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Disclosure of potential conflict of interest: R. P. Schleimer has consultant arrangementswith

Interesect ENT, GlaxoSmithKline, Allakos, and Aurasense. L. C. Grammer has received

grants and travel support for meetings or other purposes from the National Institutes of

Health (NIH); has received grants from the Bazley Foundation; has consultant arrange-

ments with Atellas Pharmaceuticals; is employed by Northwestern University and the

Northwestern Medical Faculty Foundation; has grants/grants pending with the NIH,

the Food Allergy Network, and S&C Electric; has received payment for lectures,

including service on speakers’ bureaus, from theAmericanAcademy ofAllergy, Asthma

& Immunology and Mount Sinai; and receives royalties from Lippincott, UpToDate,

BMJ, and Elsevier. B. K. Tan has received grants from the NIH and the Triological

Society/American College of Surgeons; has consultant arrangements with Acclarent,

Inc, and has received travel/accommodations/meeting expenses from the Foundation

for Innovation, Education, and Research in Otorhinolaryngology. T. R. Torgerson has

consultant arrangements with Baxter Biosciences and BDBiosciences; has grants/grants

pending with Baxter Biosciences and CSL Behring; has received payment for lectures,

including service on speakers’ bureaus, from Baxter Biosciences; receives royalties

from New England Biolabs; and receives payment for the development of educational

presentations from Baxter Biosciences. K. E. Harris has received grants from the NIH/

National Heart, Lung and Blood Institute (grant no. R37L068546 and grant no.

R01HL078860), the NIH/National Institute for Allergy and Infectious Disease (grant

no. T32A1083216), and the Ernest S. Bazley Trust. A. T. Peters has received payment

for lectures, including service on speakers’ bureaus, from Baxter. A. Kato has received

a grant from the NIH (grant no. R21HL113913). The rest of the authors declare that

they have no relevant conflicts of interest.

REFERENCES

1. Tan BK, Schleimer RP, Kern RC. Perspectives on the etiology of chronic

rhinosinusitis. Curr Opin Otolaryngol Head Neck Surg 2010;18:21-6.

2. Meltzer EO, Hamilos DL, Hadley JA, Lanza DC, Marple BF, Nicklas RA, et al.

Rhinosinusitis: establishing definitions for clinical research and patient care.

J Allergy Clin Immunol 2004;114:155-212.

3. Peters AT, Kato A, Zhang N, Conley DB, Suh L, Tancowny B, et al. Evidence for

altered activity of the IL-6 pathway in chronic rhinosinusitis with nasal polyps.

J Allergy Clin Immunol 2010;125:397-403.e10.

4. Kato A, Peters A, Suh L, Carter R, Harris KE, Chandra R, et al. Evidence of a role for

B cell-activating factor of the TNF family in the pathogenesis of chronic rhinosinu-

sitis with nasal polyps. J Allergy Clin Immunol 2008;121:1385-92, 1392.e1-2.

5. Hulse KE, Norton JE, Suh L, Zhong Q, Mahdavinia M, Simon P, et al. Chronic

rhinosinusitis with nasal polyps is characterized by B-cell inflammation and EBV-

induced protein 2 expression. JAllergyClin Immunol 2013;131:1075-83, 1083.e1-7.

6. Babon JJ, Kershaw NJ, Murphy JM, Varghese LN, Laktyushin A, Young SN, et al.

Suppression of cytokine signaling by SOCS3: characterization of the mode of

inhibition and the basis of its specificity. Immunity 2012;36:239-50.

7. Seki Y, Inoue H, Nagata N, Hayashi K, Fukuyama S, Matsumoto K, et al. SOCS-3

regulates onset and maintenance of T(H)2-mediated allergic responses. Nat Med

2003;9:1047-54.

8. Park SJ, Kim TH, Jun YJ, Lee SH, Ryu HY, Jung KJ, et al. Chronic rhinosinusitis

with polyps and without polyps is associated with increased expression of suppres-

sors of cytokine signaling 1 and 3. J Allergy Clin Immunol 2013;131:772-80.

9. Renner ED, Rylaarsdam S, Anover-Sombke S, Rack AL, Reichenbach J, Carey JC,

et al. Novel signal transducer and activator of transcription 3 (STAT3) mutations,

reduced T(H)17 cell numbers, and variably defective STAT3 phosphorylation in

hyper-IgE syndrome. J Allergy Clin Immunol 2008;122:181-7.

