-Associated IL2RA Variation Lowers IL-2 Signaling and Contributes to Diminished CD4+CD25+ Regulatory Function This information is current as of September 28, 2021. Garima Garg, Jennifer R. Tyler, Jennie H. M. Yang, Antony J. Cutler, Kate Downes, Marcin Pekalski, Gwynneth L. Bell, Sarah Nutland, Mark Peakman, John A. Todd, Linda S. Wicker and Timothy I. M. Tree

J Immunol published online 28 March 2012 Downloaded from http://www.jimmunol.org/content/early/2012/03/28/jimmun ol.1100272 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/03/28/jimmunol.110027 Material 2.DC1

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 28, 2012, doi:10.4049/jimmunol.1100272 The Journal of Immunology

Type 1 Diabetes-Associated IL2RA Variation Lowers IL-2 Signaling and Contributes to Diminished CD4+CD25+ Function

Garima Garg,*,†,1 Jennifer R. Tyler,*,1 Jennie H. M. Yang,*,1 Antony J. Cutler,‡ Kate Downes,‡ Marcin Pekalski,‡ Gwynneth L. Bell,‡ Sarah Nutland,‡ Mark Peakman,*,† John A. Todd,‡ Linda S. Wicker,‡ and Timothy I. M. Tree*,†

Numerous reports have demonstrated that CD4+CD25+ regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including type 1 diabetes, are deficient in their ability to control autologous proinflammatory responses when compared with nondiseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the during disease development. Polymorphisms in associated with Treg function, such as Downloaded from IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in , a link between IL2RA polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with type 1 diabetes on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease- associated IL2RA haplotype correlates with diminished IL-2 responsiveness in Ag-experienced CD4+ T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FOXP3 expression by Tregs and a reduction in their ability to http://www.jimmunol.org/ suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene affect immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases. The Journal of Immunology, 2012, 188: 000–000.

ype 1 diabetes (T1D) is characterized by autoimmune a growing body of evidence to suggest that in T1D, this patho- destruction of pancreatic b cells, a process in which au- logical autoimmunity is the direct result of a failure of immune toreactive T cells play a pivotal role (1–3). There is now regulation (4). This includes the defective function of various

T by guest on September 28, 2021 populations of regulatory T cells (Tregs), especially those char- acterized as CD4+CD25hiFOXP3+. In support of this, we and *Department of Immunobiology, School of Medicine, King’s College London, Lon- † others have demonstrated that the suppression of autologous re- don SE1 9RT, United Kingdom; National Institutes of Health Research Biomedi- + hi cal Research Centre at Guy’s and St. Thomas’ National Health Service, Foundation sponder T cells by CD4 CD25 Tregs in individuals with newly Trust and King’s College London, London SE1 7EH, United Kingdom; and diagnosed T1D is reduced significantly compared with that ob- ‡ Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation served in age-matched control subjects (5–8). Importantly, we also Laboratory, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 0XY, United Kingdom demonstrated in an independent cohort of patients that defective 1G.G., J.R.T., and J.H.M.Y. contributed equally to this study. suppression is not only present close to diagnosis, but also in . Received for publication January 28, 2011. Accepted for publication February 14, individuals who have had T1D for 20 y, suggesting that the 2012. functional defect represents a phenotype that is stable over time This work was supported by Juvenile Diabetes Research Foundation U.K. Centre for and most likely under genetic control (9). Diabetes Genes, Autoimmunity, and Prevention Grant 4-2007-1003, the Juvenile A candidate gene study identified an association between T1D Diabetes Research Foundation International, Wellcome Trust Grant WT061858, the National Institute for Health Research Cambridge Biomedical Research Centre, the and the IL-2RA gene (encoding the IL-2 a-chain, CD25) National Institute for Health Research Biomedical Research Centre at Guy’s and St. (10, 11). There are three protective IL2RA haplotypes, one of Thomas’ National Health Service Foundation Trust and King’s College London, and which is marked by the single nucleotide polymorphism (SNP) the Medical Research Council Cusrow Wadia Fund. The research leading to these results received funding from European Union’s Seventh Framework Programme rs12722495, where the protective allele confers a relative risk for Grant FP7/2007-2013 under Grant Agreement 241447 (Natural Immunomodulators T1D of 0.65. Recently, the disease-associated IL2RA rs12722495 as Novel Immunotherapies for Type 1 Diabetes). J.R.T. is the recipient of a Diabetes haplotype was correlated with several distinct cellular immuno- U.K. Ph.D. studentship. The Cambridge Institute for Medical Research is the recip- ient of Wellcome Trust Strategic Award 079895. phenotypes, most notably the higher expression of CD25 on + Address correspondence and reprint requests to Dr. Timothy Tree, Department of memory CD4 T cells and higher levels of IL-2 secretion from Immunobiology, King’s College London, School of Medicine, 2nd Floor, New Guy’s these memory T cells (12). IL-2RA is part of the high-affinity IL-2 House, Guy’s Hospital, London SE1 9RT, United Kingdom. E-mail address: timothy. receptor complex and is constitutively expressed at high levels on [email protected] both naturally occurring and peripherally induced FOXP3+ Tregs The online version of this article contains supplemental material. (13, 14). Numerous lines of evidence from both mice and humans Abbreviations used in this article: aTreg, activated regulatory T cell; iTreg, induced regulatory T cell; MFI, median fluorescence intensity; mTconv, conventional mem- have demonstrated that IL-2 plays a key role in both the genera- + ory T cell; mTreg, memory regulatory T cell; nTconv, conventional ; tion and function of FOXP3 Tregs (15–20). Studies in vitro have rTreg, resting regulatory T cell; SNP, single nucleotide polymorphism; Tconv, con- demonstrated that activation of CD4+CD25+ T cell suppressor ventional T cell; T1D, type 1 diabetes; Treg, regulatory T cell. function requires IL-2 (21) and in vivo that IL-2 is required for + + Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 peripheral survival and expansion of CD4 CD25 Tregs (reviewed

