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Regulatory Suppress T Cells Partly through -10 Christine Lingblom, Jennie Andersson, Kerstin Andersson and Christine Wennerås This information is current as of September 27, 2021. J Immunol published online 17 May 2017 http://www.jimmunol.org/content/early/2017/05/16/jimmun ol.1601005 Downloaded from

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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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 17, 2017, doi:10.4049/jimmunol.1601005 The Journal of Immunology

Regulatory Eosinophils Suppress T Cells Partly through Galectin-10

Christine Lingblom, Jennie Andersson, Kerstin Andersson, and Christine Wenneras˚

Eosinophils have the capacity to regulate the function of subsets. Our aim was to test the hypothesis of the existence of a regulatory subset of eosinophils. Human eosinophils were incubated with T cells that were stimulated with allogeneic leukocytes or CD3/CD28 cross-linking. After 2 d of coculture, 11% of the eosinophils gained CD16 expression. A CD16hi subset of eosinophils, encompassing 1–5% of all eosinophils, was also identified in the of healthy subjects. FACS sorting showed that these CD16hi eosinophils were significantly stronger suppressors of T cell proliferation than were conventional CD16neg eosinophils. Human eosinophils contain stores of the immunoregulatory protein galectin-10. We found that Ab-mediated neutralization of galectin-10 partially abrogated the suppressive function of the eosinophils. Moreover, recombinant galectin-10 by itself was able to suppress

T cell proliferation. Finally, we detected galectin-10–containing immune synapses between eosinophils and . To Downloaded from conclude, we describe a subset of suppressive eosinophils expressing CD16 that may escape detection because CD16-based negative selection is the standard procedure for the isolation of human eosinophils. Moreover, we show that galectin-10 functions as a T cell–suppressive molecule in eosinophils. The Journal of Immunology, 2017, 198: 000–000.

osinophilic are involved in a range of allergic Intriguingly, the very few case reports of human subjects with a

and other inflammatory conditions that are driven by T cells, total lack of eosinophils in the blood and indicate that http://www.jimmunol.org/ E such as atopic , allergic and , true deficiency is associated with dysregulated immu- eosinophilic esophagitis, , Churg–Strauss syn- nity (11). These patients appear to have an increased risk for drome, and graft-versus-host disease (1–3). The role of eosinophils developing thymoma, , and allergic and in these conditions has not been precisely defined; however, lately it autoimmune disorders, such as asthma, urticaria, drug , has become evident that one activity of eosinophils is to regulate the vitiligo, hemolytic anemia, and even (11); a possible function of T cells. Roufosse and coworkers (4) have demonstrated explanation for this is that eosinophils regulate function that eosinophils can inhibit the activation and expansion of pre- and/or development. In fact, Moqbel and coworkers (12) proposed malignant T cells associated with the lymphocytic variant of that thymic eosinophils are involved in the establishment of tol- hypereosinophilic syndrome. Murine eosinophils suppress the erance during infancy. by guest on September 27, 2021 differentiation of Th17 cells in the small intestine and Th2 cells in Regulatory T cells (Tregs) are essential for the maintenance of Peyer’s patches (5, 6). We have recently shown that eosinophils ; when they are functionally impaired, a severe from transplant recipients with chronic autoimmune and allergic disease (immunodysregulation poly- graft-versus-host disease, as well as eosinophils from healthy endocrinopathy X-linked syndrome) develops (13). adults, are able to suppress allogeneic proliferation of CD4+ and Interestingly, Kubach et al. (14) showed that galectin-10 was + CD8 T cells (7). Earlier studies indicate that eosinophils can necessary for the suppressive function of human Tregs. Notably, selectively inhibit the activation of Th1 cells (8) and that purified galectin-10 is considered a signature molecule of eosinophils, eosinophilic granule proteins can reduce cell proliferation in MLRs because no other cell in the human body contains more of this (9). However, eosinophils have also been shown to boost the pro- protein; it is estimated that galectin-10 accounts for 7–10% of the duction of by polyclonally activated T cells (10) and to protein contents of eosinophils (15). Despite this, no function has + enhance the proliferation of human CD4 T cells (4). been ascribed to galectin-10 in eosinophils, and it is not known how this intracellular protein is secreted. Importantly, eosinophils of mice and other rodents are devoid of galectin-10, precluding the Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Go¨teborg SE-413 46, Sweden use of murine models to investigate its function (16). ORCIDs: 0000-0001-6860-9355 (J.A.); 0000-0002-0950-1165 (C.W.). The main objective of this study was to test the hypothesis of the existence of a regulatory subset of eosinophils. Our idea was that Received for publication June 16, 2016. Accepted for publication April 17, 2017. this might explain the divergent reports regarding the regulatory This work was supported by ALF Go¨teborg (Grant 71580), Strategic ALF/Transplan- tation (Grant 74080), the and Foundation (Grant 149781), the Health and stimulatory effect of eosinophils on activated T cells (4–10). and Medical Care Committee of the Regional Executive Board of Region Va¨stra Another objective was to assess the capacity of eosinophil galectin-10 Go¨taland (96490), the Inga Britt and Arne Lundberg Research Foundation, and the to mediate T cell suppression. University of Gothenburg. Address correspondence and reprint requests to Prof. Christine Wenneras,˚ Depart- ment of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, Uni- Materials and Methods versity of Gothenburg, Guldhedsgatan 10A, Floor 6, Go¨teborg SE-413 46, Sweden. Human samples E-mail address: [email protected] Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; FMO, fluores- Healthy adults donated blood for the purpose of this study and completed a cence minus one; FPR1, formyl -1; median-FI, median fluorescence form disclosing all diseases and ongoing medication. Volunteers were not intensity; Treg, . allowed to donate blood if they had current symptomatic allergic disease, other symptomatic inflammatory processes, and/or were being treated Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 with or other immunosuppressive and/or antiflogistic drugs.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601005 2 CD16hi/GAL-10hi EOSINOPHILS ARE POTENT T CELL SUPPRESSORS

