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Functionally Significant Differences in Expression of Disease-Associated IL-7 Receptor α Haplotypes in CD4 T Cells and Dendritic Cells This information is current as of October 2, 2021. Edwin Hoe, Fiona C. McKay, Stephen D. Schibeci, Kaushal Gandhi, Rob N. Heard, Graeme J. Stewart and David R. Booth J Immunol 2010; 184:2512-2517; Prepublished online 22

January 2010; Downloaded from doi: 10.4049/jimmunol.0902900 http://www.jimmunol.org/content/184/5/2512

References This article cites 41 articles, 14 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/184/5/2512.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Functionally Signiﬁcant Differences in Expression of Disease-Associated IL-7 Receptor a Haplotypes in CD4 T Cells and Dendritic Cells

Edwin Hoe,1 Fiona C. McKay,1 Stephen D. Schibeci, Kaushal Gandhi, Rob N. Heard, Graeme J. Stewart, and David R. Booth

Common genetic variants of IL-7 receptor a (IL-7Ra) have recently been shown to affect susceptibility to multiple sclerosis (MS) and type 1 diabetes, and survival following bone marrow transplantation. Transcription of the gene produces two dominant isoforms, with or without exon 6, which code for membrane-bound or soluble IL-7Ra, respectively. The haplotypes produce different isoform ratios. We have tested IL-7Ra mRNA expression in cell subsets and in models of T cell homeostasis, activation, tolerance, and differentiation into regulatory T cell/Th1/Th2/Th17, memory, and dendritic cells (DCs) under the hypothesis that the conditions in which haplotype differences are maximal are those likely to be the basis for their association with disease Downloaded from pathogenesis. Maximal differences between haplotypes were found in DCs, where the ligand is mainly thymic stromal lymphopoietin (TSLP). The MS-protective haplotype produces a much lower ratio of soluble to membrane-bound receptor, and so potentially, DCs of this haplotype are more responsive to TSLP. The TSLP/IL-7Ra interaction on DCs is known to be critical for production of thymic regulatory T cells, and reduced production of these cells in MS susceptibility haplotypes may be a basis for its association with this disease. IL-7Ra mRNA expression varies greatly through cell differentiation so that it may be a useful marker for cell states. We also show that serum levels of soluble receptor are much higher for the MS susceptibility haplotype (p = http://www.jimmunol.org/ 4 3 10213). Because signaling through IL-7Ra controls T cell regulation, this haplotype difference is likely to affect the immunophenotype and disease pathogenesis. The Journal of Immunology, 2010, 184: 2512–2517.

ultiple sclerosis (MS) is the most common chronic represents the next major challenge in diseases of complex genetics. neurologic disease of young adults, affecting .2 million Haplotypes contain genetic variants that potentially affect regulation M people worldwide. Extensive studies of population ge- of transcription and mRNA processing, especially splicing. This is netics have shown a major heritable component to the disease, with the first level at which genetic variance can produce functional an increasing risk with genetic closeness to an index case (1). Until differences for genes and the level where there is the least noise from by guest on October 2, 2021 2007, only one inherited risk factor had been validated across other processes. For IL-7Ra, four common haplotypes have been multiple populations, the MHC class II DRB1p1501 allele, which described previously (6). Haplotype 2 is protective against the confers a 3- to 4-fold relative risk, and implicates Ag presentation to disease and is tagged by a T allele in exon 6 (rs6897932). This CD4 T cells in MS pathogenesis. Several publications in 2007 and single nucleotide polymorphism (SNP) is associated with reduced subsequently confirmed our earlier reports (2) of the gene encoding exon splicing and the production of less of the gene encoding the IL-7 receptor a-chain (IL-7Ra, also known as CD127) as the