Real- intracellular cytokine staining (representative gating strategies are time PCR was performed using SensiFAST SYBR Green PCR Master shown in Supplemental Fig. 1). We observed a transient increase Mix (Bioline) with 10 mM primers (Table I). Reactions were performed + + in multiple technical replicates, and results were calculated using the in the frequency of IL-10 cells when CD4 T cells were stimu- D cycle threshold method. lated with anti-CD3/CD28 mAb, which was maintained by TNF-a blockade (Fig. 1A, 1B). To rule out a possible artifact due to the Actinomycin D assay PMA/ionomycin restimulation, we examined the expression of CD4+ T cells were stimulated with anti-CD3/CD28 mAb and cultured in IL10 mRNA levels by qPCR in CD4+ T cells stimulated with anti- the presence or absence of 1 mg/ml ADA for 72 h. After stimulation, the cells CD3/CD28 mAb with or without anti-TNF (Table I). We observed were treated for 2 h with either 1 mg/ml actinomycin D (Cambridge Bio- a similar pattern, namely a transient increase of IL10 expression science) or an equivalent volume of DMSO. Cells were subsequently har- vested for RNA and assayed for gene expression by quantitative PCR (qPCR). upon stimulation, which was maintained in the presence of ADA at day 3 (Fig. 1C). We also observed IL-10 secretion in the cell + Viral transduction of CD4 T cells culture supernatant upon 3 d of anti-CD3/CD28 mAb stimulation The plasmids pCSIG-IKZF3-GFP (lenti-IKZF3) and pCSIG-GFP (lenti- that was significantly increased in the presence of anti–TNF-a EV) were packaged into lentiviral particles by transfecting (Fig. 1D). HEK293T cells with a pCSIG vector, pSPAX2, and pMD2.G. Viral particles IL10 mRNA has been shown previously to be controlled at the were concentrated using PEG-it (Cambridge Bioscience) according to the manufacturer’s instructions. posttranscriptional level (26). To determine whether IL10 mRNA Primary CD4+ T cells were activated with plate-bound anti-CD3 and was stabilized by TNF-a blockade, we performed an actinomycin anti-CD28 mAb (2 mg/ml) with 20 U/ml recombinant human IL-2 D assay on CD4+ T cells stimulated with anti-CD3/CD28 mAb for The Journal of Immunology 3

FIGURE 1. TNF-a blockade maintains the expression of IL-10 in CD4+ T cells, which involves active transcription. (A and B) Primary CD4+ T cells from healthy do- nors were stimulated with anti-CD3/CD28 mAb for 0, 1, 2 or 3 d in the absence (blue bars) or presence (red bars) of 1 mg/ml ADA. Cells were restimulated with PMA and ionomycin and assessed for IL-10 ex- pression. Representative (A) and cumulative (n =7)(B) data showing the frequency of IL-10–expressing cells within CD4+ T cells. (C) IL10 mRNA expression was analyzed by Downloaded from qPCR after 1, 2, or 3 d in culture without restimulation (n = 6). Data in (B) and (C) were analyzed by two-way ANOVA. (D) Quantification of IL-10 in cell culture su- pernatants from CD4+ T cells stimulated as above for 3 d (n=7; Wilcoxon test). (E) After 3 d of culture as above, CD4+ T cells http://www.jimmunol.org/ were treated with either DMSO or 1 mg/ml actinomycin D for 120 min to block tran- scription. mRNA abundance was assessed by qPCR (n = 5; two-way ANOVA with multi- ple comparisons, comparing DMSO and actinomycin D treatment conditions within each group as well as actinomycin D–treated cells between cells stimulated in the absence

or presence of 1 mg/ml ADA). *p , 0.05, by guest on September 28, 2021 **p , 0.01.