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100272 2 IL2RA HAPLOTYPE AFFECTS CD4+CD25+ TREG FUNCTION in Ref. 13). Furthermore, signaling via common g-chain cyto- Flow cytometric analysis for pSTAT5a kines, of which IL-2 is a key member, is required for the main- Analysis of pSTAT5a in PBMCs was performed using BD Phosflow tained expression of FOXP3 by Tregs, which is essential for their reagents (BD Biosciences) according to the manufacturer’s instructions. suppressive function (19, 22). Briefly, 1 3 106 PBMCs were incubated with various concentrations of These findings invoke the hypothesis that gene polymorphisms IL-2 for 10 min at 37˚C, fixed with Phosflow buffer I, permabilized with in the IL-2/IL-2RA pathway exert their influence on T1D risk via Perm Buffer III, stained with anti–CD4-FITC, anti–CD25-PE, anti–CD127- + PerCP-Cy5.5, anti–CD45RA-eFluor 450, and anti–STAT5a-pY694-Alexa effects on the number or functional ability of FOXP3 Tregs. In Fluor 647 and analyzed in a BD FACSCanto II (BD Biosciences). Be- support of this, recent studies show that Tregs from individuals cause of a technical failure in one sample, only nine pairs of PBMC samples with T1D are more prone to and more easily lose ex- were analyzed for expression of pSTAT5a. For simultaneous detection of pression of FOXP3 and that these phenotypes may be linked to pSTAT5a and FOXP3, 500 ml fresh blood was incubated with 500 mlX- Vivo media containing various concentrations of IL-2 for 10 min at 37˚C, a relative defect in signaling via the IL-2 pathway (7, 23, 24). To fixed with warm BD Lyse/Fix buffer (BD Biosciences), and permeabilized date, however, direct evidence linking polymorphisms in IL2RA with 100% methanol for 20 min on ice. After extensive washing with PBS with altered Treg function is lacking. To address this knowledge containing 0.2% BSA, cells were stained with anti–CD4-Alexa Fluor 700, gap, we examined whether the T1D susceptibility allele defined by anti–CD25-allophycocyanin, anti–CD45RA-Pacific Blue, anti–FOXP3-PE, the IL2RA SNP rs12722495 is associated with a reduction in the and anti–STAT5a-pY694-Alexa Fluor 488 and analyzed using a BD For- tessa (BD Biosciences). Results are expressed as the MFI of pSTAT5a functional capacity of Tregs. To avoid any potential confounding staining for all cells within a particular T cell subset. effects of disease on Treg phenotype, we conducted these studies in individuals without disease. Our studies show that the T1D- Isolation and analysis of cell populations for functional studies susceptibility IL2RA haplotype identified by rs12722495 is asso- PBMCs were stained with anti–CD4-eFluor 450, anti–CD25-PE, anti– Downloaded from ciated with decreased signaling via the IL-2 pathway in both CD127-PerCP-Cy5.5, anti–CD45RA-Alexa Fluor 700, and anti–CD14- memory T cells and Tregs and that this is linked to diminished allophycocyanin-Cy7 and control populations stained with anti–CD4- Treg function. eFluor 450, IgG1-PE, IgG1-PerCP-Cy5.5, anti–CD45RA-Alexa Fluor 700, and anti–CD14-allophycocyanin-Cy7. were identified based on forward and side scatter parameters and populations isolated for Materials and Methods functional analysis using a BD FACSAria II flow cytometer and FACSDiva