soluble IL-7Ra in PBMCs of healthy control individuals (7, 8). The first non-HLA gene influencing the risk of MS in studies involving putative proximal promoter contains several SNPs, and we have .20,000 patients from Australia, the United States, the U.K., and found evidence of haplotypic differences in expression and re- Europe (3). Recently published genome-wide association studies sponse to IFN-b (E. Hoe, F.C. McKay, S. Schibeci, R. Heard, G.J. by us (4) and others (5) have identified several further genes af- Stewart, and D.R. Booth, submitted for publication). The hap- fecting MS susceptibility. lotypes are also tagged by codon-changing SNPs whose functional With gene identification progressing rapidly, determination of the effects, if any, are yet to be determined (6). The haplotypes have functional effects of haplotypes influencing disease susceptibility also been associated with other diseases: haplotype 2 decreases risk of type 1 diabetes (9), and haplotype 1 is associated with an in- Westmead Millennium Institute, University of Sydney, Westmead, New South Wales, creased risk of death following matched unrelated bone marrow Australia transplantation (10). 1E.H. and F.C.M. contributed equally. IL-7Ra is expressed in multiple immune cell types, as a subunit Received for publication September 1, 2009. Accepted for publication December 5, of the heterodimeric receptors for IL-7 (with the common cyto- 2009. kine g-chain) and for thymic stromal lymphopoietin (TSLP; with This work was supported by an Australian Research Council Industry Linkage grant the TSLP receptor). IL-7 plays a key and nonredundant role in T with Biogen Idec (to E.H., K.G., and F.C.M.). lymphocyte differentiation, survival, and proliferation (11), and its Address correspondence and reprint requests to Dr. David Booth, Westmead Millen- nium Institute, Darcy Road, Westmead, New South Wales, Australia. E-mail address: heterodomeric receptor is predominantly expressed on T cells. [email protected] TSLP promotes Th2 differentiation in the periphery and the de- Abbreviations used in this paper: DC, dendritic cell; MS, multiple sclerosis; sIL-7Ra, velopment of regulatory T cells (Tregs) in the thymus (11). Its serum IL-7Ra; SNP, single nucleotide polymorphism; Treg, regulatory T cell; TSLP, heterodomeric receptor is expressed predominantly on dendritic thymic stromal lymphopoietin. cells (DCs). Inherited loss of function of IL-7Ra in humans leads Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 to a form of SCID (12), underlying its key role. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902900 The Journal of Immunology 2513

To further characterize the functional effects of MS susceptibility CD4CD45RA cells were cultured for 4 d with 2.5 anti-biotin microbeads haplotypes, we have taken two approaches: first, to determine the (Miltenyi Biotec) loaded with 0.5 pg anti-CD3 beads (UCHT1 from eBio- contexts in which the IL-7Ra haplotype may affect immune function, science, San Diego, CA) per cell, rested for 2 d, then restimulated in wells coated with anti-CD3 (OKT3) at a concentration of 0.1 mg/ml for an additional we selected two major cell types of potential importance in MS 4 d (21), determined intitration experiments toinduce relatively low levels ofT pathogenesis, namely T cells and DCs (13), and examined regulation cell proliferation by [3H]thymidine incorporation. of IL-7Ra under various conditions to determine whether the hap- Central memory T cells were generated by 5-d coculture of CD4CD45RA lotypes are differentially regulated in response to varying stimuli or cells with mature autologous monocyte-derived DCs (described below; at a ratio of 10 T cells to 1 DC) and a nonspecific Ag, staphylococcal exotoxin A cellular transcriptional programs. We found a significant reduction in (1 ng/ml). IL-2 (20 IU/ml) was added for an additional 9 d with replenishment splicing out of exon 6 in the IL-7Ra MS-protective haplotype mRNA every 2–3 d (22), and CD45RO+CCR7+ phenotype was confirmed by flow in whole blood, and this difference