EDTA-anticoagulated venous blood was used for flow cytometry analyses, freshly isolated eosinophils. Results are expressed as median fluorescence and heparin-anticoagulated blood was used for the T cell–suppression intensity (median-FI) or the percentage of positive cells. assays. The study was approved by the Regional Ethical Review Board of Gothenburg, Sweden. FACS Eosinophil purification Granulocytes were freshly isolated from heparinized venous blood by Ficoll separation following removal of erythrocytes by dextran sedimentation and Eosinophils were freshly isolated from heparinized venous blood by hypotonic lysis. The cells were incubated with 1 mg/ml Vivaglobin before negative immunomagnetic depletion using a mixture of magnetic beads the addition of fluorochrome-conjugated mAbs against Siglec-8, CD16, and conjugated to mAbs against CD3, CD14, CD16, and CD19, as previously CD193 (CCR3) (Table II). A SY3200 flow cytometer sorter (Sony Bio- described (17), and were used directly in T cell–suppression assays. technology) equipped with a 70-mm nozzle was used to separate cells at a Suppression assays of CD3/CD28-stimulated PBMCs or T cells PBMCs (105 cells) or T cells (105 cells) purified using the Pan T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) were added to anti-CD3 mAb-coated (1 mg/ml, OKT-3; Affymetrix) round-bottom 96-well plates (TPP, Trasadingen, Switzerland) together with anti-CD28 mAb (2 mg/ml, clone CD28.2; Affymetrix) in X-VIVO 15 buffer (Lonza, Basel, Switzerland) supplemented with 50 mg/ml gentamicin (Sigma- Aldrich). Three hours later, homologous eosinophils were added to the CD3/CD28-stimulated cell cultures: conventional (i.e., immunomagnetically purified eosinophils; 105 cells) or FACS-sorted eosinophils (i.e., CD16neg, dim hi 4 CD16 ,orCD16 eosinophils; 10 cells) were used. The cocultures were Downloaded from incubated for 2 d at 37˚C and then pulsed with [3H]thymidine (1 mCi per well; PerkinElmer) for 6 h to assess T cell proliferation. In the neutralization experiments, mAbs against CD16 (4 mg/ml), galectin-10 (8–10 mg/ml), CD18, CD54, CD274, CD279 (all at 10 mg/ml) or an control (10 mg/ml) were added to the cultures (Table I). Various concentrations of recombinant galectin-10 (NBP1-51096; Novus Biologicals, Abingdon, U.K.) dissolved in 20 mM Tris-HCl (pH 8) supplemented with 1 mM 1,4-DTT, 10% glycerol, and 0.1 M NaCl and further diluted in cell culture medium http://www.jimmunol.org/ were tested for their ability to suppress CD3/CD28-stimulated proliferation of PBMCs. Kinetics of eosinophil CD16 expression Eosinophils were analyzed for CD16 expression using flow cytometry before and after 4, 14, and 40 h of coculture with CD3/CD28-stimulated PBMCs. Cells were stained with mAbs against CCR3 (Brilliant Violet 421), CD16 (FITC), and isotype control (FITC) (Table II). Dead eosinophils were identified by staining for Annexin V and 7-aminoactinomycin D (7-AAD) (PE Annexin V Detection Kit I; BD Biosciences); only Annexin by guest on September 27, 2021 Vneg 7-AADneg CCR3+ eosinophils were analyzed for CD16 expression. An isotype-control Ab (FITC) was used to monitor background staining (Table II). CD16 isoform Eosinophils were stained using two clones of anti-CD16 mAbs: one rec- ognizing the A and B isoforms (3GA) and one specific for the B isoform (clone CLB-gran11.5) (Table II). Suppression assay of allogeneically activated PBMCs Eosinophils were exposed to PBMCs activated by HLA-mismatched leukocytes in a one-sided MLR, as previously described (7). In short, eosinophils and PBMCs (responder cells; 105) were added to wells at a ratio of 1:1; 1:3 or 1:10. A pool of gamma-irradiated PBMCs from 11 healthy adult donors was used as trigger cells at a 1:1 ratio with responder cells. The cocultures were incubated for 6 d in RPMI 1640 buffer supple- mented with 10% human AB serum, 2 mM L-glutamine, 50 mg/ml gentamicin, and 0.05 mM 2-ME. T cell proliferation was determined by [3H]thymidine incorporation. Galectin-10 neutralization was attempted by the addition of anti–galectin-10 mAb (Table I). An isotype-matched Ab was used as a control (Table I). Flow cytometry EDTA-anticoagulated blood was depleted of erythrocytes by repeated osmotic lysis. The remaining unfractionated leukocytes were stained with Fixable Viability Dye eFluor 780 (Affymetrix) and incubated with 1 mg/ml Vivaglobin (CLS Behring, King of Prussia, PA) to block Fc receptors, followed by the addition of fluorochrome-conjugated mAbs (Table II). For FIGURE 1. Eosinophils suppress the proliferation of PBMCs and purified subsequent intracellular staining, cells were fixed and permeabilized using T cells. (A) Eosinophils at a 1:1 PBMC ratio suppress the proliferation of the Foxp3/ Factor Staining Buffer Set (Affymetrix). Imme- PBMCs stimulated by CD3/CD28 cross-linking (n =6).(B) Dose-response diately after staining, 10,000 cells were acquired with a FACSCanto II flow curve of the capacity of eosinophils to suppress the allogeneic proliferation cytometer (BD Biosciences) and FACSDiva Software 8.0.1 and were an- C alyzed with FlowJo software 7.6.5 (TreeStar). Fluorochrome minus one of PBMCs (n =6).( ) Comparison of the capacity of eosinophils to sup- (FMO) and isotype controls were used to monitor background surface and press the polyclonal proliferation of purified T cells and PBMCs evoked by intracellular staining, respectively. Eosinophils in T cell cocultures were CD3/CD28 stimulation (n =2).*p , 0.05, ***p , 0.001, paired Wilcoxon harvested after 16–18 h and subjected to the same staining procedure as the signed test. The Journal of Immunology 3 concentration of 107 granulocytes per milliliter. Diff-Quik staining was ventional CD16neg eosinophils (Fig. 2C), because galectin-10 took used to ascertain eosinophil purity (Dade Behring). part in the T cell–suppressive process. We sought to neutralize the Imaging flow cytometry T cell–suppressive function of eosinophils by adding anti–galectin- 10 mAbs to the eosinophil:T cell cocultures (Fig. 3A). Eosinophils Cells from 2-d-old eosinophil:PBMC cocultures were incubated with incubated with proliferating T cells in an MLR at a 1:1 ratio allophycocyanin-conjugated anti-CCR3 mAb, PE-conjugated anti–galectin- 10 mAb, and the DNA stain DAPI (140 nM) (Table II). One thousand cells inhibited 80% of the T cell proliferation (MLR without eosinophils, were collected in the imaging flow cytometer (ImageStream X Mark II; median cpm = 55,427; MLR with eosinophils, median cpm = 11,327). Amnis, Seattle, WA) and analyzed with IDEAS software 6.0. When the anti–galectin-10 mAb was added to the MLR:eosinophil Statistical analyses cocultures, T cell proliferation was only inhibited by 60% (MLR with anti–galectin-10 mAb, median cpm = 59,115; MLR with eosin- The Wilcoxon matched-pairs signed-rank test and Kruskal–Wallis one-way ophils and anti–galectin-10 mAb, median cpm = 23,764). This resulted ANOVA were used for paired and multiple comparisons, respectively (Graph- Pad Prism 6.0 Software). The p values ,0.05 were considered statistically in a partial restoration of the ability of T cells to proliferate, such significant. Multivariate analyses were performed using Orthogonal-Projection that a doubling of the T cell proliferation was achieved (Fig. 3A); to Latent Structures with variable importance in the projection application no such effect was seen for the isotype control (Fig. 3B). We also (SIMCA 14.1 software; MKS Data Analytics Solutions, Malmo¨,Sweden). tested the capacity of recombinant galectin-10 to suppress T cell proliferation and found that galectin-10 suppressed CD3/CD28- Results induced PBMC proliferation in a dose-dependent manner (Fig. 3C). Eosinophils suppress the polyclonal proliferation of PBMCs On average, the highest concentration of galectin-10 that was tested and purified T cells (10 mg/ml) inhibited proliferation by 26% (Fig. 3C). Downloaded from First, we show that human eosinophils suppressed the activation of Galectin-10–containing immune synapses between eosinophils PBMCs triggered by ligation of CD3/CD28 or by allogeneic T cells and lymphocytes (Fig. 1A, 1B). The eosinophil-mediated suppression was dose de- pendent and maximal at a 1:1 ratio of eosinophils/PBMCs (Fig. 1B). Because our earlier work had shown that cell–cell contact is re- Eosinophils were also able to suppress the proliferation of purified quired for eosinophils to suppress lymphocytes (7), a logical step

