Thymopoiesis, Regulatory T Cells, and TCRVβ Expressionin Thymoma With and Without Myasthenia Gravis,and Modulatory Effects of Steroid Therapy

Andrea Fattorossi & Alessandra Battaglia &

Alexia Buzzonetti & Giacomo Minicuci & Raffaella Riso &

Laura Peri & Giovanni Scambia & Amelia Evoli

Received: 12 July 2007 /Accepted: 16 October 2007 /Published online: 14 November 2007# Springer Science + Business Media, LLC 2007

Abstract We analyzed thymocyte and thymic regulatory Tcell (CD4SPCD25+Foxp3+cells, Treg) development inthymoma with and without myasthenia gravis (MG, MG-thymoma, non-MG-thymoma) and in MG-associated non-neoplastic thymus (MG-NNT). An increased number ofimmature CD4+CD8−CD3− thymocytes through theCD4+CD8+ to CD4+CD8− transition and an abnormal Tcell receptor Vβ (TCRVβ) development through theCD4+CD8+ to CD4−CD8+ transition were seen both inMG-and non-MG-thymomas. Terminal thymopoiesis, i.e.,CD45RA+ cells within the CD4+CD8−CD3+ andCD8+CD4−CD3+ subsets, was skewed towards the CD4+

compartment in MG-thymoma and CD8+ compartment innon-MG-thymoma, but thymic export was increased onlyin the latter in keeping with the hypothesis that CD8+

lymphocytes may play a role in the initial stages ofautosensitization and in disagreement with the relevanceof an increased output of CD4+ T lymphocytes in paraneo-plastic MG. Treg level in normal thymus and MG-NNT andboth MG- and non-MG-thymoma was similar, and TCRVβdevelopment in Treg cells was slightly altered in thymomabut irrespective of MG presence. Thus, the relevance of adefective Treg development in MG context remains to be

established. Most alterations in thymopoiesis were cor-rected by therapeutic corticosteroid administration, and theeffects of steroid administration may be mediated by thymicmicroenvironment.

Keywords Myasthenia gravis . thymopoiesis . steroids .

recent thymic emigrants . thymoma . regulatory Tcells .

Foxp3 . TCRVβ repertoire

Introduction

Myasthenia gravis (MG) is an antibody-mediated disorderof neuromuscular junction targeting, in most cases, thenicotinic acetylcholine receptor (AChR) [1]. Several obser-vations point to a pivotal role of the thymus in thepathogenesis of the disease.

Thymic alterations are very common in MG. Theyinclude lympho-follicular hyperplasia (LFH) with germinalcenters in the thymic perivascular areas and thymoma, atumor of thymic epithelial cells harboring a variable amountof non-neoplastic lymphocytes [2–3]. Thymic LFH iscurrently considered as the site of the ongoing immuniza-tion against the AChR and a relevant source of specificantibodies (abs) [4]. This view is supported by theobservation that, in these patients, thymectomy induces asignificant clinical improvement and a marked reduction inserum anti-AChR ab level [1, 3, 5–6]. On the other hand,thymectomy appears to be less effective when MG isassociated with thymoma [7] consistently with the scarcityof B lymphocytes in the neoplastic thymus. It is wellknown that thymic epithelial tumors can associate withmany paraneoplastic diseases, of whichMG is by far the mostcommon [8]. Although the various thymoma-associated

J Clin Immunol (2008) 28:194–206DOI 10.1007/s10875-007-9147-2

A. Fattorossi (*) :A. Battaglia :A. Buzzonetti :L. Peri :G. ScambiaLaboratory of Immunology, Oncology Department,Catholic University,Campobasso, Italye-mail: cytogyn@rm.unicatt.it

G. Minicuci :R. Riso :A. EvoliNeuroscience Department, Catholic University,Rome, Italy

autoimmune diseases are sustained by different pathogenicscenarios, a thymoma-induced defect in self-tolerance mayplay a role in all these conditions. So far, these aspects havebeen investigated mainly in relation to MG, but they havenot been completely clarified.

Earlier studies in MG-associated thymoma pointed to analtered CD4+ T lymphocyte development and an increasedexport of mature, potentially autoreactive, CD4+ T lym-phocytes [9–13]. Other reports [7, 14] proposed that thefirst alteration in intratumorous thymopoiesis—before MGonset—may involve the CD8 compartment via the gener-ation of muscle-specific cytotoxic T lymphocytes thatwould be responsible for the initial phase of autosensitiza-tion. According to this hypothesis, once tolerance is broken,autoreactive CD4+ T lymphocytes would expand to provideB cell help for autoantibody production, and overt MGwould then ensue.

