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Clinical Immunology (2011) 140, 71–83

Regulatory T cellswith reduced repressor capacities areextensively amplified in pulmonary sarcoid lesions andsustain granuloma formationGunter Rappl a,h,⁎,1, Stefan Pabst b,1, Dagmar Riemannc, Annette Schmidt d,Claudia Wickenhausere, Wolfgang Schütte f, Andreas A. Hombacha,Barbara Seliger c, Christian Grohég, Hinrich Abkena,h

a Department of Internal Medicine I, Laboratory for Tumorgenetics, University Hospital Cologne, Cologne, Germanyb Department of Internal Medicine II, University Hospital Bonn, Bonn, Germanyc Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle/Saale, Germanyd Institute for Circulation Research and Sports Medicine, German Sports University, Cologne, Germanye Department of Pathology, University Hospital Cologne, Cologne, Germanyf Martha-Maria Hospital, Halle-Dölau GmbH, Halle/Saale, Germanyg Evangelische Lungenklinik Berlin-Buch, Berlin, Germanyh Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany

Received 9 August 2010; accepted with revision 18 March 2011Available online 24 March 2011

Abbreviations: BALF, broncho-alvehybridization; FVC, basic forced volumlatency-associated peptide; MACS, maoncostatin M; Q-FISH, quantitative flregulatory T; VIP, vasoactive intestina⁎ Corresponding author at: Center fo

Fax: +49 221 478 88723.E-mail address: gunter.rappl@uni-k

1 Authors G.R. and S.P. contributed e

1521-6616/$ – see front matter © 201doi:10.1016/j.clim.2011.03.015

KEYWORDSSarcoidosis;Treg cells;Chronic inflammation;Immunosenescence;CD7

Abstract Sarcoidosis can evolve into a chronic disease with persistent granulomas accompa-nied by progressive fibrosis. While an unlimited inflammatory response suggests an impairedimmune control in sarcoid lesions, it stands in contrast to the massive infiltration withCD4+CD25highFoxP3+ regulatory T cells. We here revealed that those Treg cells in affected lunglesions were mainly derived from activated natural Treg cells with GARP (LRRC32)-positivephenotype but exhibited reduced repressor capacities despite high IL-10 and TGF-beta 1 levels.The repressive capacity of blood Treg cells, in contrast, was not impaired compared to age-
matched healthy donors. Treg derived cells in granuloma lesions have undergone extensiverounds of amplifications indicated by shortened telomeres compared to blood Treg cells of thesame patient. Lesional Treg derived cells moreover secreted pro-inflammatory cytokines
olar lavage fluid; CFSE, 5-carboxylfluorescein diacetate succinimidyl ester; FISH, fluorescence in situe vital capacity; iTreg, induced regulatory T cells; KLRG-1, killer cell lectin-like receptor G1; LAP,gnetic activated cell sorting; MFI, mean fluorescence intensity; nTreg, natural regulatory T cells; OSM,uorescence in situ hybridization; Th3, type 3 regulatory T cells; TLC, total lung capacity; Treg,l peptider Molecular Medicine Cologne, University of Cologne, Robert-Koch-Str. 21, D-50931 Köln, Germany.

oeln.de (G. Rappl).qually.

1 Elsevier Inc. All rights reserved.

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http://dx.doi.org/10.1016/j.clim.2011.03.015

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72 G. Rappl et al.

including IL-4 which sustains granuloma formation through fibroblast amplification and theactivation of mast cells, the latter indicated by the expression of membrane-bound oncostatin M.© 2011 Elsevier Inc. All rights reserved.

1. Introduction

Sarcoidosis is a granulomatous disease of unknown etiologycharacterized by the formation of non-caseating granulomasin a variety of organs, most commonly the lung. Althoughgranulomas may regress in nearly half the cases withoutmajor signs of tissue damage, sarcoidosis can evolve into achronic disease with persistent granulomas accompanied byprogressive fibrosis, the main cause of morbidity andmortality [1]. However, progression to fibrosis cannot bepredicted from the initial stages of the disease raising theneed to understand immunological processes in order topredict prognosis and to apply appropriate treatment.