10. Seshadri S, Rosati M, Lin DC, Carter RG, Norton JE, Choi AW, et al. Regional

differences in the expression of innate host defense molecules in sinonasal mucosa.

J Allergy Clin Immunol [Epub 2013 Jul 26] http://dx.doi.org/10.1016/j.jaci.2013.

05.042.

11. Zhang N, Van Zele T, Perez-Novo C, Van Bruaene N, Holtappels G, DeRuyck N,

et al. Different types of T-effector cells orchestrate mucosal inflammation in

chronic sinus disease. J Allergy Clin Immunol 2008;122:961-8.

Available online September 29, 2013.http://dx.doi.org/10.1016/j.jaci.2013.08.015

Differential homeostatic dynamics of humanregulatory T-cell subsets following neonatalthymectomy

To the Editor:FOXP3-expressing CD41 regulatory T (Treg) cells are

important in the maintenance of self-tolerance and immune

homeostasis. There are 2 possible origins for Treg cells:1 (1)thymus-derived natural Treg (nTreg) cells and (2) peripherallyinduced Treg (iTreg) cells. Although both Treg cell populationsexpress similar phenotypic proteins, it has been proposedthat they exert different functions in maintaining immune homeo-stasis. While nTreg cells have been shown to be essential inself-tolerance, iTreg cells may be important in tolerance tononpathogenic foreign antigens.2 Disturbing the production ofeither Treg cell population may affect immune regulation laterin life. Recently, it has been shown, for example, that alterationsat neonatal age in thymic Treg cell maturation affects clinicaloutcome.3 It was demonstrated that in atopic children, thymicTreg cell function is significantly delayed early on in life. Howev-er, further data on human Treg cell development early in life arescarce.

The functional Treg cell population contains 2 distinct pop-ulations, a naive CD45RA1RO2FOXP3low fraction and an acti-vated/memory CD45RA2RO1FOXP3high fraction, both equallycapable of suppressive activity.4 Although both subpopulationsare true Treg cells, they have distinct differentiation dynamics.We hypothesized that Treg cell population dynamics would beaffected in patients who undergo neonatal thymectomy duringcardiac surgery. Previously, we showed the effect of neonatal thy-mectomy on long-term restoration of the naive T-cell compart-ment.5 In the present study, we evaluated the dynamics ofdistinct Treg cell subpopulations in the first 3 years followingneonatal thymectomy.

Twenty-six children with a median age of 11.4 months (range,2.5-34.7 months) whowere previously thymectomized during thecorrection of a cardiac defect were included (see Table E1 in thisarticle’s Online Repository at www.jacionline.org). The studywas approved by the medical ethical committee of the UniversityMedical Center Utrecht (METC 05-041 and 06-149), and writteninformed consent was obtained. Thymectomy was performedwithin the first month of life (10.06 9.0 days) in all participants.At the time of blood sampling, all children showed no sign ofinfection or immune dysregulation. For full information on thestudy population and flow cytometry staining protocols, see thisarticle’s Methods section in the Online Repository at www.jacionline.org.

First, we determined the impact of thymectomy on the total,peripheral CD41 T-cell population. Following thymectomy, ab-solute CD4 counts dropped significantly compared with those inhealthy infants (Fig 1, A; also see Fig E1, A, in this article’sOnline Repository at www.jacionline.org). Similarly, totalFOXP31CD41 T-cell numbers were significantly lower in thy-mectomized patients than in healthy age-matched controls (Fig1, B, and Fig E1, B). At a young age, CD311 (PECAM-1) Tcells represent recent thymic emigrants.6 Compatible with aloss of thymic production of Treg cells, thymectomized patientsshowed a significantly lower percentage of CD311FOXP31

T cells than did age-matched controls (Fig 1, C). Takentogether, neonatal thymectomy results in a loss of thymus-derived Treg cells and a reduced number of circulating Tregcells.

Interestingly, after thymectomy, the percentage ofFOXP31CD41 T cells was increased compared with that in con-trols (Fig 1,D, and Fig E1,C). Therefore, we investigated whethertherewas an increase in peripheral proliferation to compensate forthe loss of thymic output. Compared with the FOXP32CD41

population, the FOXP31CD41 Treg cell population had a higher

FIG 1. CD41 and FOXP31 T-cell dynamics and expression of CD31 and Ki67 after thymectomy. A, CD41

T-cell count. B, FOXP31CD41 Treg-cell count. C, Percentage of CD311 (FOXP31) Treg cells. D, Percentage

of FOXP31 (CD41) cells. E, Percentage of FOXP32 and FOXP31 T cells in cell cycle (Ki671) in thymectomized

subjects (TX) (:) and age-matched controls (HC) (B). Horizontal line represents median value per group.