T cells, in a dose-dependent manner, in the absence of the accessory was to investigate whether eosinophil suppression entailed the http://www.jimmunol.org/ cells present in the PBMC fraction (Fig. 1C). formation of immune synapses. Cell clusters of two or more cells hi lo in cocultures of eosinophils with CD3/CD28-activated PBMCs CD16 /galectin-10 subset of eosinophils appears after were stained with mAb against galectin-10 and analyzed by im- coculture with proliferating PBMCs aging flow cytometry. Close apposition of eosinophils (CCR3+ Following coculture of eosinophils with proliferating PBMCs for cells with lobulated nucleus) and lymphocytes (round nucleus) 2 d, we discovered that the eosinophils could be divided into two was seen with bridge formation (Fig. 4) and apparent transfer of populations, one of which expressed CD16 de novo. At the start of galectin-10 from the eosinophils to the lymphocytes (Fig. 4A, 4D). neg the culture, all eosinophils were CD16 , because CD16 expression hi hi

CD16 /galectin-10 subset identified among blood by guest on September 27, 2021 is the basis for the negative immunoselection performed to isolate eosinophils eosinophils from in the fraction. A repre- sentative flow cytometry plot is shown in Fig. 2. The CD16hi Next, we examined the blood of healthy individuals for the subgroup of eosinophils expressed lower levels of galectin-10 than presence of CD16-expressing eosinophils. Eosinophils were gated did conventional CD16neg eosinophils (Fig. 2C). After 2 d of in- based on high forward and side scatter and high CCR3 expression + cubation with proliferating T cells, 11% of eosinophils expressed (Fig. 5A, 5B). CCR3 eosinophils were characterized further hi CD16 (median; range 3–15%) and 89% were conventional CD16neg based on CD16 expression. Indeed, a subset of CD16 eosinophils eosinophils (median; range 85–97%) (n = 6 eosinophil donors). was identified in the blood of healthy adults (Fig. 5C); however, compared with the CD16hi subset of eosinophils evoked by ex- Galectin-10 mediates part of the T cell–suppressive activity of posure to proliferating T cells, these eosinophils had increased eosinophils expression of galectin-10 (Fig. 5D). This CD16hi/galectin-10hi We reasoned that CD16hi eosinophils that arose after exposure to subset constituted ∼5% (median; 25–75th percentiles, 3–7%) of proliferating T cells contained less galectin-10 than did the con- the eosinophils in the blood of adults (n = 9). When vital dye was

FIGURE 2. A CD16hi/galectin-10lo eosinophil subset develops after coculture with CD3/CD28-stimulated PBMCs. (A and B) Gating strategy used for defining CD16neg and induced CD16hi CCR3+ eosinophils. (A) FMO control. (B) CD16 expression. (C) Representative dot plot (of n = 6 eosinophil donors) depicting the two CCR3+ eosinophil populations (CD16hi/galectin-10lo and CD16neg/galectin-10+) that appear after coculture for 2 d with proliferating T cells. 4 CD16hi/GAL-10hi EOSINOPHILS ARE POTENT T CELL SUPPRESSORS

tional CD16neg eosinophils. We were also curious to determine whether eosinophils expressing moderate levels of CD16 (CD16dim) would be superior T cell suppressors compared with eosinophils that expressed no CD16. Our theory was that this subset might be a “resting regulatory subset of eosinophils” that became activated when the cells were incubated with proliferating T cells. Using flow cytometry–activated cell sorting, we were able to separate CD16neg, CD16dim, and CD16hi eosinophils from the blood of seven healthy individuals. The gating strategy was to first identify eosinophils based on high forward and side scatter, followed by expression of Siglec-8 and CCR3 (Fig. 6A). Next, the eosinophils were subgated and sorted into CD16neg, CD16dim, and CD16hi subsets (Fig. 6B). Sorting of CD16dim eosinophils was based on eosinophils expressing higher levels of CD16 compared withanFMOcontrol.Sortedcells were .98% eosinophils, as determined by eosin staining (Fig. 6C). Because of the paucity of CD16hi eosinophils, sorted eosinophils were cocultured with CD3/CD28-activated T lymphocytes at a 10-fold lower ratio (1:10) than the ratio normally used in cocul-

tures with conventional nonsorted eosinophils (1:1). CD16hi eosino- Downloaded from phils inhibited T cell proliferation by a median of 72% (25–75th percentiles: 65–80%), CD16dim eosinophils inhibited T cell proliferation by a median of 53% (25–75th percentiles: 40–58%), and CD16neg eosinophils inhibited T cell proliferation by a median of 46% (25–75th percentiles: 37–51%) (Fig. 6D). CD16hi eosin-