was much greater in DCs. Second, cytometry. DCs were generated from CD14 monocytes (purified from we examined the effect of haplotype on the concentration of circu- PBMCs by positive selection; Miltenyi Biotec) by culture with IL-4 (17 ng/ lating soluble receptor and demonstrate that all peripheral leukocytes ml) and GM-CSF (67 ng/ml) over 5 d, with or without maturation with LPS (1 mg/ml) for an additional 2 d (23). The mature DC phenotype was con- are potentially exposed to lower effective signaling through the re- firmed by flow cytometry (CD83+ and HLA-DR+) (24). For each model, ceptor in all individuals carrying the MS-susceptibility haplotypes. cells were washed briefly in ice-cold PBS and harvested immediately in Cells-to-signal lysis buffer (Ambion, Austin, TX) to stabilize labile mRNAs and stored at 280˚C until RNA extraction (RNeasy; Qiagen, Valencia, CA). Materials and Methods In vitro generation of cell subsets Genotyping and gene expression Peripheral blood was collected in EDTA from healthy control subjects of IL-7Ra haplotype was determined from DNA by RFLP as described pre- known IL-7Ra haplotype and mononuclear cells (PBMCs) isolated by Ficoll viously (6). Total gene expression was measured by quantitative RT-PCR. Downloaded from density gradient separation. Naive T helper cells (CD4CD45RA) were pu- Relative IL-7Ra haplotype expression was measured in heterozygotes by rified from PBMCs by magnetic separation (Miltenyi Biotec, Auburn, CA). SNaPSHOT analysis (2, 25, 26) where the relative abundance of two codon For the activation model, CD4CD45RA cells were coincubated with anti- region haplotype-tagging SNPs is quantitated in cDNA. The ratio of full-length CD3 and anti-CD28 beads (Dynabeads; Invitrogen, Carlsbad, CA) for 48 h to soluble isoforms was determined from specifically amplified IL-7Ra gene (14) at a ratio of 0.2 beads/cell. Th1 and Th2 subsets were generated by products (7) using an Agilent Bioanalyzer (Waldbronn, Germany). IL-2Ra activation as above in the presence of IL-12 (30 ng/ml) and anti–IL-4 Ab mRNA expression was measured by using TaqMan Universal PCR Mastermix (0.5 mg/ml) for Th1 cells, or IL-4 (20 ng/ml) and anti–IFN-g Ab (2.5 mg/ml) and commercially available TaqMan primers (TaqMan gene expression for Th2 cells. IL-2 (200 IU/ml) was added to the culture on day 2, and all assay, accession number Hs 00907778; Applied Biosystems, Foster City, CA). http://www.jimmunol.org/ cytokines and Abs were replenished every 2 d until cell harvest on day 8 (15). For Th17 polarization, T cells were activated for 3 d in the presence of IL-1b Soluble IL-7Ra ELISA (10 ng/ml), IL-6 (50 ng/ml), with anti–IL-4 and anti–IFN-g Abs as above. On Sera were collected from people with MS (n = 115) and healthy control day 3, IL-2 (20 IU/ml) and IL-23 (20 ng/ml) were added, and all cytokines subjects (n = 100), each of whom had been genotyped for IL-7Ra, and and Abs replenished every 2–3 d until cell harvest on day 12 (16). Tregs were soluble IL-7Ra levels were determined by sandwich ELISA using a cap- generated by activation of T cells in the presence of TGF-b (10 mg/ml) for 4 d ture Ab (5 mg/ml, MAB306) and a biotinylated detection Ab (50 mg/ml, (17). Phenotypes were confirmed by flow cytometry (CD45RO+ for cellular high high BAF306; both from R&D Systems, Minneapolis, MN) incubated with activation; CD25 FoxP3 for Tregs) or RT-PCR (upregulation of T-bet, HRP-conjugated streptavidin and tetramethylbenzidine as substrate. GATA-3, and RORgt for Th1, Th2, and Th17, respectively). Standard curves were generated from a recombinant soluble IL-7Ra-Ig To deliver an activation signal intermediate between media alone and the conjugate (Apollo Biosciences, Sydney, Australia). by guest on October 2, 2021 activation model (as assessed by downregulation of IL-7Ra gene expression; “signal strength” model), anti-CD3 and anti-CD28 beads were used at a ratio Cell surface IL-7Ra expression of 1 bead per 40 cells. Conditions of peripheral homeostasis were modeled by culture of CD4CD45RA cells with IL-7 (10 ng/ml) for 7 d with cytokine re- Whole blood was collected in EDTA from 18 people with MS and 20 age- plenishment every 2–3 d (18, 19), and IL-7–mediated upregulation of IL-2Ra and gender-matched healthy control subjects. MS subjects had not been (20) was confirmed by RT-PCR (see below). To model T cell tolerance, treated with immunomodulatory therapy in the previous 3 mo. Blood was