*P < .05 and **P < .001.

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proportion of proliferation marker Ki671 cells, with a significantincrease following thymectomy (Fig 1, E). Thus, these data sug-gest that there is a compensatory, selective expansion of Tregcells, leading to an increased percentage of Foxp31CD41 T cells.

Next, we studied the dynamics of 2 distinct Treg cell sub-populations; gating of the FOXP31 fractions is illustrated in FigsE2 and E3 in this article’s Online Repository at www.jacionline.org. The naive subpopulation CD45RO2FOXP3low (fraction I)and the memory population with the highest FOXP3 expressionCD45RO1FOXP3high (fraction II) have been shown to be‘‘true’’ Treg cells, whereas the memory population with lowFOXP3 expression (fraction III) is nonsuppressive.4 We observedthat the percentage of FOXP3 cells expressing the naive CD45isoform (fraction I) remained stable, whereas both memory frac-tions increased significantly in thymectomizd patients (Fig 2, A).The relative increase in the heterogeneous and nonsuppressiveCD45RO1FOXP3low population (fraction III) may be a reflec-tion of activation-induced transient upregulation of FOXP3, ashas been shown in vitro.7 Absolute numbers of both ‘‘true’’Treg cell fractions decreased marginally, though not significantly(Fig 2, B). While numbers of CD45RO2FOXP3low Tregcells (fraction I) were within the range of healthy controls, themajority of the patients had low numbers (Fig 2, B). Thus,neonatal thymectomy affects the composition of the Treg cellpopulation.

When we examined the proliferation of the 2 ‘‘true’’Treg cell subpopulations, a clear hierarchy was observed,with a 10-fold higher percentage of Ki67-expressing cellsin CD45RO1FOXP3high (fraction II) than in the naiveCD45RO2FOXP3low (fraction I) Treg cell population in both

thymectomized and healthy individuals. Following thymectomy,an increase in proliferation of both subpopulations was foundcompared to the healthy controls, reaching statistical significancein the naive Treg cell fraction (fraction I) (Fig 2, C). Together,these data suggest that peripheral proliferation of both naiveand activated Treg cells compensated for the loss of thymicoutput, resulting in maintenance of and increase in percentagesof naive Treg and activated Treg cell populations, respectively(Fig 2, A). Although it is most likely that increased expansionof the activated Treg cell population is responsible for maintain-ing the number of activated Treg cells in these thymectomizedchildren (Fig 2,B), we cannot exclude the possibility of additionalincreased conversion of naive Treg cells to activatedCD45RO1FOXP3high Treg cells.4 In a subgroup of patients,the number of naive CD45RO2FOXP3low Treg cells was lowdespite increased proliferation, which appeared most prominentin the children older than 6 months (see Fig E4 in this article’sOnline Repository at www.jacionline.org). A shift in the balancebetween naive and memory Treg cells has been associated withseveral pathological conditions. Reduced naive Treg cells witha compensatory increase in memory Treg cells has beenassociated with multiple sclerosis8 and sarcoidosis,4 while anincrease in naive Treg cells, albeit with impaired suppressivefunction, has been observed in active systemic lupus erythemato-sus.4,9 Thus, removal of the thymus in the first month of life mayaffect immune regulation later in life. Overall, it is prudent tospare thymic tissue in patients requiring congenital heart surgerywhen technically possible.

This study demonstrates the specific homeostatic control of 2distinct FOXP31 Treg-cell populations. Peripheral proliferation

FIG 2. FOXP31 subpopulation dynamics after thymectomy. A, Percentage of the 3 FOXP31 subpopulations

within CD4 T-cell population. B, Cell count of the 2 FOXP31 Treg-cell populations in healthy controls (HC)

(B) and thymectomized patients (TX) (:), and ratio of fraction ii:i. C, Expression of Ki67 per FOXP31

Treg-cell population. n.s., No significant difference. **P < .001.

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of Treg cells counteracted the effect of loss of thymopoiesis,which illustrates the relative plasticity of the human immunesystem. However, changes in composition of the Treg cellpopulation dowarrant further investigation of the long-term func-tional effects of neonatal thymectomy following cardiac surgery.