ophils were significantly more potent T cell suppressors compared http://www.jimmunol.org/ with CD16neg (p = 0.016) and CD16dim (p = 0.016) eosinophils (Fig. 6D). Phenotype of CD16hi eosinophils in the blood of adults Next, we wanted to characterize the phenotype of the CD16hi eosinophils in peripheral blood in more detail. To this end, we compared the expression of 40 surface markers on CD16hi eosin- ophils and conventional CD16neg eosinophils in five healthy adults. We selected a broad range of molecules encompassing activation by guest on September 27, 2021 markers, adhesion molecules, and receptors, and immunoregulatory molecules known to be expressed by eo- sinophils or other types of suppressive cells (e.g., Tregs, myeloid- derived suppressor cells, and suppressive neutrophils) (Tables I, II). Multivariate analysis revealed that 19 of the tested molecules were differentially expressed on CD16hi eosinophils in compari- son with conventional CD16neg eosinophils: 11 molecules were upregulated on CD16hi eosinophils, and 8 molecules were down- regulated (Fig. 7A). Hence, CD274 (PD-), CD40, CD194 (CCR4), CD183 (CXCR3), TSLPR, CD54 (ICAM-1), CD4, formyl FIGURE 3. Eosinophils from healthy subjects partially suppress T cell peptide receptor-1 (FPR1), CD199 (CCR9), CD44, and CD66c hi proliferation via galectin-10. Conventional CD16neg eosinophils were added at were more highly expressed on the surface of CD16 eosinophils a 1:1 ratio to PBMCs activated allogeneically in an MLR. Cocultures were relative to conventional eosinophils, whereas the surface levels allowed to proceed for 6 d. (A) T cell proliferation after 6 d of coculture with of Siglec-8, CD49d (a4-chain of VLA-4), CD45, CD294 or without eosinophils and with or without addition of anti–galectin-10 mAb (CRTH2), CD9, CD71, CD66b, and CD11a were depressed on (n = 18). Bars represent median values with interquartile ranges. (B)An CD16hi eosinophils compared with conventional eosinophils isotype-matched control mAb had no impact on T cell proliferation (n =5; (Fig. 7A). The median-FI values for these surface markers on C pairedWilcoxonsignedranktest).( ) Recombinant galectin-10 was added CD16hi eosinophils and conventional eosinophils are shown in to cultures of CD3/CD28-stimulated PBMCs from four blood donors. The Fig. 7B and 7C. percentage inhibition of T cell proliferation evoked by different concentra- tions of galectin-10 is shown. The p value was determined using Kruskal– Eosinophil CD16 expression is triggered by proliferating , Wallis one-way ANOVA. **p 0.01, paired Wilcoxon signed-rank test. T cells, but CD16 does not mediate T cell suppression The kinetics of CD16 expression by eosinophils induced by co- used to exclude dead or dying eosinophils, CD16-expressing culture with CD3/CD28-activated PBMCs were studied (n = 4). eosinophils constituted 1.1% of the total blood eosinophils Only viable eosinophils (i.e., cells that were negative for 7-AAD (median; 25–75th percentiles, 0.57–1.3; n =5). and Annexin V) were analyzed for expression of CD16 (Fig. 8A). hi Maximal CD16 expression was already reached after 4 h, such that CD16 subset is a superior suppressor of T cell proliferation an average of 22% of the eosinophils expressed CD16; thereafter, Subsequently, we assessed whether CD16hi eosinophils were more the proportion of CD16-expressing eosinophils declined over the potent suppressors of T cell proliferation compared with conven- 40-h incubation period (Fig. 8B). However, eosinophils that were The Journal of Immunology 5

FIGURE 4. Eosinophils form galectin-10–con- taining synapses with lymphocytes. (A–D) Image- Stream photographs of eosinophils cocultured with CD3/CD28-activated PBMCs for 2 d, showing bright- field (BF) images, CCR3 staining, DNA staining (DAPI), galectin-10 expression, and an overlay image of the BF and galectin-10 images. DAPI stains show the round nucleus of lymphocytes and the bilobed nucleus of eosinophils. Arrows indicate synapses between lymphocytes and eosinophils. Downloaded from incubated in the absence of PBMCs also began to express CD16, subset in blood (n = 2), as well as the CD16hi eosinophils in- and to an even greater extent: an average of 42% of eosinophils duced after coculture with activated PBMCs (n = 4), expressed expressed CD16 after 4 h, and this fraction of CD16-expressing the B isoform. eosinophils remained elevated for a longer period of time com- hi Comparison of the phenotype of naturally occurring CD16 http://www.jimmunol.org/ pared with eosinophils incubated with activated PBMCs (Fig. 8B, eosinophils with induced CD16hi eosinophils inset). Neutralization experiments revealed that CD16 itself was Six of the molecules whose expression differed the most between not directly involved in eosinophil-mediated T cell suppression, conventional CD16neg eosinophils and the naturally occurring because blocking of CD16 did not abolish the suppressive ca- CD16hi eosinophils in the blood of healthy subjects were inves- pacity of eosinophils (data not shown; n = 5). We also analyzed tigated to determine whether eosinophils whose CD16 expression which of the two CD16 isoforms (A or B) that the regulatory was induced by coculture with CD3/CD28-activated eosinophils eosinophils expressed by staining the cells with two clones of had a similar molecular profile. A direct comparison of CD16hi CD16 mAbs: one recognizing both isoforms and the other spe-

eosinophils before (day 0) and after overnight incubation (day 1) by guest on September 27, 2021 cific for the B isoform. We found that the naturally occurring with activated PBMCs (n = 5–6) showed that the patterns were similar, but not identical: CD54, CD66c, CD199, CD274, and FPR1 were expressed by naturally occurring and induced CD16hi eosinophils (Fig. 9). A few differences were noted between the two cell subsets: CD44 was barely detectable on resting CD16hi eosinophils, and the levels of CD54 and CD274 (PD-L1) were borderline and significantly increased on the induced eosinophils compared with the naturally occurring cells (p = 0.062, n = 5 for CD54; p = 0.031, n = 6 for CD274) (Fig. 9). Neutralization experiments Lastly, we decided to neutralize surface markers on eosinophils to better understand the suppressive mechanisms used by eosinophils. We selected the two molecules whose expression increased the most on the surface of CD16hi eosinophils induced by proliferating PBMCs: CD54 and PD-L1 and their respective counter-receptors, LFA-1 (CD11a, CD18) and PD-1. We discovered that the addition of a neutralizing Ab against CD54 to CD3/CD28-activated PBMCs diminished their proliferative capacity by a median of 30% compared with the median reduction of 61% accomplished by eosinophils (Fig. 10A). When eosinophils and the anti-CD54 Ab were combined, an additive effect was seen, resulting in FIGURE 5. A CD16hi/galectin-10hi subset of eosinophils is found in the 83% reduction of proliferation (Fig. 10A). Similar to the CD54- peripheral blood of healthy adults. (A) Representative dot plot of whole- neutralization experiments, the anti-CD18 mAb on its own dimin- blood leukocytes depicting the gating strategy used for defining gran- ished the proliferation of activated PBMCs and had an enhancing B ulocytes. ( ) Eosinophils are distinguished from neutrophils based on their effect on the eosinophil-mediated suppression of PBMC prolifera- high expression of CCR3. (C) Gating of CD16hi and CD16neg eosinophils. (D)CD16hi eosinophils in the blood of healthy control subjects (HC) have tion (Fig. 10B). In contrast, anti–PD-L1 and anti-PD1 Abs on their higher intracellular levels of galectin-10 than do conventional CD16neg eo- own did not decrease the proliferative capacity of activated PBMCs; sinophils, measured as the percentage increase in median-FI. The p value instead they gave rise to a slight increase in PBMC proliferation was determined using a paired Wilcoxon signed-rank test. above baseline (Fig. 10C, 10D). Although addition of eosinophils 6 CD16hi/GAL-10hi EOSINOPHILS ARE POTENT T CELL SUPPRESSORS