FIGURE 1. IL-7Ra gene expression in in vitro- generated T cell subsets and monocyte-derived DCs. During CD3/CD28 stimulation of naive CD4 T cells gene expression of IL-7Ra (A) was downregulated with reciprocal upregulation of IL-2Ra over 48 h compared with culture in media alone (B), determined by quantitative RT-PCR. Error bars represent SEM from triplicate cultures. Total IL-7Ra expression varies in T cell subsets generated in vitro from naive CD4 T cells (n = 18) or for remaining T cell models (n = 2 per model), all from heterozygous individuals, each cultured in triplicate (C), and in monocyte-derived DCs with or without maturation with LPS as determined by quantitative RT-PCR (from 10 individuals of varying haplotypes) (D). Donors were healthy controls. 2514 EXPRESSION DIFFERENCES BETWEEN IL-7Ra HAPLOTYPES

T cell activation, differentiation, tolerance, and homeostasis, and DC maturation and measured total and relative haplotype expression in these models. We also determined the effect of haplotype on soluble and cell surface IL-7Ra protein expression. IL-7Ra total expression in CD4 T cells and DCs We first measured total expression of IL-7Ra mRNA in each of the cellular models. IL-7Ra mRNA was expressed at a high level in resting naive CD4 T cells and was downregulated over time of activation by CD3 and CD28 stimulation, with reciprocal upregulation of the IL-2Ra (Fig. 1A,1B). Downregulation of IL-7Ra was observed in all models of CD4 T cell differentiation (Th1, Th2, Th17, Treg, central memory), during strong (activation) and intermediate (signal strength) levels of CD3/CD28 stimulation, and in response to consumption of IL-7 (homeostasis) (Fig. 1C). Weak CD3 stimulation in the absence of CD28 costimulation, mimicking T cell tolerance to low-affinity Ag (e.g., self-Ag) did not down- regulate IL-7Ra (Fig. 1C). IL-7Ra was expressed at a very low level on immature, monocyte-derived DCs; it was significantly upregulated upon maturation with LPS but only to levels ∼20-fold Downloaded from lower than those expressed on naive CD4 T cells (Fig. 1D). IL-7Ra relative haplotype expression in CD4 T cells and DCs To determine whether particular IL-7Ra haplotypes are preferentially expressed in certain cellular contexts, we measured ex-

pression of each haplotype in heterozygote cells under different http://www.jimmunol.org/ conditions. Heterozygotes are particularly useful for this purpose, because both haplotypes are in identical conditions (the same FIGURE 2. Relative IL-7Ra haplotype gene expression in in vitro- cells) but can respond differently if their regulatory sequences are generated T cell subsets and monocyte-derived DCs. A, Relative expres- differentially controlled. For each condition, we examined at least sion of transcripts of the IL-7Ra haplotypes determined by SNaPshot analysis in hap1/hap2 heterozygotes and hap4/hap2 heterozygotes, given four individuals. The MS-protective haplotype, haplotype 2, was as a ratio compared with haplotype 2 in T cell subsets and mature DCs. In never preferentially expressed in CD4 T cells: comparisons in Hap mature DCs haplotype 2 is expressed at a higher level than the other 1/Hap 2 and Hap 4/Hap 2 heterozygotes demonstrate that haplotype haplotypes (pp , 0.05; Mann-Whitney U test; n = 6). For each T cell model, four individuals were tested, each in triplicate cultures. B, Relative by guest on October 2, 2021 expression of the haplotypes was stable over time (48 h) in the activation model (representative experiment showing expression of haplotype 4 and haplotype 2 transcripts in a hap2/hap4 heterozygote determined in triplicate). Error bars represent SEMs. Donors were healthy controls.