3 d. This assay is frequently used to determine the relative stability mRNA IL2RA (Fig. 1E). We did not observe a significant differ- of mRNA species between treatments or cell types (27). The ence between control- and ADA-treated CD4+ T cells. These re- treatment of cells with actinomycin D inhibits mRNA transcrip- sults indicate that the increase in IL10 mRNA is due to active tion. Once blocked, unstable mRNA transcripts are degraded by transcription. cellular machinery over time and not replenished. Comparing + mRNA levels between actinomycin D and vehicle control-treated IKZF3 is enriched in IL-10–producing CD4 T cells cells gives an indication of mRNA stability. IL10 mRNA in acti- Our previous gene expression analysis indicated that IKZF3 was vated CD4+ T cells was sensitive to the addition of actinomycin D upregulated in Th17 cells in response to TNF-a blockade and could and therefore unstable, similar to and unlike the more stable bind at the IL10 locus in these cells (10). To examine whether

Table I. qPCR primer sequences used in this study

Gene Name Forward Primer Sequence Reverse Primer Sequence UBC 59-CGGGATTTGGGTCGCAGTTCTTG-39 59-CGATGGTGTCACTGGGCTCAA-39 GAPDH 59-GTCAGCCGCATCTTCTTTTGC-39 59-AATCCGTTGACTCCGACCTTCC-39 B2M 59-GTATGCCTGCCGTGTGAAC-39 59-AAAGCAAGCAAGCAGAATTTGG-39 IL10 59-GCCTAACATGCTTCGAGATC-39 59-TGATGTCTGGGTCTTGGTTC-39 IKZF3 59-AGCAGGCCAACCAGTGGAAAGA-39 59-TGGGCGTTCACCAGTATGGCT-39 IL2RA 59-ACAAGCTCTGCCACTCGGAAC-39 59-AGCCCTGTATCCCTGGACG-39 MYC 59-TAGTGGAAAACCAGCCTCCC-39 59-GGCAGCAGCTCGAATTTCTT-39 4 IKZF3 IS ASSOCIATED WITH IL-10+ CD4+ T CELLS

IKZF3 was associated with IL-10 production in CD4+ T cells, In our previous work, we observed an increase in IKZF3 ex- we performed a combined intracellular cytokine staining and an pression in Th17 cells following TNF-a blockade using a CD14+ intranuclear stain for IKZF3 to determine the expression of IKZF3 monocyte/CD4+ T cell coculture system. To determine whether within CD4+ T cells expressing IL-10, IL-17A, IFN-g, or TNF-a, the increase in IKZF3 upon TNF-a blockade occurred in the ab- either ex vivo or after 3 d of anti-CD3/CD28 mAb stimulation. sence of monocytes and in all T cell subsets, we compared IKZF3 IKZF3 was expressed at higher levels in IL-10+ CD4+ T cells expression in CD4+ T cells cultured alone versus CD4+ T cells compared with the total CD4+ T cell population and the IL- cocultured with CD14+ monocytes in the absence or presence of 17A+ and TNF-a+ subsets ex vivo (Fig. 2A, 2B). Upon anti-CD3/ ADA (Supplemental Fig. 2). We previously established that IL-10 CD28 mAb stimulation, a significant increase was observed in expression is increased upon TNF blockade in both culture sys- IKZF3 expression in IL-102expressing cells compared with the tems (10, 25). In agreement with our previous results, upon T cell total CD4+ and the TNF-a+ cell populations. However, there was stimulation in the presence of CD14+ monocytes and anti-TNF, no longer a significant difference between IL-10+ and IL-17A+ IKZF3 expression was increased in the total CD4+ T cell pop- CD4+ T cells (Fig. 2C, 2D). Because IL-10 can be expressed by ulationaswellasintheIL-10+ and IL-17A+ subsets (Supplemental multiple cytokine-producing CD4+ T cell subsets (especially after Fig. 2A). In the absence of CD14+ monocytes, IL-10+ CD4+ Tcells stimulation), we compared IKZF3 expression in the IL-17A+, had high expression of IKZF3 in both control- and ADA-treated TNF-a+,andIFN-g+ cells that coproduced IL-10 and those that samples, but TNF-a blockade did not alter IKZF3 expression in did not (Fig. 2E, 2F). In all subsets analyzed, a significantly these cells (Supplemental Fig. 2B, 2C). These data indicate that in higher expression of IKZF3 was observed in IL-10–coproducing CD4+ T cell–only cultures, the anti–TNF-a–mediated increase of

CD4+ T cells compared with cells that did not produce IL-10 IL-10 can occur in the absence of a concomitant increase in IKZF3 Downloaded from (Fig. 2F). expression. http://www.jimmunol.org/