FIGURE 6. The eosinophil CD16hi subset is a superior T cell suppressor. (A) Eosinophils were distinguished from neutrophils based on their high expression of CCR3 and Siglec-8. (B) Gating strategy used for defining CD16hi, CD16dim, and CD16neg eosinophils. (C) Cyto- spun CD16hi eosinophils stained with Diff-Quik (original magnification 320). (D) FACS-sorted CD16hi, CD16dim, and CD16neg eosinophils from healthy individuals were cocultured at a 1:10 ratio with CD3/CD28-stimulated T cells for 2 d (n = 7). Each symbol with a connecting line denotes the T cell–inhibitory effect of hi dim neg

CD16 ,CD16 and CD16 eosinophils, Downloaded from respectively. *p , 0.05, paired Wilcoxon signed- rank test. http://www.jimmunol.org/

and the anti–PD-L1 mAb to the cocultures resulted in more de- pression and found that the higher the CD16 levels, the more pressed proliferation than when eosinophils were added alone suppressive the eosinophils. However, this T cell–suppressive (Fig. 10C), addition of the anti-PD1 mAb did not boost the anti- capacity was not directly dependent on CD16, because neutrali- proliferative effect of eosinophils (Fig. 10D). zation of this surface Ag had no impact on suppression. This was to be expected because eosinophils express the B isoform of CD16 Discussion that is loosely attached to the plasma membrane via a GPI anchor by guest on September 27, 2021 We identified a CD16hi subset of eosinophils in the blood of (19, 20) and, hence, is unable to transmit signals into the cell on its healthy adults that are potent T cell suppressors in vitro. This own (21). Eosinophils contain intracellular stores of CD16 that discovery originated from our finding that a CD16hi subset of can be mobilized within minutes to the cell surface by in vitro eosinophils appeared after coincubation with proliferating T cells. exposure to given chemoattractants (fMLF, C5a, PAF) and cyto- There were no CD16hi eosinophils at the start of the cocultures, kines (IFN-g) (19, 20). The broad range of in vitro stimuli capable because eosinophils are routinely purified from the granulocyte of triggering CD16 expression may explain why we found that fraction of leukocytes by negative immune selection based on 20–40% of the eosinophils that were incubated on their own (e.g., their supposed lack of CD16 expression. But after 2 d of coculture, in the absence of growth factors) or in the presence of activated 11% of the eosinophils gained CD16 expression. This led us to lymphocytes upregulated CD16. Our results agree with those of search for CD16hi eosinophils in blood and, indeed, we found such Hartnell et al. (19), who showed that CD16 is never expressed by a population. CD16-expressing eosinophils have been described all of the in vitro–activated eosinophils, but only by 5–34% of them. previously in the blood of persons with allergic asthma, rhinitis, However, it has proven difficult to transfer the in vitro findings to and hypereosinophilia due to , but the significance the in vivo situation: despite IFN-g being a potent in vitro upre- of CD16 expression by eosinophils was not determined (18). We gulator of CD16, no correlation was found between the serum decided to sort eosinophils from healthy persons with no evidence concentrations of IFN-g and the levels of CD16-expressing eo- of allergic or other inflammatory processes based on CD16 ex- sinophils in the circulation of allergic patients (18). We found that CD16hi eosinophils in the blood expressed higher neg Table I. Neutralizing mAbs levels of galectin-10 than did conventional CD16 eosinophils, whereas CD16hi eosinophils that appeared after coculture with Marker (Clone) proliferating T cells contained less galectin-10. This led us to hypothesize that galectin-10 was upregulated in naturally occur- a CD16 (3G8) hi b ring CD16 eosinophils in the circulation but had been consumed CD18 (TS1/18) hi CD54/ICAM-1 (HA58)a by the “culture-induced” CD16 eosinophils. This, together with CD274/PD-L1 (MIH1)c the demonstration by Kubach et al. (14) that galectin-10 functions CD279/PD-1 (J116)c as a suppressive molecule for Tregs, prompted us to explore whether Galectin-10 (B-F42)d a this function was shared by eosinophil galectin-10. The suppres- Isotype (107.3) sion of T cells by eosinophils was indeed partially mediated by aBD Biosciences. galectin-10, because 20% of the eosinophil-mediated suppression bBioLegend. cAffymetrix eBioscience was abrogated when galectin-10 was neutralized. This was reinforced dDiaclone SAS. by our discovery that recombinant galectin-10 on its own also exerted The Journal of Immunology 7