software (BD Biosciences). Flow cytometry data were analyzed using http://www.jimmunol.org/ Subjects and study design FlowJo software (Tree Star, Ashland, OR). Individuals homozygous for the T1D protective rs12722495 IL2RA haplotype and the fully susceptible IL2RA haplotype (denoted as P1P1 Flow cytometric analysis for FOXP3 and Helios and SS, respectively) were recruited for the present study. The levels of FOXP3 and Helios staining was performed on cells immediately postsorting + CD25 expression on CD4 T cell subsets have previously been studied in and after 48 h culture with various concentrations of IL-2 using the Bio- these individuals, and pairs of P1P1 and SS individuals were preselected Legend FOXP3 Fix/Perm buffer set according to the manufacturer’s who showed typical haplotype-specific patterns of CD25 expression on instructions. For analysis of FOXP3 and Helios in cultured cells, 104 sorted + the conventional memory CD4 T cell (Tconv) subset (i.e., a relatively Tregs were incubated in complete media supplemented with IL-2 for 48 h higher level of CD25 expression in P1P1 individuals compared with SS). prior to analysis. For PBMC isolation, blood samples were collected in vacutainers con- by guest on September 28, 2021 taining sodium heparin anti-coagulant, diluted 1:1 with RPMI 1640 sup- In vitro coculture suppression assays plemented with 100 mg/ml penicillin/streptomycin (Invitrogen, Paisley, U.K.) and stored overnight under constant rotation. The following day, Suppression assays were performed by culturing memory or naive Tconv 3 PBMCs were isolated from whole blood by density gradient centrifu- populations (2.5 3 10 /well) in the presence or absence of either autologous gation (Lymphoprep; Axis-Shield, Oslo, Norway). For whole blood Tregs or a third party Treg cell line at the ratios indicated. Cells were activated staining, blood was collected into vacutainers containing sodium heparin by the addition of Dynabeads Human T-Activator anti-CD3/anti-CD28 anti-coagulant and used on the day of collection. Because of the day- beads (Invitrogen) at a bead/conventional cell ratio of 1:1. All conditions to-day variation inherent in intracellular staining protocols, it was not were conducted in triplicate. The third party Treg cell line was generated by possible to normalize pSTAT5a and CD25 median fluorescence intensity expanding FACS sorted Tregs from a single donor for 14 d in 600 U/ml IL-2. (MFI) values through time as we were able to do previously using a cell Expanded Tregs were cryopreserved and a single aliquot was thawed for use surface staining protocol (12). Therefore, all analyses were performed in with each pair of samples analyzed. After 5 d culture, 100 ml supernatant was a paired manner with one P1P1 and one SS individual analyzed on a removed and stored at 280˚C for later analysis. Proliferation was 3 single day. Ethical approval for this study was granted by the local assessed by the addition of 0.5 mCi/well [ H]thymidine (PerkinElmer, Wal- Ethics Committee and informed consent was obtained. All experiments tham, MA) for the final 18 h coculture. The percentage of suppression was were performed in a blinded manner without knowledge of genotype of calculated using the following formula: % suppression = 100 2 [(cpm in the the individual. presence of Tregs 4 cpm in the absence of Tregs) 3 100]. mAbs and reagents Statistics The following Abs were used in these studies as indicated: PE-conjugated The normality of all datasets was tested using the D’Agostino–Pearson monoclonal anti-CD25 (clones M-A251 and 2A3), FITC-conjugated anti- omnibus normality test. Where data did not significantly deviate from the CD4 (clone SK3), Alexa Fluor 647-labeled anti-STAT5a (pY694) and normal distribution, either an independent or paired Student t test was used Alexa Fluor 488-labeled anti-STAT5a (pY694) were obtained from BD to test for significance as indicated. Where one or more datasets were Biosciences (Oxford, U.K.); eFluor 450-labeled anti-CD4 (clone SK3), found to significantly deviate from the normal distribution, statistical anti-CD45RA (clone HI100), and PerCP-Cy5.5-conjugated anti-CD127 significance was determined using Wilcoxon matched-pairs signed (clone eBioRDR5) were obtained from eBioscience (Hatfield, U.K.); and test for paired data. All statistical analyses were performed using Graph- Alexa Fluor 700-labeled anti-CD45RA (clone HI100), allophycocyanin- Pad Prism (GraphPad Software, La Jolla, CA). Cy7-conjugated anti-CD14 (clone HCD14), FITC-conjugated anti-Helios (clone 22F6), Alexa Fluor 647-labeled anti-FOXP3 (clone 259D), Alexa Results Fluor 700-labeled anti-CD4 (clone RPA-T4), Pacific Blue-conjugated anti- CD45RA (clone H100), and PE-conjugated anti-FOXP3 (clone 259D) were We have previously reported that the IL2RA rs12722495 pro- obtained from BioLegend (Cambridge, U.K.). Ab concentrations used were tective haplotype is associated with significantly higher levels based on manufacturers’ recommendations and optimization studies. of expression of CD25 on conventional memory CD4+ Tcells Complete media for functional studies was X-Vivo 15 media (Lonza, (mTconv) and we observed a similar trend for FOXP3+ Tregs Wokingham, U.K.) supplemented with 5% human pooled AB+ sera (PAA Laboratories, Lutterworth, U.K.) and 100 mg/ml penicillin/streptomycin (12). Therefore, we first sought to investigate whether increased (Invitrogen). Proleukin (Chiron, Emeryville, CA) was used as a source levels of CD25 expression from individuals with the P1P1 IL2RA of human rIL-2. haplotype resulted in altered responsiveness to IL-2 signaling, The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 1. Example of the gating used for the identification of T cell populations for STAT5a phosphorylation studies using PBMCs. (A) Lymphocytes identified by their forward and side scatter properties were gated for CD4+ expression and then examined for expression of CD25. Tregs were identified using two different gating strategies: 1) based on a high level of CD25 staining and reduced CD4 staining (CD4loCD25+ Tregs), and 2) gating on the top 1% of CD25-staining CD4+ cells (CD25hi Tregs). Tconv were identified by low/intermediate levels of CD25 staining. (B–D) All populations were analyzed for expression of CD45RA to delineate populations of CD45RA+ and CD45RA2 Tconv and Tregs. by guest on September 28, 2021 measured via phosphorylation of STAT5a in all T cell popula- using this method and via CD25+CD127lo expression was highly tions after brief in vitro exposure to IL-2. Because fixation pre- correlated; R2 = 0.87, p , 0.0001; Supplemental Fig. 1). Addi- cluded the use of CD127 as a surface marker of Tregs and FOXP3 tionally, a more stringent definition was applied to identify Tregs costaining was found to be unreproducible using the pSTAT5a expressing very high levels of CD25 (CD25hi Tregs) by gating on staining protocol for PBMC samples, Tregs were initially identi- the top 1% of CD25-staining CD4+ T cells as previously described fied based on a high level of CD25 staining and reduced CD4 by other investigators (23, 25) (gating on an identical population in staining as previously described by other investigators and as il- unfixed cells confirmed that .98% of these cells were CD127lo/2 lustrated in Fig. 1A (the frequency of CD4loCD25+ Tregs defined in all individuals examined). Tconv were subdivided based on ex-