collected between 8 AM and 2 PM, and samples were processed exactly 2 h postcollection. Buffy coat was stained either with CD45RA-FITC, CrTh2- PE, CD4-PerCP, IL-7Ra-APC, CCR7-PECy7, and CD25-APC-Cy7 or CD4-PerCP with isotype controls for FITC, PE, APC, PECy7, and APC- Cy7. Lymphocytes were gated on forward and side scatter parameters, and CD4 T cells were gated as a subset of lymphocytes from a plot of CD4 against side scatter. Mean fluorescence intensity for IL-7Ra-APC on all CD4 T cells is presented, corrected by subtraction of IgG-APC mean fluorescence intensity of CD4 T cells. Only the results for the CD4 subset are presented in this study; other Ab details are included for accurate re- porting of the Ab panel from which the data were collected. All Abs were from BD Biosciences (San Jose, CA), except APC-conjugated Abs (from R&D Systems). Red cells were lysed with BD-Lyse (BD Biosciences), and the leukocytes were fixed, permeabilized, and stained with FoxP3-Pacific Blue (eBioscience) using the manufacturer’s buffer set and staining pro- tocol. Samples were analyzed using an LSRII (BD Biosciences) on the day of staining, and all experiments were performed within a 1-mo window. Automatic compensation generated using FACSDiva (BD Biosciences) on the first day of staining was applied to all subsequent data files. FIGURE 3. Haplotype differences in soluble IL-7Ra gene expression This study was approved by the Sydney West Area Health Service are manifest in T cells but magnified in DCs. Representative electro- Human Research Ethics Committee HREC2002/9/3.6(1425), and all par- pherograms (Agilent Bioanalyzer) of soluble (arrow) and full-length ticipants gave written informed consent. (higher peak; no arrow) IL-7Ra gene expression determined in mRNA from whole-blood (A) or monocyte-derived (B) DCs from individuals Results homozygous for haplotype 1 or haplotype 2 or haplotype 1/2/hetero- We used in vitro modeling of CD4 and DC subsets to define zygotes. C, Full-length to soluble ratio of IL-7Ra mRNA isoforms in contexts in which the MS-susceptibility haplotypes of IL-7Ra may monocyte-derived DCs for Hap1 (n = 3) and Hap2 (n = 2), respectively. be differentially regulated and thus potentially relevant in MS Differences significant by Mann-Whitney U test. Error bars represent pathogenesis. We used different conditions designed to model SEMs. Donors were healthy controls. The Journal of Immunology 2515

2 is expressed in equimolar proportions, with the MS-susceptibility haplotypes in CD4 T cells, including following Th1 or Th17 differentiation or IL-7 consumption (homeostasis) for haplotype 1, and following differentiation to a Th2 or central memory phenotype for haplotype 4 (Fig. 2A). In mature DCs, the converse was observed: the MS-protective haplotype was preferentially expressed (at ∼30% higher level) over the MS-susceptibility alleles. Relative expression was examined over time in the activation model, and equal expression of haplotypes 2 and 4 in resting naive T cells was main- tained over 48 h of activation-induced downregulation (Fig. 2B). IL-7Ra splicing and protein expression of receptor isoforms We and others have found previously that the MS-protective haplotype is associated with reduced splicing of exon 6 and the production of less of the gene encoding soluble IL-7Ra in PBMCs of healthy control individuals (7, 8) and MS subjects (E. Hoe, F.C. McKay, S. Schibeci, R. Heard, G.J. Stewart, and D.R. Booth, submitted for publication). However, in whole blood, where the majority of the IL-7Ra transcript is T cell derived, the soluble isoform represented only a minor component of the total gene Downloaded from RNA in all haplotypes. To determine whether exon 6 splicing is similar for the haplotypes in other cell types, full-length and soluble isoforms were compared in whole blood (Fig. 3A) and mature monocyte-derived DCs (Fig. 3B). Lower expression of the soluble isoform was observed in whole-blood RNA from haplotype 2 than haplotype 1, with hap- http://www.jimmunol.org/ lotype 1/2 heterozygotes showing intermediate levels of exon splicing. As for whole-blood RNA, the MS-protective haplotype showed reduced splicing of exon 6 in mature monocyte-derived DCs. However, the transcript encoding soluble IL-7Ra isoform was expressed at a much higher level relative to the full-length isoform in DCs, such that full-length and soluble transcripts are equimolar for the MS-susceptibility haplotype. DCs have both increased expression (Fig. 2A) and a higher proportion of the isoform by