Alvin W. L. Schadenberg, MDa,b

Theo van den Broek, MDa,b

Marten A. Siemelink, MDc

Selma O. Algra, MDb

Petrus R. de Jong, MDa,b

Nicolaas J. G. Jansen, MD, PhDb

Berent J. Prakken, MD, PhDa*

Femke van Wijk, PhDa*

From athe Department of Pediatric Immunology and the Laboratory of Translational

Immunology, bthe Department of Pediatric Intensive Care/Pediatric Cardiothoracic

Surgery, and cthe Deparment of Experimental Cardiology, University Medical Center

Utrecht, Utrecht, The Netherlands. E-mail: F.vanwijk@umcutrecht.nl.

*These authors contributed equally to this work.

Disclosure of potential conflict of interest: F. van Wijk has received the Veni

personal fellowship from the Netherlands Organization for Scientific Research.

A. W. L. Schadenberg has received grants from the Wilhelmina Kinderziekenhuis

Fonds. B. J. Prakken is the coinventor of and receives royalties from a patent of

UCSD on Pan-DR Binder peptides. The rest of the authors declare that they have

no relevant conflicts of interest.

REFERENCES

1. Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune

tolerance. Cell 2008;133:775-87.

2. Long SA, Rieck M, Tatum M, Bollyky PL, Wu RP, Muller I, et al. Low-dose an-

tigen promotes induction of FOXP3 in human CD41 T cells. J Immunol 2011;187:

3511-20.

3. Tulic MK, Andrews D, Crook ML, Charles A, Tourigny MR, Moqbel R, et al.

Changes in thymic regulatory T-cell maturation from birth to puberty: differences

in atopic children. J Allergy Clin Immunol 2012;129:199-206.

4. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional

delineation and differentiation dynamics of human CD41 T cells expressing the

FoxP3 transcription factor. Immunity 2009;30:899-911.

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5. Gent van R, Schadenberg AW, Otto SA, Nievelstein RA, Sieswerda GT, Haas F,

et al. Long-term restoration of the human T-cell compartment after thymectomy

during infancy: a role for thymic regeneration? Blood 2011;118:627-34.

6. Braber den I, Mugwagwa T, Vrisekoop N, Westera L, M€ogling R, de Boer AB,

et al. Maintenance of peripheral naive T cells is sustained by thymus output in

mice but not humans. Immunity 2012;36:288-97.

7. Wang J, Ioan-Facsinay A, van der Voort E, Huizinga TW, Toes RE. Transient

expression of FOXP3 in human activated nonregulatory CD41 T cells. Eur J Im-

munol 2007;37:129-38.

8. Haas J, Fritzsching B, Trubswetter P, Korporal M, Milkova L, Fritz B, et al. Prev-

alence of newly generated naive regulatory T cells (Treg) is critical for Treg sup-

pressive function and determines Treg dysfunction multiple sclerosis. J Immunol

2007;179:1322-30.

9. Pan X, Yuan X, Zheng Y, Wang W, Shan J, Lin F, et al. Increased CD45RA1 Fox-

P3(low) regulatory T cells with impaired suppressive function in patients with sys-

temic lupus erythematosus. PloS One 2012;7:e34662.

Available online October 18, 2013.http://dx.doi.org/10.1016/j.jaci.2013.08.030

South African amaXhosa patients with atopicdermatitis have decreased levels of filaggrinbreakdown products but no loss-of-functionmutations in filaggrinq

To the Editor:Loss-of-function (LOF) mutations in the filaggrin gene (FLG)

are the strongest known genetic risk factors for atopic dermatitis(AD). The genetic architecture of FLG mutations is well estab-lished in European, Japanese, and selected Chinese populations,but their contribution to AD in African populations is not well un-derstood. The only data on FLGmutations in Africans come froma recent study conducted in Ethiopia1 that studied 103 patientswith AD, 7 patients with ichthyosis vulgaris (IV), and 103 healthycontrols. This study identified only a single novel mutation (a 2-bp deletion, 632del2), by direct sequencing of FLG in a patientwith AD.

To investigate the role of filaggrin in the etiology of AD inSouth Africa, we studied 69 children with AD from the amaXhosacommunity along with 81 age-, ethnic- and sex-matched controls,with no history of AD. The patients (n5 69) and controls (n5 81)were recruited from tertiary referral AD clinics in Cape Town.Clinical and demographic characteristics of control subjects andpatients with AD are outlined in Table I. The study was conductedin accordance with the Helsinki Declaration and was approved bythe Human Research Ethics Committee of the Faculty of HealthSciences of the University of Cape Town. Written consent/ascentin the amaXhosa language was obtained from the patients or theirparents.