FIGURE 2. Relationship between IL2RA haplotype and CD25 expression on CD4+ Tconv and Treg populations from PBMCs. PBMCs from donors with the protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS) were isolated and analyzed as matched pairs by flow cytometry as shown in Fig. 1. Each symbol type represents a pair of individuals analyzed on the same day and are joined by a horizontal line in paired analysis. MFI is shown of CD25 staining on (A) mTconv, (B) CD4loCD25+ Tregs, (C) CD4loCD25+CD45RA2 Tregs, and (D) CD4loCD25+CD45RA+ Tregs. Statistical significance was determined using a two-tailed paired Student t test. 4 IL2RA HAPLOTYPE AFFECTS CD4+CD25+ TREG FUNCTION

FIGURE 3. Example of pSTAT5a staining in Tconv and Treg populations from PBMCs. PBMCs were incubated with various concentrations of IL-2 for 10 min, fixed, and stained for CD4, CD25, CD45RA, and pSTAT5a (Y694). (A and B) Representative plot of CD4+ cells from one individual incubated with (A) 0 U/ml and (B) 10 U/ml IL-2. (C) Representative example of a dose response curve in Tconv and Treg populations from one individual: 4, nTconv; :, mTconv; n, CD4loCD25+CD45RA+ Tregs; d, CD4loCD25+CD45RA2 Tregs; N, represent CD25hi Tregs. pression of CD45RA to delineate memory (CD45RA2,mTconv) the gender and age bands of subjects and the symbols used to denote and naive (CD45RA+, nTconv) cells (Fig. 1B). Similarly, Tregs the results are shown in Supplemental Table I. Downloaded from were subdivided into CD45RA2 Tregs and CD45RA+ (Fig. 1C). hi . 2 Relationship between IL2RA haplotype and expression of Because CD25 Tregs were 90% CD45RA (Fig. 1D; i.e., con- + sisted mainly of Ag-experienced Tregs), this population was not CD25 on CD4 Tconv and Treg populations subdivided for analysis. In all data presented, a pair of subjects dif- Analysis of isolated PBMCs indicated that individuals with the fering at the rs12722495 SNP has been studied in a discrete, simul- P1P1 IL2RA haplotype express significantly more CD25 on mTconv taneous experiment; each pair is denoted by a distinct symbol that is (p = 0.0002) and Treg (p = 0.01) compared with individuals with http://www.jimmunol.org/ consistent throughout all graphs in the results section. Details of the SS haplotype (Fig. 2A, 2B). The higher levels of CD25 were by guest on September 28, 2021