To gain a better understanding of thymopoiesis andperipheral thymic export in thymomas with and withoutMG (MG- and non-MG-thymomas), we analyzed thymo-cytes for thymopoietic differentiation markers and distribu-tion of T cell receptor Vβ (TCRVβ)-chain variable region,using MG non-neoplastic thymus (MG-NNT) as compari-son. We found that although thymopoiesis was altered inboth MG and non-MG-thymomas, there were some differ-ences: terminal thymocyte maturation was skewed towardsCD4+ lymphocytes in MG-thymoma and towards CD8+

lymphocytes in non-MG-thymoma, but this biased thymicproduction was reflected in the periphery only in the latter.The thymus can also contribute to MG pathogenesisthrough maturation and export of regulatory T (Treg) cells.Data on possible alterations of this subset in myasthenicpatients are controversial. One paper reported circulatingTreg in MG patients, but the authors found no differencebetween MG cases and healthy subjects [15], while in ourexperience, the number of these cells was lower inuntreated patients and increased after disease stabilizationfollowing immunosuppressive treatment [16]. Treg werefound reduced in thymoma irrespective of MG presence[17], although a trend towards a more pronounced decreasein MG-associated thymoma was later described [18].Thymic Treg have been found nonfunctional in hyperplasticMG thymus, albeit present at a normal level, in one paper[19], while no conclusive data are available on their activityin MG-thymoma. In this paper, we conclude that grossalterations in thymic Treg development are not evident inMG inasmuch as Treg frequency in normal thymus andMG-NNT and both in thymoma with and without MG wassimilar, and TCRVβ development in Treg cells was slightlyaltered in thymoma but irrespective of MG presence. Inaddition, we show that therapeutic steroid administrationrestored thymopoiesis and Treg development and postulatethat thymic stroma intervenes in mediating these effects.

Methods

Patients

Thymus samples and/or peripheral blood (PB) T lympho-cytes were evaluated in 79 subjects who underwentthymectomy in the course of the study. Their clinicalcharacteristics are reported in Table I. Patients weregrouped as follows: non-MG-thymomas; MG-thymomas(treated and untreated with immunosuppressants beforethymectomy); and MG-NNT treated and untreated withimmunosuppressants. MG-NNT included LFH and normal/involuted thymus. Two thymuses from adult individualswho underwent elective cardiac surgery were available forthe study.

Thymoma histology (Table II) was evaluated accordingto the W.H.O. classification [20]. The diagnosis of MG wasbased on conventional criteria. Anti-AChR antibodies werepresent in all MG-thymoma, 30/33 MG-NNT and 2/9 non-MG-thymoma patients. No patient had antimuscle-specifictyrosine kinase (MuSK) antibodies [21]. MG treatment wasadministered according to the accepted guidelines [22]. Inparticular, steroid therapy with oral prednisone was per-formed in 31 patients with disabling disease not satisfacto-rily controlled with anticholinesterase drugs; in 13 of thesepatients, prednisone was associated with azathioprine.Thymectomy was always performed in the presence of athymoma. Otherwise, it was carried out in patients withearly-onset (age at onset ≤45 years) generalized MG.

As all patients were not evaluated in each immunophe-notypic analysis, the actual numbers of thymus and PBsamples tested are indicated in the appropriate sections.

Isolation and Flow Cytometry Analysis of Lymphocytesfrom PB and Thymus Samples

Mononuclear cell suspension from thymus samples wasobtained immediately after surgery, as described previously[16]. Briefly, thymus tissue was mechanically disaggre-

Table I Patients’ Characteristics

Thymoma(N=46)

Non-neoplasticthymus (N=33)

Sex (M/F) 22/24 4/29Age range (mean) 26–79 years (52) 16–51 years (28)Presence of MG 37/46 33/33Immunosuppressive treatmenta

None 18/46 17/33Steroids 17/46 14/33Steroids + Azathioprine 11/46 2/33

MG Myasthenia gravisa At thymectomy

J Clin Immunol (2008) 28:194–206 195195

gated using a scalpel and needle followed by syringingthrough a 22-gauge needle under sterile conditions. PBsamples were stained by the routine whole blood techniqueusing a lyse-then-wash step, as described previously [23].Four-color flow cytometry was performed using monoclo-nal antibodies (mAb) to: CD3, CD4, CD8, CD45RO, andCD45RA, (all from Beckman Coulter, Miami, FL, USA).Foxp3 had to be determined by intracellular staining [24].To this end, cells were stained for surface antigens, washed,and then fixed and permeabilized using the staining kitprovided by eBioscence according to manufacturer’sinstructions. With permeabilized lymphocytes, mAb can giveincreased background fluorescence, possibly due to entry offree fluorochrome and/or mAb reactivity with charged orpolar internal molecules that cannot be correctly evaluated bythe conventional isotype staining. In this paper, we overcomethis complication by first incubating cells with an eightfoldmolar excess of unlabeled anti-Foxp3 mAb PCH101 clone tocompletely saturate the specific binding sites and finally withthe fluorescein isothiocyanate (FITC)-conjugated anti-Foxp3mAb. All the mAb were purchased as conjugates with thefluorescent dyes FITC, phycoerythrin (PE), phycoerythrin-Texas Red (ECD), and phycoerythrin-Cyanin 5.1 (PC5) andappropriately combined to assess the cell subset of interest inthymus and PB. Additionally, CD7 and CD19 mAb wereroutinely used in dual-color fluorescence to evaluate thepossible presence of cells other than T lymphocytes andinfiltration of thymus from peripheral B lymphocytes. The Vβusage was assessed by the mAb Vβ3.1, Vβ5.1, Vβ5.2,Vβ6.7, Vβ7, Vβ12, Vβ13.1 Vβ14, Vβ17, Vβ20, Vβ21.3,and Vβ22. When a sufficient number of cells was recovered,Vβ 8 and/or Vβ 5.3 were also analyzed. All Vβ-specific mAbwere FITC conjugated and were used together with CD4,CD8, and CD3 or CD25 in thymus. Flow cytometry wasperformed using a Beckman Coulter XL flow cytometerequipped for four-color immunofluorescence. A minimum of5,000 cells of interest were acquired for each sample.Typically, this required at least 200,000 events to be acquired.List mode data were then analyzed using Expo 32™ (Beck-man Coulter) software. Automated white blood cell count anddifferential were obtained for all PB samples.