The entire coding sequence of the FLG gene was directlysequenced (as described previously2) in 31 patients with ADwith prominent features of IV, that is, those who should mostlikely have FLGmutations. Sequencing of PCR products was per-formed by a core facility (DNA Sequencing and Services, Univer-sity of Dundee, Dundee, United Kingdom) according to ourstandard operating procedures. The entire collection was addi-tionally typed for the previously known FLG mutations R501X,2282del4, R2447X, and S3247X by using custom-made Taqmanallelic discrimination assays.2 Although the primers used for theamplification of the FLG gene were originally optimized for the

qThis is an open-access article distributed under the terms of the Creative Commons

Attribution License, which permits unrestricted use, distribution, and reproduction

in any medium, provided the original author and source are credited.

sequencing of European populations,2 in the 31 amaXhosa pa-tients with AD who were sequenced, we identified 124 mutations(synonymous and nonsynonymous) throughout exon 3 of theFLGgene by using these methods. Identification of these silent andnonpathogenic missense mutations in the FLG gene indicatesminimal allele dropout using these primer sets. None of theseidentified mutations was predicted to lead to loss of filaggrin atthe protein level. Screening of the entire collection of patientsfor the FLG mutations R501X, 2282del4, R2447X, and S3247Xshowed that all samples were wild type for these mutations.

In addition to gene sequencing, in all patients with AD andcontrols, we determined the concentrations of filaggrin break-down products in the stratum corneum (SC). Filaggrin is degradedin the later stages of epidermal differentiation into free aminoacids (FAA) and their derivatives; a major proportion of the totalSC FAA (70% to 100%) is derived from filaggrin.3 Themost com-mon amino acid residues in filaggrin repeats are basic amino acidssuch as histidine (413 of 4061 residues; 10.17%) and arginine(440 of 4061 residues; 10.83%) and the polar residue glutamine(367 of 4061; 9.04%) (see Fig E1 in this article’s Online Repos-itory at www.jacionline.org). Histidine is enzymatically deami-nated to trans-urocanic acid (trans-UCA).3 Trans-UCA, whichis converted to cis-UCA on ultraviolet irradiation, functions asa major chromophore and exerts immunomodulatory effects inthe skin.3,4 UCA maintains an acidic pH in the skin, which iscrucial for the optimal function of several enzymes in the SCand antimicrobial defence.3,4 Another abundant amino acid gluta-mine is further converted into pyrrolidone-5-carboxylic acid(PCA). PCA is highly hygroscopic and is one of the major com-ponents of the natural moisturizing factor, thus providing a hu-mectant effect by retaining water in the SC.3,4 Filaggrindegradation products thus have multiple functions.

FLG mutations lead to reduced levels of filaggrin degradationproducts in the SC in a dose-dependent fashion.5 It has beenshown that moderate-to-severe AD also has an effect on SC filag-grin expression6 as well as on the levels of filaggrin degradationproducts5 possibly due to the systemic TH2 immune response.6

In the present study, the levels of both PCA and UCA and theirsum were significantly decreased in the SC of patients with ADthan in control subjects (Fig 1; see Table E1 in this article’s OnlineRepository at www.jacionline.org). The magnitude of reductionbetween cases and controls in this study was approximately20%. Similar results were obtained when comparing total FAAcontent as well as total FAA including their derivatives, PCA,UCA, citrulline, and ornithine (Fig 1; Table E1). While FLGmu-tations are the major determinants of the level of filaggrin break-down products in the SC, it has been previously shown that theirlevels are significantly reduced in European populations both innonlesional skin of patients with AD with FLG mutations andin patients without FLG mutations.5 In this study, we have repli-cated these filaggrin breakdown product findings in patients withAD without FLG mutations in an African population. This isconsistent with the in vitro findings of Howell et al6 and Pellerinet al7 and highlights the fact that there is an interplay betweenthe skin barrier and a systemic immunologic process, with sys-temic TH2 inflammation causing a decrease in SC filaggrinexpression.

The SC profiles of filaggrin breakdown products are highlyinformative in this African population because they provide asecond look, in addition to direct sequencing, for FLGmutations.In our study, we demonstrate that the diminution of filaggrin

METHODSStudy population and blood specimens

Twenty-six patients who all had undergone complete thymectomy at

neonatal age during surgical correction for a congenital heart defect at the

children’s heart center, University Medical Center Utrecht, The Netherlands,

were included in this study. The thymus is routinely removed during surgery

involving the major vessels, such as transposition of the great arteries,

hypoplastic heart syndrome, and hypoplastic aortic arch, because of its

anatomical obstruction in relation to the heart. A healthy, age-matched,

control group was included from both patients admitted for correction of a

heart defect that did not necessitate removal of the thymus such as ventricular

septum defects (n 5 9) and healthy children who visited the University

Medical Center Utrecht to undergo elective urologic or plastic surgery (n5 8).