An uncontrolled Th1/Th17 polarized immune response isone of the central mechanisms, which maintain chronicinflammation in sarcoidosis [2]. A characteristic ring at theperiphery of granulomas is formed by B cells, mast cells andpredominantly CD4+ and CD8+ T lymphocytes, most of themsecreting pro-inflammatory cytokines, such as IL-2, TNF-αand IFN-γ, which attract additional inflammatory cells [3].Cytokines may also sustain fibrogenesis in chronic diseasestages by stimulating mesenchymal cell proliferation andsecretion of inflammatory cytokines by surrounding cells[4,5]. The unlimited inflammatory process in sarcoidosisimplies an impaired immune repression in those patients.Investigators highlighted the “immune paradox” of sarcoid-osis, which is characterized by an intense immune responsein the affected organs including the lungs and a relativeanergy in non-affected organs associated with a negativeresponse to the Mantoux test [6]. Although a disequilibriumbetween effector and regulatory T cells has been postulatedfor years, the cellular basis remains unclear so far. Thedisequilibrium seems to occur during transition from activeto chronic inflammation in affected lesions. In activesarcoidosis high numbers of Treg cells accumulate in theaffected lung where they dampen the inflammatory T cellresponse and facilitate remission of the disease [7]. Inchronic stages, however, Treg cells seem to be incompetentin repression or the effector cells resistant to repressionleading to prolonged inflammatory reactions associated withprogression in fibrosis. Recent analyses revealed that Tregcells can repress the initial steps of granuloma formation,but have no impact on established sarcoid lesions [8,9]. Wetherefore asked whether lesional Treg cells in establishedlesions exhibit altered functional capacities compared toTreg cells in the periphery of the same patients or of healthydonors. We particularly addressed whether Treg cells insarcoid lesions are more progressed in terminal T celldifferentiation that is associated with altered functionalcapacities. Our data draw the concept that Treg derived cellsin sarcoid lesions, but not peripheral Treg cells, are impairedin repression, have undergone extensive amplification, andsecrete pro-inflammatory cytokines capable to promotegranuloma formation.

2. Material and methods

2.1. Patient characteristics

For this study, 45 patients (20male) in the age of 24 to 78 years(median 51 years) with sarcoidosis defined by clinical pheno-type, histopathology and lymphocyte counts in broncho-alveolar lavage fluid (BALF) and/or peripheral blood wererecruited. This study was performed in conformity with theDeclaration of Helsinki of the World Medical Association andapproved by the Ethics Committee of the School of Medicine,Bonn University (reference no 080/06). Written informedconsent was obtained from each sarcoid patient and healthydonor prior to their enrolment. Patient characteristics aresummarized in “Supplemental information”.

2.2. T cell characterization

Peripheral blood mononuclear cells and BALF cells and theirsubsets were isolated by magnetic activated cell sorting(MACS) as described [10]. Freshly isolated biopsy cells werecharacterized by flow cytometry using a FACSCalibur orFACSCanto flow cytometer (BD Biosciences, Heidelberg,Germany) equipped with the Cell Quest or DIVA software,respectively. Suppressor activity was determined usingstandard procedures. Telomere lengths were determinedby fluorescence in situ hybridization (FISH) techniques. Fordetails we refer to the “Supplemental information”.

2.3. Immunohistochemistry

Lung biopsies from sarcoidosis patients of stage II (n=5) andstage III (n=1) and of healthy donors (n=5) were snap frozenand stored at −80 °C. Control samples of healthy human lungwere obtained from fresh surgical specimens.

2.4. Statistical analysis

Results are presented as means±SDs unless otherwise stated.A paired and an unpaired two-tailed t-test were used tocompare the mean values between patients and controls andbetween different cell culture conditions. pb0.05 wasconsidered statistically significant.

3. Results

3.1. Treg cells in sarcoid lesions express thephenotype of late memory T cells

We characterized in detail the CD4+ T cell subpopulations inboth BALF and peripheral blood of sarcoid patients by flowcytometry. Nearly all CD4+CD45RO+ T cells expressed reduced