Table II. mAbs used for flow cytometry and FACS (CD11a/CD18), which is essential for the formation of immuno- logical synapses between APCs and T cells (29). CD54 has been FITC-conjugate (clone) Brilliant Violet 421-conjugate (clone) CD3 (UCHT1)a CD193/CCR3 (5E8)b shown to mediate part of the lymphocyte-suppressive capacity of CD8 (RPA-T8)a CD274/PD-L1 (29E.2A3)b murine mesenchymal stem cells, which is contact dependent (30). CD16 (3G8)a APC-conjugate (clone) We have previously reported that cell–cell contact is required for CD66b (80H3)c CD4 (RPA-T4)a a eosinophils to suppress T cell proliferation (7). In the current study, CD66c (KOR- CD11c (B-ly6) we describe the formation of synapses between eosinophils and SA3544)c CD70 (Ki-24)a CD18 (6.7)a lymphocytes. Cellular proximity is also needed for suppressive Isotype (MOPC-21)a CD23 (EBVCS-5)a neutrophils to exert their function, and they too form synapses with PE-conjugate (clone) CD25 (2A3)a lymphocytes (24). A Japanese study showed that CD54 blockade a a CD9 (M-L13) CD40 (5C3) reverted ∼60% of the suppression exerted by eosinophils on PHA- CD11a (HI111)a CD44 (G44-26, C26)a CD11b (D12)a CD54/ICAM-1 (HA58)a induced T cell proliferation (31). We were puzzled by our opposite CD11c (B-ly6)a CD69 (FN50)a results: neutralization of CD54 amplified the suppressive effect of CD16 (3G8)a CD71 (M-A712)a CD16 (CLB-gran11.5)a CD86 (2331 (FUN-1))a CD31 (WM59)a CD183/CXCR3 (1C6/CXCR3)a CD35 (E11)a CD184/CXCR4 (12G5)a CD40 (5C3)a CD193/CCR3 (5E8)a CD49d (9F10)a CD195/CCR5 (2D7/CCR5)a a b CD80 (L307.4) CD199/CCR9 (L053E8) Downloaded from CD90 (5E10)a CD274/PD-L1 (29E.2A3)b CD193/CCR3 (5E8)a Galectin-10 (B-F42)d,e CD194/CCR4 (1G1)a Siglec-8 (7C9)b CD274/PD-L1 Isotype (MOPC-21)a (MIH1)a TSLPR (1F11/ AF647-conjugate (clone) TSLPR)a FPR1 (350418)d CD105 (266)a http://www.jimmunol.org/ HLA-DR (G46-6)a CD127 (HIL-7R-M21)a Galectin-10 (B-F42)d,e CD193/CCR3 (5E8)a CD294/CRTH2 (BM16)a PerCP-Cy5.5-conjugate (clone) CD45 (HI30)a aBD Biosciences. bBioLegend. cBeckman Coulter. dDiaclone SAS. by guest on September 27, 2021 a T cell–suppressive effect that was of similar magnitude as the suppression mediated by galectin-10 released from eosinophils. To our knowledge, this is the first time that a function has been assigned to galectin-10 in eosinophils. In addition, we found that immune synapses containing galectin-10 formed between eo- sinophils and lymphocytes, suggestive of intracellular transfer of galectin-10. This might indicate that the neutralization ex- periments underestimated the suppressive effect of eosinophil galectin-10, because neutralizing Abs would not access intracellu- larly deposited galectin-10. However, much remains to be elucidated with regard to galectin-10, especially how it is secreted from eosin- ophils and the mechanisms behind its immune-suppressive function. Another objective of the study was to phenotype the surface of the regulatory eosinophils, including those in the blood of healthy individuals (naturally occurring CD16hi eosinophils) and those that were induced by incubation with actively dividing T cells (“in- duced” CD16hi eosinophils). To this end, we selected a broad panel of mAbs directed at molecules expressed by eosinophils, as well as molecules expressed by other regulatory cells (i.e., Tregs, sup- FIGURE 7. Surface phenotype of regulatory CD16hi eosinophils in pressive neutrophils, and myeloid-derived suppressor cells) (22–25). peripheral blood. The surface molecular profile of CD16hi eosinophils and neg The resting CD16hi eosinophils resembled suppressive neutrophils conventional CD16 eosinophils was compared. (A) Loading plot ob- based on their expression of CD16 and upregulation of CD54 (24). tained using Orthogonal-Projection to Latent Structures analysis, followed They were also found to have some markers in common with Tregs: by Variable Importance in the Projection analysis with a cut-off = 1. The upper shaded bars represent molecules that are relatively increased on the the adhesion molecule CD44, the homing markers CD194 (CCR4) surface of the CD16hi eosinophils, whereas the lower shaded bars represent and CD199 (CCR9), the suppressive marker galectin-10, as well molecules that are relatively decreased on the surface of CD16hi eosinophils. as depressed levels of CD49d (14, 26–28). Univariate analyses indicating the surface molecules that are upregulated (B) CD54 and CD274 were strongly upregulated on eosinophils and downregulated (C)onCD16hi eosinophils compared with conventional hi exposed to proliferating T cells (induced CD16 eosinophils). eosinophils. Box-and-whisker plots show medians and 10th to 90th percentiles. CD54 (ICAM-1) is an adhesion molecule whose receptor is LFA-1 The p values were determined using Kruskal–Wallis one-way ANOVA. 8 CD16hi/GAL-10hi EOSINOPHILS ARE POTENT T CELL SUPPRESSORS

FIGURE 9. Comparison of the molecular patterns displayed by naturally occurring CD16hi eosinophils (Day 0) and induced CD16hi eosinophils (Day 1). Blood eosinophils from five or six healthy donors were analyzed by flow cytometry for the expression of six surface molecules before and after overnight incubation with CD3/CD28-stimulated PBMCs. Only viable CD16hi eosinophils, determined using eFluor 780 stain, were analyzed. Bars represent the median-FI levels + range for the six molecules that were Downloaded from expressed by CD16hi eosinophils. *p , 0.05, paired Wilcoxon signed- rank test.

regulatory capacity was not investigated (33). We found that the

anti–PD-L1 mAb enhanced the antiproliferative effect of eosino- http://www.jimmunol.org/ phils on CD3/CD28-stimulated PBMC proliferation. This is op- posite from what has been described for Tregs, in which addition of a PD-L1–inhibitory Ab boosted the proliferation of effector T cells activated by CD3/CD28, requiring higher ratios of Tregs/ effector T cells to maintain suppressive capacity (34). A similar role for PD-L1 was reported for a subset of suppressive neutro- phils (i.e., neutralization of PD-L1 resulted in a partial reversal of FIGURE 8. Kinetics of CD16 expression induced by exposure of eo- neg the capacity of the cells to inhibit T cell proliferation) (35). Thus,

sinophils to proliferating T cells. Conventional CD16 eosinophils were by guest on September 27, 2021 with regard to the effect of PD-L1 neutralization on suppressive cocultured with CD3/CD28-activated PBMCs for 40 h. Only viable eosino- hi phils, as determined by Annexin V and 7-AAD staining, were studied. (A) capacity, regulatory CD16 eosinophils differ from Tregs and Representative dot plots from one individual show gating of live eosinophils suppressive neutrophils. hi (7-AADneg,AnnexinVneg) and their CD16 expression and binding of an The frequency of CD16 eosinophils in the blood of healthy isotype-matched control mAb after 4, 14, and 40 h of coculture with pro- persons was estimated to be ∼1% of the total number of eosino- liferating T cells. (B) The percentages of eosinophils that expressed CD16 phils when we considered only live cells. This frequency is on the were determined by flow cytometry after 4, 14, and 40 h of coculture with same order as the original estimates of the frequency of Tregs in CD3/CD28-stimulated PBMCs (n = 4). The inset shows CD16 expression by human blood (1–2%), when these cells were defined as CD25hi single cultures of eosinophils. CD4+ T cells (34). The frequency of CD16hi eosinophils was 5-fold higher if we took live and dead cells into consideration. The “dead eosinophils on PBMC proliferation. The answer to this conundrum CD16hi eosinophils” might be an artifact (i.e., dead conventional probably lies in the complexity of the suppression assays, which eosinophils that became autofluorescent and were misinterpreted involve several types of leukocytes (T cells, accessory cells, eosin- to bind the fluorochrome-labeled anti-CD16 mAb). Alternatively, ophils). Our results indicate that CD3/CD28-stimulated PBMCs also these were true CD16hi eosinophils that had died following their upregulated CD54, because addition of the neutralizing Ab (in the activation by proliferating T cells. Human Tregs have a high turnover absence of eosinophils) diminished their proliferative capacity by and rapidly undergo apoptosis in vitro once they have accomplished 30%. Shevach and coworkers (32) elegantly resolved the dilemma of their task (36), but they maintain their suppressive capacity even how to define whether it is the suppressor cell or the target cell that when they are dead (e.g., fixed in paraformaldehyde) (37). In expresses a particular molecule by setting up suppression assays contrast, suppressive neutrophils cannot be identified in the blood of consisting of murine Tregs combined with human effector T cells, or healthy individuals and are only detectable when the host is afflicted human Tregs combined with murine effector T cells. They found that by severe conditions, such as invasive or major injury the suppressive capacity of human Tregs relied on the expression of (24). A goal for future studies will be to determine the frequency of CD11a/CD18, which paired with CD54 on effector T cells (32). This regulatory CD16hi eosinophils in the blood of patients with allergic may explain why we observed enhanced suppression when eosino- and other inflammatory processes driven by T cells. phils and the neutralizing CD54 Ab were combined. In contrast, To conclude, we identified a highly T cell–suppressive subset of when the anti–galectin-10 mAb was combined with eosinophils, their eosinophils in the blood of healthy subjects. Similar to Tregs and suppressive capacity decreased, which is logical in view of the fact suppressive neutrophils (24, 34), these regulatory eosinophils were that only eosinophils express galectin-10; neither effector T cells nor able to inhibit T cells directly in the absence of APCs. We have accessory cells do so. previously reported the necessity of cell–cell contact for eosino- Eosinophils isolated from the sputum of asthmatic patients were phils to be suppressive (7), a feature shared by Tregs (34) and sup- recently shown to express PD-L1 (CD274), but their immune- pressive neutrophils (24); we now expand this knowledge by the The Journal of Immunology 9