All included patients were considered immunologically healthy because they

did not have a recent history of infectious disease or a hematologic or

immunologic disorder. Patients with a known syndrome or genetic disorder

were excluded (eg, 22q11 deletion and trisomy 21). Characteristics of the 26

included patients and healthy controls are depicted in Table E1. Because cell

counts were not available for all samples, absolute numbers of cell populations

are not shown for all study subjects.

Cell preparation and flow cytometryPBMCs were isolated from heparinized blood samples by using the Ficoll

Isopaque density gradient centrifugation (Amersham Pharmacia Biotech,

Uppsala, Sweden), and viably frozen and stored in liquid nitrogen until

further processing. Characterization of the T-cell compartment was

performed on thawed cryopreserved PBMCs that were washed in

fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% FCS

and 0.1% sodium azide) and blocked with normal mouse and rat serum. The

cells were incubated in 50 mL FACS buffer containing the appropriately

diluted antibodies against human CD3, CD4, CD45RO, and CD31. For

intracellular staining of Ki-67 and FOXP3, the cells were first surface stained,

followed by fixation and permeabilization according to the manufacturer’s

protocol. Antibodies against CD4 (clone SK3) and CD31 (WM59) were

obtained from BD Biosciences (San Jose, Calif), against CD45RO (UCHL1)

from Caltag (Buckingham, United Kingdom), against Ki67 (MIB-1) from

Immunotech (Marseilles, France), and against FOXP3 (PCH101) from

eBioscience (San Diego, Calif). Finally, stained mononuclear cells were

washed twice in FACS buffer and run on an LSRII and analyzed by using

FACSDiva software (BD Biosciences). The gates for the different populations

were kept identical for each experiment containing both thymectomized

patients and healthy controls.

StatisticsTo analyze the quantitative differences between thymectomized patients

and healthy age-matched controls, data only after thymectomy were included.

Statistical significance between the 2 groups was assessed by using the

Mann-Whitney U test for unpaired data and the x2 test for dichotomous data.

Statistical difference is indicated as *P < .05 and **P < .001.

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FIG E1. FOXP3 percentages and counts in the first years after neonatal thymectomy. A, Absolute CD41

T-cell counts per microliter of blood. B, FOXP31CD41 Treg-cell counts per microliter of blood. C, Percentage

of FOXP31 cells in CD41 T-cell populations. :, values after thymectomy; D, samples taken just before

thymectomy; B, healthy controls. Lines connect longitudinal samples.

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FIG E2. Gating strategy of subpopulations of FOXP31 T cells. FOXP31

subpopulations after gating for CD41 lymphocytes. Fraction I,

CD45RO2FOXP3low; fraction II, CD45RO1FOXP3high; fraction III,

CD45RO1FOXP3low.

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FIG E3. CD45RO2Foxp31 T cells represent CD45RA1Foxp31 T cells. A, Dot plot of CD45RO (memory) and

CD45RA (naive) expression on CD31CD41 T cells. B, Dot plot of CD45RO and CD45RA expression on

CD31CD41Foxp3 (Treg) cells. C, Expression of CD45RA1 in the CD45RO2FOXP31 subpopulation. Healthy

control group (HC), n 5 7, and thymectomized patients (TX), n 5 8. Data represented as mean

percentage 6 SD.

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FIG E4. Subgroup analysis of naive and memory Treg-cell populations in 0- to 6-month-old and more than

6-month-old subjects. CD45RO2FOXP3low naive Treg (A) and CD45RO1FOXP3high memory Treg-cell (B)

numbers in the subgroups less than 6 months and more than 6 months of age in healthy controls (HC)and thymectomized patients (TX). Data represented as median, 25% and 75% percentile boxes, and range.

*P < .05.

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TABLE E1. Characteristics of the included patients and healthy

controls

Thymectomy Control P value

No. of subjects 26 17

Age (mo) 11.4 6 10.9 10.8 6 12.6 .62

Age at TX (d) 10.0 6 9.0 –Female:male 9:17 6:11 .96

TX, Thymectomy.

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