Figure 1 The CD7− subset of CD4+CD45RO+ T cells accumulates in peripheral blood and BALF of sarcoid patients and mainlyrepresents regulatory T cells. (A) Expression of T cell differentiation markers on CD4+CD45RO+ T cells from the peripheral blood andBALF of 10 healthy donors and 39 patients with sarcoidosis was evaluated by flow cytometry. Dot plots from one representative healthydonor and sarcoid patient in disease stage III are presented. Data summarize all samples and represent the means±standard error ofthe mean (SEM). (B) CD4+CD45RO+ T cells from the peripheral blood or BALF of 10 healthy donors or 15 patients (stage I: n=8, stage II:n=6, and stage III: n=1) with sarcoidosis were simultaneously stained with monoclonal antibodies specific for CD4 plus CD25, CD7,FoxP3 and CD127. Summary of immunofluorescence analysis of blood lymphocytes or BALF cells of sarcoid patients and healthy donorsis shown. Data of Teff (FoxP3lowCD127+) and Treg (FoxP3highCD127−) cells in the CD4+CD45RO+ T cell population are presented. Datarepresent the means±SEM. *pb0.05. (C) T cells from BALF of 6 healthy donors or 6 sarcoidosis patients in stage II (n=5) and stage III(n=1) were simultaneously stained for CD4 plus CD25, CD7, FoxP3, CD127, CD45RO, Helios, GARP, and LAP, respectively, and recordedby flow cytometry. A representative analysis of one sarcoid patient in stage II is shown.

73T cells with repressor capacities are amplified in lesions and sustain granuloma formation

Figure 1 (continued).

74 G. Rappl et al.

levels of CD27, CD28, and CD62L, elevated levels of CD57 andKLRG-1, and lack of CD7 (Fig. 1A). T cells with same phenotypewere found accumulated in the peripheral blood of sarcoid

patients as well compared to healthy, age-matched volun-teers. Themajority (75±3%) of CD4+CD45RO+ T cells fromBALFof sarcoid patients were Treg cells based on high FoxP3expression and lack of IL-7 receptor (CD127) compared to 24±3% Treg cells in BALF of healthy donors (Figs. 1B and C). Thenumber of Treg cells in the peripheral blood of sarcoid patientswas also increased compared to that of healthy donors(Fig. 1B). Most FoxP3highCD127− Treg cells from BALF of sarcoidpatients exhibit a late memory phenotype indicated byreduced levels of CD27, CD28, and CD62L, lack of CD7, andelevated levels of CD57 and KLRG-1 whereas less than 5% ofBALF Treg cells with this phenotype were found in healthydonors. The phenotype of sarcoid T cells is in contrast to BALFand peripheral blood Treg cells of healthy donors whichpredominantly express the “younger” CD7+ memory pheno-type (Fig. 2A). The majority of CD7− Treg cells from BALF ofsarcoid patients are of CD45RO+ phenotype, whereas the fewCD7+ Treg cells harbor some naïve Treg cells (35±3%)(Fig. S1B). About 2±1% of CD7− Treg cells expressed theCCR7−CD45RA+ phenotype, which is in accordance withterminal differentiation of those Treg cells [11].

The frequency of CD7− Treg cells in sarcoid BALFspecimens increased during the progression of the disease,i.e., from 21±1% in stage I to 81±4% in stage III, whereasCD7− Treg cell numbers in peripheral blood decreased from25±1% in stage I to 14±4% in stage III (Fig. 2B).

Treg cell populations recorded in BALF specimens reflectthe situation in lung sarcoid lesions as monitored byimmunostaining of biopsies (Fig. S2). FoxP3high T cells co-expressed CD4 and CD25, but lacked CD127 (IL-7Rα)expression assigning these cells as Treg cells. About 80% ofinfiltrating CD4+FoxP3highCD127− Treg cells was of CD7−

phenotype in sarcoid lesions compared to 2% in the healthylung. Taken together, Treg cells of CD7− phenotypesubstantially accumulate in lung sarcoid lesions duringdisease progression representing the vast majority of lesionalCD4+CD45RO+ T cells.

To discriminate FoxP3+ Treg cells from freshly activatedeffector T cells we recorded GARP (LRRC32) and latency-associated peptide (LAP), both exclusively expressed onactivated natural Treg cells [12], and Helios expressed onthymic derived natural FoxP3+ Treg cells [13]. The number ofGARP+FoxP3high and of LAP+FoxP3high Treg cells was increasedin BALF compared to peripheral blood of sarcoid patients andcompared to healthy donors (Fig. 2C) implying their activa-tion in the periphery. This was found for both CD7− and CD7+

Treg cells. The expression levels of GARP as well as of LAP,however, were lower in CD7− compared to CD7+ Treg cells. Inline with increased numbers of GARP+ cells, the number ofHelios+ decreased in both BALF and blood of sarcoid patientscompared to healthy donors. We conclude that natural Tregcells accumulate and activate in the periphery of sarcoidpatients.