FIGURE 10. Additive suppressive effect of eosinophils combined with neutralizing Abs. PBMCs were induced to proliferate by CD3/ CD28 cross-linking. Eosinophils (1:3 PBMCs) and/or neutralizing mAbs (8–10 mg/ml) were added to the cultures, and their capacity to in- hibit proliferation was evaluated after 2 d. The proliferation of PBMCs in the absence of Abs and eosinophils was set as 100% (dotted line). Reduction in PBMC proliferation in the pres- ence of mAbs to CD54 (A), CD18 (B), PD-L1 (C), and PD1 (D), with or without added eo- sinophils. PBMCs and eosinophils from n = 7 donors. Box-and-whiskers plots show medians Downloaded from and 10th to 90th percentiles. The p values were determined using a paired Wilcoxon signed- rank test. http://www.jimmunol.org/

demonstration of immune synapses between eosinophils and 11. Gleich, G. J., A. D. Klion, J. J. Lee, and P. F. Weller. 2013. The consequences of not having eosinophils. Allergy 68: 829–835. lymphocytes. We also provide evidence that galectin-10 medi- 12. Tulic, M. K., P. D. Sly, D. Andrews, M. Crook, F. Davoine, S. O. Odemuyiwa, ates the suppressive function of eosinophils. Given the superior A. Charles, M. L. Hodder, S. L. Prescott, P. G. Holt, and R. Moqbel. 2009. T cell–suppressive capacity of CD16hi eosinophils, we propose Thymic indoleamine 2,3-dioxygenase-positive eosinophils in young children: potential role in maturation of the naive . Am. J. Pathol. 175: to name this immune-regulatory subset of eosinophils suppres- 2043–2052. by guest on September 27, 2021 sive eosinophils, in analogy with suppressive neutrophils. 13. Bacchetta, R., F. Barzaghi, and M.-G. Roncarolo. 2016. From IPEX syndrome to FOXP3 : a lesson on . Ann. N. Y. Acad. Sci. Feb. 25. doi:10.1111/nyas.13011. Disclosures 14. Kubach, J., P. Lutter, T. Bopp, S. Stoll, C. Becker, E. Huter, C. Richter, The authors have no financial conflicts of interest. P. Weingarten, T. Warger, J. Knop, et al. 2007. Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function. Blood 110: 1550–1558. 15. Ackerman, S. J., S. E. Corrette, H. F. Rosenberg, J. C. Bennett, D. M. Mastrianni, References A. Nicholson-Weller, P. F. Weller, D. T. Chin, and D. G. Tenen. 1993. Molecular 1. Cromvik, J., M. Johnsson, K. Vaht, J. E. Johansson, and C. Wenneras.˚ 2014. cloning and characterization of human eosinophil Charcot-Leyden crystal pro- Eosinophils in the blood of hematopoietic stem cell transplanted patients are tein (lysophospholipase). Similarities to IgE binding proteins and the S-type J. Immunol. activated and have different molecular marker profiles in acute and chronic graft- animal lectin superfamily. 150: 456–468. 16. Lee, J. J., E. A. Jacobsen, S. I. Ochkur, M. P. McGarry, R. M. Condjella, versus-host disease. Immun. Inflamm. Dis. 2: 99–113. A. D. Doyle, H. Luo, K. R. Zellner, C. A. Protheroe, L. Willetts, et al. 2012. 2. Lee, N. A., E. W. Gelfand, and J. J. Lee. 2001. Pulmonary T cells and eosino- Human versus mouse eosinophils: “that which we call an eosinophil, by any phils: coconspirators or independent triggers of allergic respiratory pathology? other name would stain as red”. J. Allergy Clin. Immunol. 130: 572–584. J. Allergy Clin. Immunol. 107: 945–957. 17. Svensson, L., E. Redvall, C. Bjo¨rn, J. Karlsson, A. M. Bergin, M. J. Rabiet, 3. Simon, D., and H. U. Simon. 2007. Eosinophilic disorders. J. Allergy Clin. C. Dahlgren, and C. Wenneras.˚ 2007. House dust mite activates human Immunol. 119: 1291–1300, quiz 1301–1302. eosinophils via formyl peptide receptor and formyl peptide receptor-like 1. Eur. 4. Harfi, I., L. Schandene´, S. Dremier, and F. Roufosse. 2013. Eosinophils affect J. Immunol. 37: 1966–1977. functions of in vitro-activated human CD32CD4+ T cells. J. Transl. Med. 11: 112. 18. Davoine, F., S. Lavigne, J. Chakir, C. Ferland, M. E. Boulay, and M. Laviolette. 5. Sugawara, R., E. J. Lee, M. S. Jang, E. J. Jeun, C. P. Hong, J. H. Kim, A. Park, 2002. Expression of FcgammaRIII (CD16) on human peripheral blood eosino- C. H. Yun, S. W. Hong, Y. M. Kim, et al. 2016. Small intestinal eosinophils regulate phils increases in allergic conditions. J. Allergy Clin. Immunol. 109: 463–469. Th17 cells by producing IL-1 receptor antagonist. J. Exp. Med. 213: 555–567. 19. Hartnell, A., A. B. Kay, and A. J. Wardlaw. 1992. IFN-gamma induces ex- 6. Strandmark, J., S. Steinfelder, C. Berek, A. A. Kuhl,€ S. Rausch, and S. Hartmann. pression of Fc gamma RIII (CD16) on human eosinophils. J. Immunol. 148: 2017. Eosinophils are required to suppress Th2 responses in Peyer’s patches 1471–1478. during intestinal by nematodes. Mucosal Immunol. 10: 661–672. 20. Zhu, X., K. J. Hamann, N. M. Mun˜oz, N. Rubio, D. Mayer, A. Hernrreiter, and 7. Andersson, J., J. Cromvik, M. Ingelsten, C. Lingblom, K. Andersson, A. R. Leff. 1998. Intracellular expression of Fc gamma RIII (CD16) and its J. E. Johansson, and C. Wenneras.˚ 2014. Eosinophils from hematopoietic stem mobilization by chemoattractants in human eosinophils. J. Immunol. 161: 2574– cell recipients suppress allogeneic T cell proliferation. Biol. Blood Marrow 2579. Transplant. 20: 1891–1898. 21. Wirthmueller, U., T. Kurosaki, M. S. Murakami, and J. V. Ravetch. 1992. Signal 8. Odemuyiwa, S. O., A. Ghahary, Y. Li, L. Puttagunta, J. E. Lee, S. Musat-Marcu, transduction by Fc gamma RIII (CD16) is mediated through the gamma chain. J. Exp. Med. 175: 1381–1390. A. Ghahary, and R. Moqbel. 2004. Cutting edge: human eosinophils regulate 22. Mason, G. M., K. Lowe, R. Melchiotti, R. Ellis, E. de Rinaldis, M. Peakman, T cell subset selection through indoleamine 2,3-dioxygenase. J. Immunol. 173: S. Heck, G. Lombardi, and T. I. Tree. 2015. Phenotypic complexity of the human 5909–5913. regulatory T cell compartment revealed by mass cytometry. J. Immunol. 195: 9. Peterson, C. G., V. Skoog, and P. Venge. 1986. Human eosinophil cationic 2030–2037. proteins (ECP and EPX) and their suppressive effects on lymphocyte prolifer- 23. Santegoets, S. J., E. M. Dijkgraaf, A. Battaglia, P. Beckhove, C. M. Britten, ation. Immunobiology 171: 1–13. A. Gallimore, A. Godkin, C. Gouttefangeas, T. D. de Gruijl, H. J. Koenen, et al. 10. Liu, L. Y., S. K. Mathur, J. B. Sedgwick, N. N. Jarjour, W. W. Busse, and 2015. Monitoring regulatory T cells in clinical samples: consensus on an es- E. A. Kelly. 2006. Human airway and peripheral blood eosinophils enhance Th1 sential marker set and gating strategy for regulatory T cell analysis by flow and Th2 cytokine secretion. Allergy 61: 589–597. cytometry. Cancer Immunol. Immunother. 64: 1271–1286. 10 CD16hi/GAL-10hi EOSINOPHILS ARE POTENT T CELL SUPPRESSORS