FIGURE 2. IKZF3 is associated with IL- 10–producing CD4+ T cells. (A and B) Pri- mary CD4+ T cells from healthy donors were stimulated with PMA and ionomycin and assessed for frequency of cytokine- by guest on September 28, 2021 producing cells and IKZF3 expression. Representative gMFI of IKZF3 expression shown on the right of each histogram (A) and cumulative data (n = 8) shown (B). (C–F)CD4+ T cells were stimulated with anti- CD3/CD28 mAb for 3 d and subsequently restimulated with PMA and ionomycin and assessed for frequency of cytokine-producing cells and IKZF3 expression. Expression of IKZF3wascalculatedwithintotalpopulations of cytokine-producing cells (C and D)or within IL-10+ or IL-102 subsets within those populations (E and F). Representative gMFI of IKZF3 expression shown on the right of each histogram (C) and cumulative (n=13) (D) data of total cytokine-producing pop- ulations are shown. Representative gMFI of IKZF3 expression shown on the right of each histogram (E) and cumulative (n = 8–11) (F) data for IKZF3 expression within IL-10+ and IL-102 subsets are shown. Data in (B)and(D) were analyzed by ANOVA, data in (F) was analyzed by Wilcoxon test. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 5

IKZF3 degradation by lenalidomide does not alter IL-10 frequencies, a decrease in IL-17A+ frequencies, and a slight de- expression ex vivo but disrupts anti-CD3/CD28 mAb–mediated crease in cell viability (median viability: 88.9–82.75%, control IL-10 production versus lenalidomide, respectively; Supplemental Fig. 3B). Treat- ment of CD4+ T cells with lenalidomide also consistently in- We sought to determine whether IKZF3 is required for IL-10 + expression. We first attempted to deplete IKZF3 from CD4+ creased secretion of IL-2 by CD4 T cells after 3 d of anti-CD3/ T cells using small interfering RNA in primary CD4+ T cells. CD28 mAb stimulation (Supplemental Fig. 3C, n = 5). However, this approach did not work because of the stability of IKZF3 is not sufficient to drive expression of IL10 in CD4+ the IKZF3 protein (as shown by cycloheximide assays; data not T cells at the mRNA or protein level shown) and its upregulation upon anti-CD3/CD28 stimulation We next sought to determine whether IKZF3 was sufficient to drive (required to render the cells transfectable or transducable; data not IL-10 expression in CD4+ T cells. To overexpress IKZF3, we shown). As an alternative approach, we employed the thalidomide activated CD4+ T cells and transduced the cells with an IKZF3- derivative lenalidomide, which has been shown to induce the IRES-GFP lentiviral construct (lenti-IKZF3) or an empty vector proteasomal degradation of IKZF3 (and IKZF1) and is used (lenti-EV) encoding only GFP (Fig. 4A). After transduction, live therapeutically in treating multiple myeloma (28–30). GFP+ cells were sorted for mRNA isolation or rested and stimu- + Treatment of CD4 T cells with lenalidomide overnight led to a lated with PMA/ionomycin for intracellular cytokine staining. dose-dependent decrease in IKZF3 protein levels as shown by Although cells transduced with IKZF3 showed a significant increase + Western blot (Fig. 3A) and flow cytometry (Fig. 3B). CD4 T cells in IKZF3 transcript, IL10 mRNA levels were low and not consis- were then treated with lenalidomide for 24 h in the absence of tently increased by IKZF3 overexpression (Fig. 4B). Also, at the Downloaded from T cell activation followed by intracellular cytokine staining. Al- protein level, IKZF3-transduced cells did not show a consistent though a significant reduction in the levels of IKZF3 was ob- increase in IL-10–producing cells compared with the empty vector + + served, the frequency of IL-10 cells within CD4 T cells was (Fig. 4C, 4D). A considerable proportion of cells was able to pro- + slightly increased (Fig. 3C). Ex vivo treatment of CD4 T cells duce IFN-g or IL-17A, indicating that the transduction protocol had with lenalidomide had no effect on IL-17A and IFN-g expression not affected the capacity of the cells to produce cytokines. Together, or viability (Supplemental Fig. 3A). In contrast, when CD4+