guest on October 2, 2021 encoding the membrane-bound receptor for the MS-protective haplotype (Fig. 3). We also examined haplotype differences at the level of protein expression. DCs of the MS-protective haplotype secreted ∼2-fold lower levels of soluble IL-7Ra protein upon maturation with LPS FIGURE 5. Haplotype differences in soluble and cell surface IL-7Ra in vitro, compared with the MS-susceptibility haplotype (Fig. 4). protein expression in blood. A, sIL-7Ra protein was determined by ELISA Ex vivo, soluble receptor was also measured in peripheral blood in MS (open diamonds) and control (filled diamonds) individuals for the (sera) from a large cohort of people with MS (n = 115) and healthy rs6897932 genotypes (haplotype 2 is “T,” not haplotype 2 is “C”). No controls (n = 100), including individuals who do not carry the MS- differences between MS and control sIL-7Ra levels were detected for any protective haplotype (not Hap 2; n = 132) and individuals who genotype. There was also no significant difference between MS and con- carry either one (n = 69) or two (n = 14) copies of this haplotype trols overall (45.9 and 41.7 ng/ml, respectively; p = 0.303). B, Cell surface (Hap 2). Soluble receptor was significantly lower in sera from IL-7Ra determined by flow cytometry for 18 individuals with MS and 20 individuals with the MS-protective haplotype (p , 4 3 10213; healthy control individuals who do (n = 17) or do not (n = 21) carry Fig. 5A), as well as in the MS (p , 2 3 1027) and healthy control haplotype 2. p Values were determined by Mann-Whitney U test.

cohorts (p , 6 3 1027) analyzed separately. A significant dose effect of the haplotype 2 allele was observed. There was no difference in serum IL-7Ra (sIL-7Ra) between MS and controls. There was a trend toward higher sIL-7Ra in the MS group compared with healthy controls (45.9 [95% CI: 41.50–50.37] and 41.7 [37.8–45.6] ng/ml, respectively; p = 0.303), as would be predicted from their expected lower frequency of the protective haplotype. However, haplotype 2, the protective haplotype, was as common in MS as controls in this cohort (frequency 0.23 in both MS and controls). A much larger study would be needed for a genotype FIGURE 4. Haplotype differences in soluble IL-7Ra protein expression in odds ratio of 1.2 to produce statistically significant differences in monocyte-derived DCs. IL-7Ra protein (sCD127) determined in culture su- haplotype frequency and its effect on serum sIL7Ra between the pernatants of monocyte-derived DCs from individuals of haplotype 1 (n =2) two cohorts. We also analyzed cell-surface expression of IL-7Ra or 2 (n = 1) with or without LPS stimulation by ELISA determined in triplicate on CD4 T cells of healthy controls (n = 20) and people with MS cultures. pp , 0.05; Mann-Whitney U test. Donors were healthy controls. (n = 18) and found a trend toward higher cell-surface expression 2516 EXPRESSION DIFFERENCES BETWEEN IL-7Ra HAPLOTYPES of IL-7Ra for the MS-protective haplotype (n = 17) compared and heparan sulfate components of the extracellular matrix (33) and with those who do not carry haplotype 2 (n = 21) (Fig. 5B), but no thus may be largely displayed in cell-associated form and con- differences between MS and healthy controls. We found no evi- sumed by cells at the sites of production (34). Soluble receptor dence to suggest that membrane-bound IL7Ra is different in cells may affect response to ligand both locally (around sites of pro- of people with MS, or that serum (soluble) IL-7Ra is different in duction and cellular interactions) and systemically (because of disease; nor to expect that mRNA splicing may be different in MS. binding to serum or lymph circulating ligand). The effect of increased IL-7 availability may be inferred from its Discussion effect when added experimentally or therapeutically. IL-7 injection These data demonstrate IL-7Ra mRNA expression varies .20- into mice increases T cell numbers, in thymic and nonthymic fold during CD4 T cell and DC differentiation (Fig. 1). The IL- compartments (35). It also increases immune response when used 7Ra isoform ratio and level of expression is affected by haplotype, as an adjuvant (36). In humans, it has been tested to improve especially in DCs (Fig. 3). However, the relative reduction or recovery from lymphopenia induced by drug treatment in two increase in mRNA expression for each haplotype was not different clinical trials. In both trials the numbers of CD4 and CD8 cells, throughout cell differentiation and in response to stimulation, as