3.2. CD7− Treg cells express homing receptors fortheir direction into the lung

We asked whether the CD7− Treg cells in sarcoid lung lesionsexpress homing receptors directing them to the periphery.The chemokine receptor CXCR6, associated with lung-homing[14], was expressed on 63±5% and 26±3% of BALF CD7− and


CD7+ Treg cells, respectively, from sarcoid patients which isincreased compared to the corresponding Treg cells fromhealthy donors (Fig. 3). For comparison, CXCR6was expressedby b2% of Treg cells in peripheral blood. Detailed analysesmoreover revealed that Treg cells from BALF of sarcoidpatients preferentially lack CCR7 compared to the corre-sponding cells of healthy donors. There are more CCR7− Tregcells in BALF than in the peripheral blood of sarcoid patients(Fig. 3). CCR7− T cells are prone to persist in peripheral organsdue to their inability to emigrate into lymph [15]. Takentogether, data imply that sarcoid Treg cells are preferentially

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Figure 2 Treg cells in sarcoid lesions are predominantly of CD7activated. CD4+CD45RO+ T cells from the peripheral blood or BALF ofstage III: n=1) with sarcoidosis were simultaneously stained with moCD127. A, Summary of immunofluorescence analysis of blood lymrepresenting the absolute numbers of CD4+FoxP3highCD127− T cimmunofluorescence analysis of blood lymphocytes or BALF cellsrepresenting the absolute numbers of CD4+FoxP3highCD127− T ceCD4+CD25highCD127− T cells from the peripheral blood and BALF of heaIII (n=1) were stained for CD45RO, CD7, FoxP3, Helios, GARP and LATreg cells and FoxP3highHelios+ represent natural Treg cells. Data re

targeted to the lung through CXCR6 and prevented from re-migration to the lymph through lack of CCR7 expression.

3.3. CD7− Treg cells in sarcoid lesions haveundergone extensive amplification

The assignment of lesional Treg cells to a more advancedstage in memory T cell development implies that these cellshad undergone extensive amplification. We thereforemonitored by flow-FISH the telomere lengths of CD7+ and

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− phenotype, accumulate during disease progression and are10 healthy donors or 15 patients (stage I: n=8, stage II: n=6, andnoclonal antibodies specific for CD4 plus CD25, CD7, FoxP3 andphocytes or BALF cells of sarcoid patients and healthy donorsells in BALF or peripheral blood is shown. B, Summary ofof sarcoid patients with different disease stages at diagnosislls in BALF or peripheral blood is presented. (C) Separatedlthy donors (n=6) and sarcoid patients in stage II (n=5) and stageP. FoxP3highGARP+ and FoxP3highLAP+ T cells represent activatedpresent the mean±SEM. *pb0.05. n.s., not significant.

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CD7− Treg cells from BALF compared to peripheral blood cellof the same individual. As summarized in Figure 4, CD7− Tregcells in BALF exhibit shorter telomeres than the correspond-ing cells in the peripheral blood of the same patient(p=0.045) which is in contrast to CD7− Treg cells of age-matched healthy controls. The mean telomere length ofCD7− Treg cells in peripheral blood and BALF of sarcoidpatients was shorter than that of the corresponding CD7+

Treg cells confirming the advanced proliferative history ofCD7− compared to CD7+ Treg cells. In situ Q-FISH analyses oftelomere lengths of Treg cells in lung biopsies confirmed ourflow-FISH results. Again, CD7− Treg cells have shortertelomeres in comparison to their corresponding CD7+ Tcells within the same lesion (Fig. S3). We conclude that themajority of Treg cells in sarcoid lesions, which express the

CD7− phenotype, have undergone further rounds of prolifer-ation compared to the corresponding cells in the peripheralblood. Thus CD7− Treg cells constituting the majority insarcoid lung lesions, have undergone more rounds ofproliferation than the same Treg cell subset in peripheralblood of the same patient and of age-matched healthydonors pointing to a more forced amplification of these cellsin sarcoid lesions.