24. Pillay, J., V. M. Kamp, E. van Hoffen, T. Visser, T. Tak, J. W. Lammers, 31. Matsunaga, Y., M. Shono, M. Takahashi, Y. Tsuboi, K. Ogawa, and T. Yamada. L. H. Ulfman, L. P. Leenen, P. Pickkers, and L. Koenderman. 2012. A subset of 2000. Regulation of lymphocyte proliferation by eosinophils via chymotrypsin- neutrophils in human systemic inflammation inhibits T cell responses through like protease activity and adhesion molecule interaction. Br. J. Pharmacol. 130: Mac-1. J. Clin. Invest. 122: 327–336. 1539–1546. 25. Pillay, J., T. Tak, V. M. Kamp, and L. Koenderman. 2013. Immune suppression 32. Tran, D. Q., D. D. Glass, G. Uzel, D. A. Darnell, C. Spalding, S. M. Holland, and by neutrophils and granulocytic myeloid-derived suppressor cells: similarities E. M. Shevach. 2009. Analysis of adhesion molecules, target cells, and role of and differences. Cell. Mol. Life Sci. 70: 3813–3827. IL-2 in human FOXP3+ regulatory T cell suppressor function. J. Immunol. 182: 26. Firan, M., S. Dhillon, P. Estess, and M. H. Siegelman. 2006. Suppressor activity 2929–2938. and potency among regulatory T cells is discriminated by functionally active 33. Tak, T., B. Hilvering, K. Tesselaar, and L. Koenderman. 2015. Similar activation CD44. Blood 107: 619–627. state of neutrophils in sputum of asthma patients irrespective of sputum eosin- 27. Iellem, A., L. Colantonio, and D. D’Ambrosio. 2003. Skin-versus gut-skewed ophilia. Clin. Exp. Immunol. 182: 204–212. homing receptor expression and intrinsic CCR4 expression on human peripheral 34. Baecher-Allan, C., J. A. Brown, G. J. Freeman, and D. A. Hafler. 2001. CD4 blood CD4+CD25+ suppressor T cells. Eur. J. Immunol. 33: 1488–1496. +CD25high regulatory cells in human peripheral blood. J. Immunol. 167: 1245– 28. Sakaguchi, S., M. Miyara, C. M. Costantino, and D. A. Hafler. 2010. FOXP3+ 1253. regulatory T cells in the human immune system. Nat. Rev. Immunol. 10: 490– 35. de Kleijn, S., J. D. Langereis, J. Leentjens, M. Kox, M. G. Netea, 500. L. Koenderman, G. Ferwerda, P. Pickkers, and P. W. Hermans. 2013. IFN- 29. Fooksman, D. R., S. Vardhana, G. Vasiliver-Shamis, J. Liese, D. A. Blair, g-stimulated neutrophils suppress lymphocyte proliferation through expression J. Waite, C. Sacrista´n, G. D. Victora, A. Zanin-Zhorov, and M. L. Dustin. 2010. of PD-L1. PLoS One 8: e72249. Functional anatomy of T cell activation and synapse formation. Annu. Rev. 36. Miyara, M., Y. Yoshioka, A. Kitoh, T. Shima, K. Wing, A. Niwa, C. Parizot, Immunol. 28: 79–105. C. Taflin, T. Heike, D. Valeyre, et al. 2009. Functional delineation and differ- 30. Ren, G., X. Zhao, L. Zhang, J. Zhang, A. L’Huillier, W. Ling, A. I. Roberts, entiation dynamics of human CD4+ T cells expressing the FoxP3 transcription A. D. Le, S. Shi, C. Shao, and Y. Shi. 2010. Inflammatory cytokine-induced factor. 30: 899–911. intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in 37. Jonuleit, H., E. Schmitt, H. Kakirman, M. Stassen, J. Knop, and A. H. Enk. 2002. mesenchymal stem cells are critical for . J. Immunol. 184: : human CD25(+) regulatory T cells convey suppressor ac- 2321–2328. tivity to conventional CD4(+) T helper cells. J. Exp. Med. 196: 255–260. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021