these data indicate that IKZF3 overexpression is not sufficient to http://www.jimmunol.org/ T cells were treated with lenalidomide for 3 d in the presence of drive IL-10 expression in CD4+ Tcells. anti-CD3/CD28 mAb stimulation, a strong reduction in both IKZF3 expression and the frequency of IL-10+ CD4+ T cells was IKZF3 is insufficient to drive the expression of enhancer or observed (Fig. 3D). These data indicate that, whereas IL-10 pro- promoter elements of IL-10 duction in unstimulated CD4+ T cells is not lenalidomide-sensitive, Our previous work showed that IKZF3 is able to bind evolutionarily the anti-CD3/CD28 mA–mediated increase in IL-10–expressing conserved regions at the IL10 locus in Th17 cells. To determine cells is lenalidomide-sensitive and thus, by extrapolation, poten- whether IKZF3 can drive transcription of IL10 via these regions, we tially regulated by IKZF3. Lenalidomide treatment for 72 h also identified 10 putative enhancer sites at the IL10 locus (Fig. 5A), as + + resulted in statistically significant increases in IFN-g and TNF-a defined by accessible chromatin (31), high H3K4me1, and low CpG by guest on September 28, 2021

FIGURE 3. Lenalidomide disrupts the anti-CD3/CD28 mAb–mediated induction of IL-10+ CD4+ T cells but does not affect ex vivo IL-10 production. (A and B) Primary CD4+ T cells from healthy donors were treated with 0.1, 1, or 10 mM lenalidomide overnight and examined for IKZF3 expres- sion by Western blot (A) or flow cytometry (B). (C) CD4+ T cells were treated with 1 mM lenalidomide overnight and then stimulated with PMA and ionomycin and assessed for IKZF3 expression and frequency of IL-10+ CD4+ T cells (n = 13). (D)CD4+ T cells were stimulated with anti-CD3/CD28 mAb for 3 d inthepresenceof1mM lenalidomide and subsequently restimulated with PMA and ion- omycin and assessed for IKZF3 expression and frequency of IL-10+ CD4+ Tcells(n =13). Data in (C)and(D)wereanalyzedby Wilcoxon test. *p , 0.05, ***p , 0.001, ****p , 0.0001. ICCS, intracellular cyto- kine staining. 6 IKZF3 IS ASSOCIATED WITH IL-10+ CD4+ T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. IKZF3 overexpression in CD4+ T cells is not sufficient to drive the expression of IL-10. (A–D) Primary CD4+ T cells from healthy donors by guest on September 28, 2021 were transduced with an IKZF3-IRES-GFP (lenti-IKZF3) or GFP-only (lenti-EV) lentivirus. Seven days posttransduction, cells were sorted on GFP ex- pression. (A) Representative GFP expression. (B) CD4+ T cells transduced with lenti-IKZF3 or lenti-EV were sorted on GFP expression, and mRNA expression of IKZF3 and IL10 was quantified by qPCR (n = 6). (C and D) Cells transduced with lenti-IKZF3 or lenti-EV were sorted on GFP expression, rested overnight, then restimulated with PMA and ionomycin and assessed for frequency of IL-10, IFN-g, or IL-17A–producing cells. Representative (C) and cumulative (n = 6–8) (D) data are shown. Data in (B) and (D) were analyzed by Wilcoxon test. *p , 0.05. methylation [from the BLUEPRINT consortium (32)]. We cloned (37, 38), and translation (39). In the innate immune system, these regions (genomic coordinates of cloned regions defined in IL-10 has been shown to be temporally regulated through Table II) and a 1.5-kb region of the IL10 promoter upstream of a regulation of transcript stability, such as through the p38/TTP Firefly luciferase open reading frame (pGL4). These vectors were axis (26, 37). then cotransfected with a control Renilla luciferase vector together We found that IL10 mRNA was maintained at higher levels with the plasmids lenti-IKZF3 (Fig. 5B)orlenti-MAF(Supplemental in the presence of anti–TNF-a mAb. This increase in IL-10– Fig. 4B, 4C), a known regulator of IL10 (33). To validate that our producing CD4+ T cells does not appear to be attributable to constructs were functional, we stained HEK293T cells transfected changes in cell survival or increased cell proliferation after TNF with lenti-IKZF3, lenti-MAF, or lenti-EV for IKZF3 or cMAF by blockade, as we showed recently (40). flow cytometry (Supplemental Fig. 4A, 4B) and