3.4. CD7− Treg cells from sarcoid patients exhibitreduced repressor capacities

We recorded increased numbers of Treg cells with IL-10 andTGF-β1 expression in both BALF and peripheral blood of

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Figure 3 CD7− Treg cells in sarcoid patients preferentiallyexpress homing receptors associated with retention in the lung.Expression of the chemokine receptors CXCR6 and CCR7 on CD7−

and CD7+ FoxP3highCD127− Treg cells from the peripheral bloodand BALF of 10 healthy donors and 24 sarcoidosis patients wasrecorded by flow cytometry. Data represent the mean±SEM.*pb0.05.


sarcoid patients compared to healthy donors (Fig. 5A). CD7−

Treg cells from sarcoid BALF, noteworthy, expressed IL-10and TGF-β1 at higher levels than in the blood of the samepatients. TGF-β1 is highly expressed by blood CD7+ Treg cellsfrom sarcoid patients, while the same cells from BALFexpressed TGF-β1 at lower levels compared to cells fromhealthy donors. Immunostainings of biopsy sections con-firmed that lesional CD7− Treg cells expressed TGF-β1 athigher levels in comparison to their corresponding CD7+ Tregcells within the same lesion. Noteworthy, CD7+ Treg cells inthe healthy lung expressed substantially more amounts ofTGF-β1 in comparison to their corresponding CD7− Treg cellswithin the same lesion (Fig. S4). Data imply that lesionalsarcoid Treg cells may exhibit reduced repressor capacities.

To address this issue, CD7− and CD7+ Treg cell subsetswere isolated from BALF and subjected to an in vitrosuppressor assay. Both Treg cell subsets displayed lowersuppressor activities compared to blood Treg cells of thesame patient, CD7− Treg cells displaying lower suppressoractivity than CD7+ Treg cells (Fig. 5B). Whereas essentiallythe same was found for Treg cells from healthy donors, CD7−

Treg cells from sarcoid patients displayed lower suppressoractivity than CD7− Treg cells from healthy donors (p=0.041).This does not reflect a general repression in suppressorcapacities since the repressor activity of blood CD7+ Treg

cells from sarcoid patients was not altered compared tohealthy donors (p=0.065). There is no significant differencein the suppressor activity of Treg cells dependent on thedisease stage (Fig. S5). Repressor capacities of Treg cellscorrelate with FoxP3 expression. Accordingly, FoxP3 expres-sion was decreased in both CD7+ and CD7− Treg cells fromsarcoid patients compared to healthy donors (Fig. 5C).Quantitative recording of FoxP3 expression in sarcoid lunglesions in situ confirmed the ex vivo analyses, in particular,that Treg cells of both subsets in sarcoid lung lesionsexpressed lower FoxP3 levels compared to Treg cells fromhealthy donors (Fig. 5D). Taken together we conclude thatrepressor activity is impaired in BALF Treg, but not inperipheral blood Treg cells of sarcoidosis patients.

3.5. CD7− Treg cells from sarcoid lesions displaypro-inflammatory IL-4

Treg cells from sarcoid BALF expressed the pro-inflammatorycytokine IL-4 in high frequencies and high levels, whereasTreg cells from BALF of healthy donors poorly express IL-4.The expression level is higher in CD7− than in CD7+ Treg cellsfrom the same BALF specimen (Figs. 6A and S6A). Immunos-taining of biopsies confirmed that CD7− Treg cells in sarcoidlesions expressed IL-4 which is in contrast to CD7+ Treg cellswhich poorly expressed IL-4 (Fig. S6B). Data point to a highpro-inflammatory capacity of CD7− Treg cells from sarcoidpatients which is not found in the healthy situation.

IL-4 is known to activate mast cells [16]. We thereforeasked whether mast cells in near vicinity to Treg cells insarcoid lesions express an activated phenotype with

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oncostatin M expression. Recording biopsies revealed thatmore than 80% of CD7− Treg cells in sarcoid lesions were inclose vicinity to oncostatin M-positive mast cells in contrastto less than 5% in the healthy lung. Mast cells moreover co-expressed IL-15 confirming their activated phenotype(Fig. 6B). The observation moreover demonstrates thatinfiltration of sarcoid lesions with Treg cells is associatedwith the activation of mast cells which is most likelymediated through the secretion of pro-inflammatory IL-4.

4. Discussion

Our data demonstrate that (i) Treg derived cells in sarcoidlesions, but not in the periphery, are deficient in repression,(ii) are progressed into a stage of late memory differentia-tion, (iii) and have pro-inflammatory capacities likely tofoster a chronic inflammatory process in the affected lung.