FIGURE 4. Relationship between IL2RA haplotype and STAT5a phosphorylation in Tconv and Treg populations from PBMCs. PBMCs from donors with the protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS) were isolated and analyzed as described in Fig. 3. Populations of conventional and regulatory cells were defined as described in Fig. 1. The percentage of cells positive for pSTAT5a in nTconv (A and B), mTconv (C and D), CD4loCD25+ Tregs (E), CD4loCD25+CD45RA+ Tregs (F), CD4loCD25+CD45RA2 Tregs (G), and CD4+CD25hi Tregs (H) following exposure to IL-2 as indicated. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line. Results are expressed as the MFI of pSTAT5a staining for all cells within a given population following exposure to IL-2 as indicated. Statistical significance was determined using a two-tailed paired Student t test. The Journal of Immunology 5 observed both in the CD45RA+ (p = 0.005) and CD45RA2 CD4lo To confirm and extend these findings, we developed a whole CD25+ Tregs (p = 0.008) (Fig. 2C, 2D). We observed no difference blood assay incorporating additional bona fide markers of Tregs, in the frequency of mTconv between the P1P1 and SS individuals that is, the FOXP3, to examine IL-2 respon- (mean, 51.5 and 42.7%, respectively), the proportion of CD4+ siveness (Supplemental Fig. 2). In addition to providing a more T cells classified as Tregs (mean, 4.28 and 4.38%, respectively), or definitive marker for Tregs, examination of the expression level of the percentage of CD45RA2 Tregs (mean, 69.2 and 65.2%, re- FOXP3 combined with expression of CD45RA also allowed the spectively). These observations are all in keeping with our previous delineation of three different populations of FOXP3+ T cells as report (12), even though CD25 levels were quantified using an in- described by Mayara and colleagues (26). As an example of the tracellular staining protocol in the present study. gating strategy used to identify FOXP3+ Tregs, the division of this population into resting Tregs (rTregs, FOXP3+CD45RA+), mem- mTconv and Tregs from individuals with the protective P1P1 ory Tregs (mTregs, FOXP3+CD45RA2), and activated Tregs IL2RA haplotype show increased sensitivity to IL-2 (aTregs, FOXP3hiCD45RA2) and the relative levels of CD25 Sensitivity to IL-2 was assessed by measuring phosphorylation of expression on these populations are shown in Fig. 5A–C. Again, STAT5a in all T cell populations after brief in vitro exposure to IL-2 sensitivity to IL-2 in this assay was related to the relative ex- (Fig. 3A, 3B). As might be predicted from their CD25 levels, pression levels of CD25 within each T cell population, especially sensitivity to IL-2 in this assay was lowest for nTconv, with at very low concentrations of IL-2 (Fig. 5D, Supplemental Fig. 2). mTconv, CD4loCD25+CD45RA+ Treg, CD4loCD25+CD45RA2 This assay was then deployed on a fresh cohort of 13 pairs Treg, and CD25hi Treg showing successively higher sensitivities, of individuals with the P1P1 or SS IL2RA haplotype consisting of especially at very low concentrations of IL-2 (Fig. 3C). 6 of the original pairs studied above and 7 new pairs. Consis- Downloaded from mTconv from individuals with the P1P1 IL2RA haplotype were tent with the results in isolated PBMCs, staining in whole blood more responsive to IL-2 at both 10 U/ml (p = 0.01) and 100 U/ml demonstrated that P1P1 individuals express significantly more (p = 0.007) compared with individuals with the SS haplotype; CD25 on mTconv (p = 0.001) and Ag-experienced FOXP3+ however, no such difference was observed in nTconv (Fig. 4A–D). mTregs (p = 0.045) and FOXP3hi aTregs (p = 0.0004) compared Additionally, we observed that at low concentrations of IL-2, with individuals with the SS haplotype; however, no such dif- lo + CD4 CD25 Tregs from P1P1 individuals were more responsive ference was observed in rTregs (Fig. 6A–D). Similarly, results http://www.jimmunol.org/ to IL-2 compared to individuals with the SS haplotype (Fig. 4E; from the whole blood assay again demonstrated that mTconv and 0.1 U/ml, p = 0.01). This difference was observed in both the FOXP3hi aTregs from individuals with the P1P1 IL2RA haplotype CD45RA+ and CD45RA2 populations (Fig. 4F, 4G; p = 0.005 and were more responsive to IL-2 at nonsaturating doses (mTconv, p = 0.03, respectively). Interestingly, these significant differences 4 U/ml, p = 0.04; aTregs, 0.3 U/ml, p = 0.02) compared with were only observed at the lowest concentration of IL-2 but not at individuals with the SS haplotype (Fig. 6E and 6H, respectively). higher concentrations of 1, 10, or 100 U/ml (data not shown). However, no significant difference was observed in these pop- Similarly, we observed that CD25hi Tregs from P1P1 individuals ulations at higher concentrations of IL-2 (mTconv, 10 or 100 U/ were more responsive to IL-2 compared to individuals with the SS ml; aTregs, 1–100 U/ml; data not shown). The lack of a significant haplotype (Fig. 4H). Again, this was true at low concentrations of difference between the P1P1 and SS donors in rTregs and mTegs by guest on September 28, 2021 IL-2 (0.1 U/ml, p = 0.02) but not at higher concentrations (data not (Fig. 6F and 6G, respectively) reflects the fact that in three pairs shown). the SS cells were more responsive to IL-2 than were the P1P1