observed at least a We also show that IL10 mRNA in anti-CD3/CD28 mAb–acti- 10-fold increase in expression in the relevant conditions. vated primary human CD4+ T cells is an unstable transcript. This The luciferase experiments showed that IKZF3 has limited ca- may represent a mechanism by which CD4+ T cells, which can pacity to drive transcription of the IL10 constructs (Fig. 5B). An transiently produce IL-10 on stimulation, eventually prevent its induction of reporter gene expression in response to IKZF3 trans- expression via negative feedback, similar to macrophages (41). fection was only seen for enhancer 10, whereas reporter gene ex- To understand what drives the transcriptional regulation of IL10,we pression for most other constructs decreased in a dose-dependent focused on IKZF3. Our previous work with a CD4+ T cell:CD14+ manner upon increasing amounts of IKZF3. In contrast, transfection monocyte coculture system showed increased IKZF3 expression upon with cMAF, a known transcriptional regulator of IL10 (34, 35), TNF-a blockade in Th17 cells, which correlated with increased IL-10 significantly upregulated multiple enhancers compared with the expression. In our current study using a T cell reductionist system, we empty vector (Supplemental Fig. 4C). saw no change in IKZF3 expression in cytokine-producing CD4+ T cell subsets upon TNF-a blockade, although we did observe an Discussion increase in IL-10+ CD4+ T cell frequency. We did observe a Regulation of IL-10 expression is a multilayered process at generally higher level of IKZF3 expression in IL-10–producing the levels of transcription (33, 36), posttranscriptional stability CD4+ T cells ex vivo and after CD3/CD28 stimulation. An The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. IKZF3 is insufficient to drive transcriptional activity via the IL10 promoter or local enhancers. (A) Chromatin immunoprecipitation se- quencing (from the BLUEPRINT consortium) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) data (from Ref. 31) from ex vivo human CD4+ T cells for the MAPKAPK2:IL10:IL19 locus was used to identify regions of putative enhancers (yellow vertical bars numbered 1–10) as well as 1.50-kb promoter region of IL10 (vertical green bar labeled Promo). (B) These promoter and enhancer regions were then cloned upstream of a by guest on September 28, 2021 luciferase reading frame and transfected along with an expression plasmid encoding IKZF3 (lenti-IKZF3) or empty vector control (lenti-EV) into HEK293T cells and assessed 48 h later for luciferase activity (n = 4). Data were analyzed by two-way ANOVA. *p , 0.05, ***p , 0.001, ****p , 0.0001. association between IKZF3 and IL-10–producing CD4+ Tcells inhibitor drug etanercept, in the absence of monocytes, showed an has been noted by other studies in human Th17 clones (42) as increased expression of IL10 upon TNF-a blockade that was not well as mouse Th1 (5) and Th17 cells (8). This association may accompanied by changes in IKZF3 expression (43). indicate common transcriptional regulators under steady-state IKZF3 (and IKZF1) has been previously described as a neg- conditions but not upon TNF-a blockade. In our study, IKZF3 ative regulator of Il2 expression in CD4+ T cells (6, 29), and our was highly expressed in IL-17A+ IL-10+ CD4+ Tcells.The findings that IL-2 secretion is increased upon lenalidomide treat- expression of IKZF3 and IL-10 in nonpathogenic Th17 cells with ment support that observation. The expression of IL-10 and a a reduced capacity to drive experimental autoimmune encepha- reduced capacity to produce IL-2 is a known hallmark of Tregs. lomyelitis has been previously noted (6, 8). Therefore, high IKZF3 expression in the IL-10+ population might Similar to our findings with CD4+ T cells cultured without be indicative of a high proportion of Tregs. However, mass monocytes, another study found that memory CD4+ T cells acti- cytometry data from our laboratory did not reveal a higher ex- vated by anti-CD3/CD28 mAb in the presence of the TNF-a pression of IKZF3 in CD25high CD127low Tregs compared with