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Figure 5 CD7− Treg cells from sarcoid patients exhibit reduced repfrom 10 healthy donors and 15 patients with sarcoidosis in stage I (n=(5 ng/ml) plus ionomycin (0.5 μM) and the expression of the cy(B) Suppressor capacities of CD7− and CD7+ subset cells of FoxP3hig

standard repressor assay. CD45RO+CD25− effector T cells were label(10 μg/ml) plus anti-CD28 mAb 15E8 (1 μg/ml) for 6 days in the presflow cytometry and reduction in proliferation calculated in compariCD7− and CD7+CD25highCD127− Treg cells from blood lymphocytes or(D) FoxP3 expression of resident CD7− and CD7+CD25highCD127− TregII (n=5) and stage III (n=1) was determined by laser-scan microscopy

The vast majority of Treg derived cells in sarcoid lesionsexhibit the CD45RO+ late memory phenotype, expressreduced levels of CD27, CD28, and CD62L, lack CD7 andshow elevated levels of CD57 and KLRG-1, all of which are incontrast to the majority of Treg cells in the peripheral blood.Associated with the altered phenotype, sarcoid Treg derivedcells express altered functional properties compared to themajority of peripheral Treg cells which are of CD7+CD45RO+

phenotype. CD7− Treg cells in sarcoid lesions are likely tohave undergone extensive rounds of amplifications sincethey have shorter telomeres compared to Treg cells in theperiphery of the same patient. This is moreover inaccordance to the observation that lasting mitogenicstimulation generates CD7− from CD7+ Treg cells, but notvice versa [10], resulting in the accumulation of CD7− T cellsin the inflamed lesion. Our data thereby extend the recentreport [8] that Treg cells of CD45RO+ phenotype proliferatein granuloma lesions in situ. According to the current

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ressor capacities. (A) CD7− and CD7+ FoxP3highCD127− Treg cells8), stage II (n=6), and stage III (n=1) were stimulated with PMA

tokines IL-10 and TGF-β1 was evaluated by flow cytometry.hCD127− Treg cells from the same donors were evaluated by aed with CFSE and stimulated with agonistic anti-CD3 mAb OKT3ence of CD7+ and CD7− Treg cells. CSFE dilution was recorded byson to effector cells without Treg cells. (C) FoxP3 expression ofBALF of the same set of donors was recorded by flow cytometry.cells in healthy human lung (n=5) and sarcoidosis lesions of stage. Data represent the mean±SEM. *pb0.05. n.s., not significant.

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concept, Treg cells are divided into thymus-derived naturallyoccurring nTregs and into induced or adaptive iTregs whichupregulate FoxP3 in the periphery upon antigen exposure inpresence of TGF-β [17,18]. High levels of TGF-β1 are presentin sarcoid lesions; infiltrating CD7− Treg cells, however,express Helios identifying those cells as natural Treg cells. Inline with that, LAP and GARP are preferentially expressed onthose Treg cells moreover indicating them as activatednatural Treg cells [19].

While Treg cells with CD7− phenotype represent less than15% in the periphery, they substantially increase in numbersin sarcoid lesions during the progression of the diseaserepresenting up to 90% of all Treg cells in stage III. Theaccumulation of CD7− Treg cells may be due to extensiveamplification of those cells after infiltrating the lesion,conversion of CD7+ to CD7− Treg cells or, in addition oralternatively, by the elimination of infiltrating CD7+ Tregcells through galectin-1 mediated apoptosis whereas CD7− T

cells are resistant to galectin-1 [20]. The picture is sustainedby a recent report on high levels of galectin-1 expression ininflamed lung lesions [21] which contributes to the elimina-tion of CD7+ while preserving CD7− Treg cells. CD7− Treg cellsare moreover rescued from apoptosis by high levels of IL-15[10] which is provided by activated fibroblasts and mast cellsin sarcoid lesions. The accumulation of CD7− Treg cells in thelung is moreover sustained by the increased expression of thelung homing receptor CXCR6 and the lack of CCR7 whichresults in the inability to emigrate from the periphery back tolymphoid organs [15]. The sarcoid environment may favorthe generation and attraction of Treg cells due to TGF-β andIL-15 produced by mast cells and fibroblasts in sarcoidlesions, both cytokines known to sustain, along with strongTCR and CD28 costimulatory signals, the generation of Tregcells from effector T cells during chronic inflammation [22–25]. During therapy with vasoactive intestinal peptideperipheral Treg cells can be induced which ameliorates

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disease severity [26]. In contrast, the accumulation of CD7−