both naive and memory, increased, but the numbers of Tregs did measured by haplotype expression in heterozygotes in T cell and not (37, 38). However, in HIV patients, a single dose of re- DC models (Fig. 2). In DCs, the haplotypes appear to be under combinant human IL-7 increased CD4 and CD8 counts but did not different regulation from each other, more so than in T cells. The change Treg frequency (39). Such effects would not be expected MS-protective haplotype has previously been reported to produce to be useful in autoimmunity, where an increase in relative Tregs significantly less soluble IL-7Ra mRNA in PBMCs and cell lines would be desirable. Relative T cell subset responses to IL-7 in (7, 8), and we demonstrate in this study that this is also so in CD4 these clinical settings were from lymphopenic individuals, the Downloaded from T cell and DC subsets. We confirm this haplotype difference ex- immunophenotype response to increased IL-7 (or TSLP) avail- tends to the protein level by demonstrating that in sera there is ability in other settings may be different. The net effect of dif- a highly significant difference in circulating sIL-7Ra, and a dif- ferential haplotype effects on expression may need to be ference in cell surface IL-7Ra associated with the haplotype 2 determined from correlations of genotype with immunophenotype. genotype (Fig. 5). In MS there is evidence of an altered immunophenotype (7, 40), The outcome of signaling from both TSLP and IL-7 is known to but as yet no correlation with IL-7Ra genotype has been described http://www.jimmunol.org/ vary according to microenvironment. The ratio of soluble to in- for these aberrant cell subsets. It is also possible that im- soluble mRNA isoforms varies greatly between cells being max- munophenotype differences are dynamic, differing at the onset of imal in DCs. These cells respond to TSLP. Studies on TSLP disease and as it progresses, so that genotype effect on it are function have so far indicated that in the periphery it promotes Th2 masked or difficult to dissect. polarization and, in the thymus, Treg differentiation (27). De- As the current genome-wide analysis studies identify additional creased signaling because of increased production of the soluble genes, the approach we have used could be useful in identifying IL-7R protein could result in fewer or less effective Tregs. In MS, the functional role of haplotypes of genes associated with MS and thymic output of Tregs is reduced (28), and diminished suppres- other common autoimmune diseases. This is especially so for by guest on October 2, 2021 sive capacity of Tregs has been described previously (reviewed in those genes expressed predominantly in T cells and DCs, such as Ref. 29). This could also be the basis for the IL-7Ra association IL-2Ra (CD25), CD6, CD40, CD58, IFN regulatory factor 8, and with type 1 diabetes, where IL-7Ra haplotype 2 is also protective CD120a, now also confirmed as associated with MS (4, 40, 41). (9). In other diseases, the clinical significance of the haplotype may have a different basis. In allogeneic bone marrow transplants Acknowledgments from matched unrelated donors, when the donor carries haplotype We thank the people with MS and healthy control participants for donating 1, the prognosis is poor (10). The haplotype 1 donor T cells may blood for the study and Najwa Marmash for excellent technical assistance. be less likely to proliferate in transplants because of decreased relative availability of IL-7. They then fail to confer protection Disclosures from infection, and so, such transplants have an increased mor- The authors have no financial conflicts of interest. tality. Haplotype 2 carriers have better recovery from lymphopenia in therapy for HIV (R. Rajasuriar et al., personal communication), References possibly as a result of improved availability of IL-7 to promote 1. Robertson, N. P., J. I. O’Riordan, J. Chataway, D. P. Kingsley, D. H. Miller, CD4 T cell proliferation. D. Clayton, and D. A. Compston. 1997. Offspring recurrence rates and clinical Soluble cytokine receptors are well-known to regulate signaling characteristics of conjugal multiple sclerosis. Lancet 349: 1587–1590. (30). Goodwin et al. (31) found evidence this was also true for sIL- 2. Booth, D. R., A. T. Arthur, S. M. Teutsch, C. Bye, J. Rubio, P.J. Armati, J. D. Pollard, 7Ra, when they demonstrated harvested supernatants from COS-7 R. N. Heard, G. J. Stewart, and Southern MS Genetics Consortium. 2005. 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