Table II. UCSC Genome Browser hg19 coordinates for regions used in cloning putative IL10 enhancer and promoter regions

Region Region Coordinates (hg19) Name Chr1 Forward Primer Sequence Reverse Primer Sequence 1 206907901–206908425 59-TTGCTAGCTTTTGCCAAATGCAGAATCA-39 59-AACTCGAGAAGGGGACAAGCAGGTCTCT-39 2 206910301–206910900 59-AACTCGAGAAGGGGACAAGCAGGTCTCT-39 59-TTGCTAGCAAAAGTGCTGGGCTTACAGG-39 3 206912926–206913525 59-TTGCTAGCAAAAGTGCTGGGCTTACAGG-39 59-AACTCGAGGGGAGCCACACTGTTCTGAC-39 4 206930851–206931750 59-AACTCGAGGGGAGCCACACTGTTCTGAC-39 59-TTGCTAGCTTTCCAGGACCCTGATATGC-39 5 206939626–206940225 59-TTGCTAGCTTTCCAGGACCCTGATATGC-39 59-AACTCGAGTGCTTTCATCCATCCAACAG-39 6 206942701–206943150 59-AACTCGAGTGCTTTCATCCATCCAACAG-39 59-TTGCTAGCTCACTGGTCACAGGACATCAC-39 Promoter 206945773–206947172 59-TTGCTAGCTCACTGGTCACAGGACATCAC-39 59-AACTCGAGATTTAGTCGGGTGTGGTGGT-39 7 206957626–206958150 59-AACTCGAGATTTAGTCGGGTGTGGTGGT-39 59-TTGCTAGCCCTGAGTAGTTCCCAGCTTGA-39 8 206964826–206965275 59-TTGCTAGCCCTGAGTAGTTCCCAGCTTGA-39 59-AACTCGAGTGAGCTAACAGGGCTTCTGG-39 9 206976226–206977275 59-AACTCGAGTGAGCTAACAGGGCTTCTGG-39 59-TTGCTAGCAGGTGCGAAGGGCAGAT-39 10 206990176–206990550 59-TTGCTAGCAGGTGCGAAGGGCAGAT-39 59-AACTCGAGAGCAGGTGAAGAATGCCTTT-39 8 IKZF3 IS ASSOCIATED WITH IL-10+ CD4+ T CELLS

CD25low CD127high effector T cells ex vivo (data not shown). drive gene expression in mouse T follicular helper cell–like cells, Furthermore, upon TNF-a blockade we did not observe an in- and it is therefore possible that this isoform could drive tran- crease in FOXP3+ Tregs (10). scription in human CD4+ T cells (13). However, we cannot rule Studies have implicated IL-2 signaling to be required for IL-10 out that other IKZF3 isoforms may differentially affect IL10 expression by multiple Th subsets in vitro (L. Gabrysova´, E.H. expression. Mann, L. Bradley, J.I. MacRae, C. Whicher, C.M. Hawrylowicz, In summary, this study shows that IKZF3 expression is asso- D. Anastasiou, and A. O’Garra, manuscript posted on bioRxiv). ciated with IL-10+ CD4+ T cells at the protein level and that Therefore, one could expect that blocking IKZF3, which is an IL2 pharmacological inhibition of IKZF3 disrupts the ability of CD4+ transcriptional repressor, would lead to an increase in IL-10 pro- T cells to produce IL-10. However, the expression of IKZF3 is duction and frequency. However, we instead observed a significant not sufficient to drive IL-10 protein or mRNA expression. We reduction in the frequency of IL-10+ CD4+ T cells in the presence also note that, whereas TNF-a blockade does lead to increased of lenalidomide. IL10 mRNA expression, this is not necessarily attributable to From our data, lenalidomide also seems to have effects on the differential expression of IKZF3. Further work is required to es- frequency of IL-17A-, TNF-a–, and IFN-g–producing cells. The tablish the transcription factors modified by TNF-a blockade that reduction in IL-17A+ CD4+ T cells could be due to the increase of lead to increased IL10 expression and whether such transcriptional IL-2 in the cell culture supernatants, which has been shown to regulation occurs in patients treated with TNF-a inhibitors. inhibit the differentiation of Th17 cells (6, 44, 45). IL-2 has also + been shown to increase the expression of IFN-g in human CD4 Acknowledgments

+ Downloaded from T cells (46, 47) and TNF-a expression in mouse CD8 T cells We thank Dr. Esperanza Perucha and Prof. Stuart Neil for helpful discus- (48). It should be noted, however, that expression of IFN-g and sions and reagents and Celine Trouillet for cell sorting. TNF-a can be suppressed by IL-10 (49). Therefore, the decrease in IL-10 expression accompanying lenalidomide treatment could Disclosures boost the induction of IFN-g–and TNF-a–producing cells. The authors have no financial conflicts of interest. It should be considered that the effect of lenalidomide on IL-10 + production in CD4 T cells may be due to off target effects. http://www.jimmunol.org/ Lenalidomide has been shown to downregulate several , References including transcription factors (50–52). Therefore, the reduction in 1. Roers, A., L. Siewe, E. Strittmatter, M. Deckert, D. Schlu¨ter, W. Stenzel, A. D. Gruber, T. Krieg, K. Rajewsky, and W. Mu¨ller. 2004. 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