FIGURE 5. Example of FOXP3-pSTAT5a staining in Tconv and Treg populations from whole blood. Whole blood was incubated with various concen- trations of IL-2 for 10 min, fixed, permeabilized, and stained for CD4, CD25, CD45RA, FOXP3, and pSTAT5a (Y694). (A and B) Example of the gating strategy to identify Tconv and FOXP3+ Treg subsets. Lymphocytes identified by their forward and side scatter properties were gated for CD4+ expression and then examined for expression of CD25 and FOXP3. FOXP3+ Tregs were identified by coexpression of CD25 and FOXP3 (A) and then divided into rTregs (FOXP3+CD45RA+), mTregs (FOXP3+CD45RA2), and aTregs (FOXP3hiCD45RA2)(B). (C) Analysis of CD25 expression levels from whole blood staining in mTconv (dotted line), FOXP3+ rTregs (dashed line), FOXP3+ mTregs (solid line), and FOXP3hi aTregs (filled histogram). (D) Representative example of a dose response curve in mTconv and FOXP3+ Treg populations from one individual: d,mTconv;n, FOXP3+ rTregs; half-open/half-closed square, FOXP3+ mTregs; N, FOXP3hi aTregs. 6 IL2RA HAPLOTYPE AFFECTS CD4+CD25+ TREG FUNCTION Downloaded from http://www.jimmunol.org/

FIGURE 6. Relationship between IL2RA haplotype, CD25 expression, and STAT5a phosphorylation in response to IL-2 in T cell subsets identified by FOXP3-pSTAT5a staining in whole blood. Whole blood from donors with the protective rs12722495 IL2RA haplotype (P1P1) and donors with the sus- ceptible IL2RA haplotype (SS) was stimulated and analyzed as described in Fig. 5. (A–D) Relationship between IL2RA haplotype and CD25 expression. MFI by guest on September 28, 2021 of CD25 staining on (A) mTconv, (B) FOXP3+ rTregs, (C) FOXP3+ mTregs, and (D) FOXP3hi aTregs. (E–H) Relationship between IL2RA haplotype and pSTAT5a expression. MFI of pSTAT5a on (E) mTconv, (F) FOXP3+ rTregs, (G) FOXP3+ mTregs, and (H) FOXP3hi aTregs following exposure to IL-2 as indicated. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line. Results are expressed as the MFI of pSTAT5a staining for all cells within a given population. Statistical significance was determined using a two-tailed paired Student t test. cells. This reflects the fact that in addition to IL2RA, many other (CD4+CD142CD25+CD1272/lo) and Tconv populations (non- genes, including PTPN2 (27), contribute to variation in the sig- Treg gate) as previously described (28) and shown in Fig. 7A. naling pathways that mediate the phosphorylation and dephos- We then measured the expression of FOXP3 in isolated Tregs both phorylation of STAT5a in response to IL-2. immediately after flow cytometric sorting and after culture for Collectively, our results show that Ag-experienced CD4+ Tconv 48 h in various concentrations of IL-2. Tregs were also stained for and Tregs from individuals with the protective P1P1 IL2RA hap- Helios, a member of the Ikaros family of zinc finger transcription lotype showed increased sensitivity to IL-2. factors that is expressed at high levels in thymic-derived Treg cells but not peripherally induced Tregs (iTregs) or activated Tconv Tregs from individuals with the protective P1P1 IL2RA haplotype (29). An example of the staining is shown in Fig. 7B. Experi- maintained higher levels of FOXP3 in the presence of IL-2 mental conditions were selected that represent nonsaturating (2 U/ Lymphocytes were gated for CD4+CD142 expression, examined ml) and saturating concentrations (20 U/ml) of IL-2 as determined for expression of CD25 and CD127, and sorted into regulatory in preliminary experiments (Fig. 8A–D). Immediately postsort