Treg cells in a more advanced differentiation stage, as foundin sarcoid lesions, is unlikely to aid recovery since those cellsexhibit a reduced repressor capacity. In contrast to aprevious report [27] accumulation of CD7− Treg cells is notassociated with an overall decline of repressor activity andan overall reduction of FoxP3, GARP, LAP and Heliosexpression in a patient's blood Treg cells. Treg cells fromthe peripheral blood of sarcoid patients show similarrepressor capacities as those Treg cells from healthy donors.The sarcoid lesion-restricted reduction in suppressor activityis in contrast to idiopathic pulmonary fibrosis where a globalreduction in both the number and the function of Treg cellswas recorded [28]. Based on the different properties of CD7−

and CD7+ Treg cells we draw the concept that in early phasesof the disease, when the frequency of CD7− Treg cells is stilllow, Treg cells may be capable to repress inflammatory T cellresponse facilitating remission of the disease. In moreadvanced stages, however, when CD7+ Treg cells extensivelyamplified and increasingly converted to CD7− Treg cells,repression becomes more and more incompetent.

Accumulation of CD7− Treg cells is moreover accompaniedby an increase in pro-inflammatory cytokine secretion. CD7−

Treg cells from sarcoid BALF secrete high amounts of IL-4along with IL-10 and TGF-β1, but no IL-5, which distinguishesthem from Th3 Treg cells. Accordingly, Treg cells with IL-4and IL-10 expression accumulate in the peripheral blood ofpatients with atopic dermatitis [29]. In early stages of thedisease the amounts of TGF-β1 and IL-10 counterbalanceTNF-α and IFN-γ levels [30]. If TNF-α and IFN-γ levels remainelevated beyond that window, however, persistently highlevels of TNF-α and IFN-γ induce IL-15 in fibroblasts and mastcells which protect newly generated CD7− Treg cells fromapoptosis. Not capable to limit the immune reaction due totheir reduced suppressor capacities, CD7− Treg cells arelikely to enhance the inflammatory reactions by IL-4secretion which leads to the recruitment of mast cells andthe expression of membrane-bound oncostatin M which inturn induces lung resident fibroblasts to proliferate andsynthesize extracellular matrix proteins [31]. TGF-β1 acti-vates lung fibroblasts and IL-10 activates alveolar macro-phages, which in turn induce lung fibroblasts to extensivelysynthesize extracellular matrix material [32]. It remainsunclear, however, whether or not programmed cell death oflesional T cells is dysregulated in chronic sarcoidosis [33].Since high IL-15 expression prevents CD7− Treg cell apoptosisand thereby amplifies the inflammatory process, persistenceof lesional Treg cells sustains granuloma formation in sarcoidlesions. IL-4 secreted by lesional CD7− Treg cells moreoveractivates mast cells as indicated by increased tryptase levelsin sarcoidosis patients with progressive disease compared tostable sarcoidosis [34], thereby promoting fibrosis.

We assume that early elimination of CD7− Treg cells oravoidance of their generation through avoiding extensiveamplification prevents progression toward chronic inflam-mation. Sarcoid patients in chronic stages of the diseasecurrently require prolonged treatment with corticosteroids,alternatives are being explored. TNF-α inhibitors wererecently reported to be beneficial in chronic stages of thedisease by inhibiting progression of granulomatosis. SinceTNF-α induces IL-15 in activated fibroblasts [35], whichprotects CD7− Treg cells from apoptosis, TNF-α neutraliza-tion is assumed to indirectly reduce the number of CD7− Tregcells. However, conflicting data on the efficacy of the anti-TNF-α therapy especially in progressive stages of the diseaseare reported [36]. It is tempting to speculate that neutral-ization of IL-15, as recently reported for rheumatoid arthritis[37], may more efficiently promote activation induced celldeath of CD7− Treg cells.

Supplementary materials related to this article can befound online at doi:10.1016/j.clim.2011.03.015.

Acknowledgments

We thank Sabrina Kießling, Bettina Hoffmann, Frank Steiger,Birgit Hops and Petra Hoffmann for technical assistance. Wethank Dr. H.P. Pircher (Institute for Immunology, Universityof Freiburg, Germany) for supplying us with the anti-KLRG-1mAb 13A2.

Funding. Our study was supported by Deutsche For-schungsgemeinschaft, Bonn, AB58/6-1, the Köln FortuneProgram of the Medical Faculty, University of Cologne andBONFOR, University of Bonn, O-139.0032.

http://dx.doi.org/10.1016/j.clim.2011.03.015


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