FIGURE 7. Example of the gating used for the iso- lation of CD4+ T cell populations for functional studies and staining of isolated Treg populations to investigate maintenance of expression of FOXP3 and Helios. (A) Lymphocytes identified by their forward and side scatter properties were gated for CD4+CD142 expres- sion and then examined for expression of CD25 and CD127 (inset plot shows isotype control staining). Tregs were isolated based on CD4+CD142CD25+ CD1272/lo.(B) Isolated Tregs were fixed, permea- bilized, and stained for expression of the transcrip- tion factors FOXP3 and Helios (dot plot) or relevant isotype control (density plot). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 8. Helios and FOXP3 staining in isolated Treg populations cultured with limiting concentrations of IL-2. (A–D) Example of Helios and FOXP3 staining in freshly isolated and cultured Tregs. Tregs were isolated by flow cytometry sorting as described in Fig. 7 and following fixation were stained for expression of the transcription factors FOXP3 and Helios either immediately postsorting (A) or following culture for 48 h in 0 U/ml IL-2 (B), 2 U/ml IL-2 (C), or 20 U/ml IL-2 (D). Quadrant gates were set based on staining with the relevant isotype controls (99th centile), and FOXP3 MFI reflects FOXP3 staining in the Helios+ Tregs. (E–H) FOXP3 expression in Helios+ Tregs under suboptimal IL-2 concentrations. Tregs were isolated from donors with the protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS) and analyzed for expression of FOXP3 and Helios either immediately postsorting (E) or following culture for 48 h in 0 U/ml IL-2 (F), 2 U/ml IL-2 (G), or 20 U/ml IL-2 (H) as described above. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line. Statistical significance was determined using a one-tailed Wilcoxon matched-pairs signed rank test. by guest on September 28, 2021 there was no significant difference in the proportion of Tregs that (data not shown). Importantly, the difference in suppression be- expressed FOXP3 (mean, 80.1 6 7.8% SD and 79.6 6 7.9% SD, tween P1P1 and SS groups only seen in cocultures containing respectively) or those that expressed Helios (mean, 73.3 6 7.4% mTconv and autologous Tregs but was not seen when a standard SD and 77.6 6 6.1% SD, respectively) between individuals with third party population of Tregs was used in the place of autologous the P1P1 or SS IL2RA haplotype (data not shown). As previously Tregs (Fig. 9B) or when nTconv were used in the place of mTconv reported, culture of Tregs in the absence of IL-2 resulted in a de- (Fig. 9C). Using data from the present study and from Dendrou crease in both the percentage of cells expressing FOXP3 and the et al. (12), we propose a model in which the rs12722495 IL2RA level of expression. FOXP3 expression can be “rescued” by ad- susceptibility genotype may influence two distinct immunophe- dition of IL-2 (22, 24). Our results indicated that FOXP3 main- notypes: less IL-2 production from mTconv and a reduced sensi- tenance under these conditions is significantly dependent on tivity to IL-2 signaling in Tregs resulting in lower levels of IL2RA haplotype. We show that Helios+ Tregs from individuals pSTAT5a and reduced expression levels of FOXP3 (Fig. 10). with the protective P1P1 IL2RA haplotype expressed significantly higher levels of FOXP3 under conditions of limiting IL-2 (0 U/ml, Discussion p = 0.03 and 2 U/ml, p = 0.04; Fig. 8E–H). Taken together, these In the present study we addressed the impact of an IL2RA hap- data indicate that Tregs from individuals with the protective P1P1 lotype associated with autoimmune T1D on Treg fitness and IL2RA haplotype maintained higher levels of FOXP3 in the suppressive function. We showed that the presence of a disease- presence of limiting concentrations of IL-2. associated (SS) IL2RA haplotype leads to diminished IL-2 re- sponsiveness, resulting in lower levels of FOXP3 expression by Tregs from individuals with the protective P1P1 IL2RA Tregs and a reduction in their ability to suppress proliferation of haplotype show increased suppression of autologous mTconv autologous mTconv cells. These data offer a rationale that po- In conventional “Shevach” suppression assays, Tregs from indi- tentially accounts for the molecular and cellular mechanisms viduals with the protective P1P1 IL2RA haplotype displayed through which polymorphisms in the IL-2RA gene affect immune higher levels of suppression of autologous mTconv compared with regulation, and consequently affect susceptibility to autoimmune the SS IL2RA haplotype (Fig. 9A). A similar difference in sup- and inflammatory diseases. pression was also observed at lower ratios of Treg/mTconv in pairs Numerous reports have demonstrated that CD4+CD25+ Tregs of individuals who demonstrated a high degree of difference at the from individuals with a range of human autoimmune diseases, 1:1 ratio (Supplemental Fig. 3); however, the difference between including T1D, are deficient either in their frequency or in their the two groups of pairs did not reach significance at lower ratios ability to control autologous proinflammatory responses when 8 IL2RA HAPLOTYPE AFFECTS CD4+CD25+ TREG FUNCTION

FIGURE 9. Percentage suppression of mTconv proliferation by autologous or third party Tregs. Tregs and Tconv were isolated from donors with the protective rs12722495 IL2RA haplotype (P1P1) and donors with the susceptible IL2RA haplotype (SS). The suppression of proliferation of Tconv by Tregs Downloaded from was measured by in vitro coculture. Plots shows (A) suppression of mTconv by autologous Tregs, (B) suppression of mTconv by standard third party Tregs, and (C) suppression of nTconv by autologous Tregs. Each symbol type represents a pair of individuals analyzed on the same day, and individuals are joined by a horizontal line. Statistical significance was determined using a one-tailed Wilcoxon matched-pairs signed rank test. compared with nondiseased, control individuals (5, 6, 30–36). detectable in the peripheral blood is not influenced by rs12722495, However, there is a major gap in present knowledge as to how the ability of Tregs to signal via IL-2 is altered by this polymor- http://www.jimmunol.org/ CD4+CD25+ Treg function relates to the development of human phism and function is thereby impaired. This constellation of autoimmune disease in terms of causality. Namely, is Treg dys- findings is resonant with observations made in patients with T1D: function a primary, causal event or is it a result of alterations in the the frequency of Treg populations is normal, but Treg function is immune system due to the disease process? We hypothesized that reduced (5, 6, 9). Additionally, a number of recent studies show if a diabetes susceptibility haplotype could be linked to altered that patients with T1D have impaired IL-2 signaling and increased (decreased) Treg function in individuals with no history of auto- Treg apoptosis (7, 24). This implies that impairment of the IL-2 immune disease, this would support the proposal that Treg dys- pathway, through its effect on Treg fitness and immune regulation, function is causal in T1D. Several genes within the IL-2/IL-2RA is a key pathway in autoimmune disease pathogenesis. pathway have been identified that influence susceptibility to hu- Binding of IL-2 to its high-affinity receptor complex leads to by guest on September 28, 2021 man autoimmune diseases (e.g., IL2, IL2RA, IL2RB, and PTPN2) a cascade of signaling events, including activation of the Ras/ (11, 37–42), and given the vital importance of IL-2 and IL-2 MAPK, JAK/STAT, and PI3K/Akt pathways. In Tregs a major signaling to the generation and function of CD4+CD25+FOXP3+ cellular consequence of IL-2 signaling is the phosphorylation and Tregs, it is likely that that these genes exert their effects by al- activation of STAT5a, which binds to the FOXP3 promoter, leading tering Treg frequency or functional ability. In this and our pre- to sustained FOXP3 expression and enhanced suppressive capacity vious report (12), the diabetes-susceptible IL2RA haplotype (19, 21, 43). IL-2 signaling and STAT5a phosphorylation are also (rs12722495) is shown to confer reduced CD25 expression and vital for the generation of induced FOXP3+ Tregs (22). In our IL-2 secretion by mTconv cells. Although the frequency of Tregs studies, mTconv from individuals with the susceptible IL2RA

FIGURE 10. A model to explain the link be- tween IL-2 pathway gene polymorphisms and the development of autoimmunity. Data from the present study and from Dendrou et al. (12) sug- gest that the rs12722495 IL2RA susceptibility genotype may influence two distinct immuno- phenotypes: less IL-2 production from mTconv and a reduced sensitivity to IL-2 signaling in Tregs resulting in lower levels of pSTAT5a and reduced expression levels of FOXP3. We propose that that these two immunophenotypes could act synergistically to reduce Treg function. The Journal of Immunology 9 haplotype had a lower level of pSTAT5a in response to IL-2 sive action of activated Tregs and a subsequent failure to suppress stimulation. A key question is the extent to which this might pathogenic autoimmunity. also affect the ability of an individual to respond to immune In summary, we have demonstrated that a disease-susceptible regulatory cues and generate iTregs. nTconv (which express lower IL2RA haplotype is associated with reduced Treg fitness and sup- levels of CD25 and in whom most IL-2–induced STAT5a phos- pressive function in vitro. To our knowledge, this represents the phorylation will occur in a CD25-independent manner) from first demonstration that a gene polymorphism that increases sus- IL2RA-susceptible individuals did not show a reduced pSTAT5a ceptibility to autoimmune diseases such as T1D is associated response. However, our in vitro studies did not take into account with altered Treg function. Further studies will be required to the fact that priming of naive T cells in vivo requires TCR ligation, determine whether other susceptibility genes also contribute to the a process that alters CD25 expression and hence IL-2 respon- defective Treg function that characterizes individuals with auto- siveness. It remains to be established, therefore, whether lower immunity. levels of IL-2 sensitivity can result in reduced ability to generate iTregs from naive T cells. Acknowledgments The definitive identification of Tregs by flow cytometry is We gratefully acknowledge the participation of all Cambridge BioResource challenging, as activated T cells share many of the phenotypic (CBR) volunteers. We thank the staff of the CBR recruitment team for as- characteristics of Tregs. Because fixation precluded the use of sistance with volunteer recruitment and K. Beer, J. Rice, P. Tagart, and CD127 when measuring pSTAT5a in PBMC samples, we adopted M. Wiesner for blood sample collection. We thank M. Woodburn and T. Att- a highly conservative approach to identify CD25hi Tregs by gating wood for contributing to sample management. We thank members of the on the top 1% of CD25-staining CD4+ T cells, an approach Cambridge BioResource Scientific Advisory Board and management com- Downloaded from adopted by other researchers (7, 25). Gating on an analogous mittee for support and the National Institute for Health Research Cambridge population in the unfixed samples demonstrated that this pop- Biomedical Research Center for funding. We also thank Richard Ellis and ulation consisted of almost exclusively CD1272 cells, and we Thomas Hayday for performing flow cytometry cell sorting. were therefore confident that the observed difference in STAT5a phosphorylation is due to a difference in sensitivity to IL-2–me- Disclosures

diated signaling in Tregs rather than in any contaminating Tconv. The authors have no financial conflicts of interest. http://www.jimmunol.org/ These findings were then confirmed in a separate cohort of indi- viduals using a whole blood assay that facilitated the simultaneous References detection of FOXP3 and pSTAT5a to allow for more definitive 1. Castan˜o, L., and G. S. Eisenbarth. 1990. Type-I diabetes: a chronic autoimmune gating of bona fide Tregs based on expression of FOXP3. Im- disease of human, mouse, and rat. Annu. Rev. Immunol. 8: 647–679. portantly, in both groups of individuals tested, this difference was 2. Tisch, R., and H. McDevitt. 1996. Insulin-dependent diabetes mellitus. Cell 85: 291–297. only observed under nonsaturating concentrations of IL-2, sug- 3. Roep, B. O. 2003. The role of T-cells in the pathogenesis of type 1 diabetes: from gesting that there was not an inherent difference in the ability of cause to cure. Diabetologia 46: 305–321. 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