Incubation of Thymocytes with Dexamethasone In Vitro

Thymocytes (106/ml) were cultured in RPMI 1640 mediumsupplemented with fetal calf serum (FCS) 5% and contain-ing 10−6 M dexamethasone at 37°C in a CO2 incubator for18 h. Thymocytes incubated with dexamethasone at 4°Cand in the absence of dexamethasone at 4 and 37°C servedas controls. Cultures were stained with appropriate combi-nations of fluorochrome-conjugated mAb to CD4 and CD8at 4°C and analyzed by flow cytometry, as described above.Data are expressed as percent reduction of the absolutenumber of double positive (DP) thymocytes cultured at 37°Cin the presence or absence of dexamethasone recovered at theend of culture.

Statistics

Student’s t test was used for the analysis of the statisticalsignificance between two groups. Analysis of variance(ANOVA), followed by post-hoc Tukeys’s multiple com-parison test, was used for the analysis of the statisticalsignificance among more than two groups. Correlationanalysis and Kruskall–Wallis test were used to evaluate Vβexpression. One-sided χ2 was used to evaluate steroid-induced effect on Vβ expression.

Results

Thymocyte Subset Distribution and Vβ Usage in UntreatedPatients

Figure 1a illustrates the relative proportions of the fourmain thymocyte subsets defined by the expression of CD4and CD8, i.e., CD4+CD8− (CD4 single positive, CD4SP),CD4-CD8+ (CD8 single positive, CD8SP), CD4+CD8+

(CD4CD8 double positive), and CD4−CD8− (CD4CD8double negative, DN) in the different patient groups. Therewas no significant difference, although CD4SP thymocytefrequency tended to be lower and that of CD8SP tended tobe higher in MG-thymomas. The percentage of CD3+ cellswithin the CD4SP subset was significantly higher in MG-NNT than in MG- and non-MG-thymomas, with nosignificant difference between the two thymoma groups(Fig. 1b). The proportion of CD3+ cells within the DPsubset was not significantly different (Fig. 1b). Consistentwith the normality of thymic tissue in MG not associatedwith a thymoma [2], thymocyte subset distribution in MG-NNT was comparable to that of normal adult thymus [25].The frequency of thymocyte subsets indicative of terminalthymopoiesis, i.e., CD3+CD45RA+CD4SP/CD8SP, tendedto be lower in all thymomas as compared to MG-NNT(Fig. 1c). However, the reduction of CD4SPCD3+

Table II Thymoma Histology

Subtypesa A AB B1 B2 B3

MG-thymomaTreatedb (n=28) – 2 1 16 9Not treatedb (n=9) – 2 1 4 2Non-MG-thymomaNot treatedb (n=9) 1 – 2 3 3

a According to WHO Classificationb Immunosuppressive treatment at thymectomy

196 J Clin Immunol (2008) 28:194–206

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Fig. 1 Phenotypic analysis ofthymocyte subsets of untreatedpatients. Thymus specimens wereassessed by flow cytometry forthe indicated surface antigens.a There was no difference amongthe diverse groups of patients(MG-thymoma, n=8; non-MG-thymoma, n=9; MG-NNT,n=13) in keeping with an overallpreserved thymopoiesis in thy-momas. b There was a signifi-cantly lower proportion of CD3expressing thymocytes in CD4SPsubset in both MG- and non-MG-thymoma compared to MG-NNT (p<0.05 by one-wayANOVA and Tukey’s test.). Theproportion of CD3 expressingthymocytes in DP subset wassimilar in all samples. c Maturethymocyte subsets indicative ofterminal thymopoiesis, i.e.,CD45RA+ thymocytes withinCD4SPCD3+andCD8SPCD3+subset, tended to belower in both MG- and non-MG-thymoma than MG-NNT.However, the lowest proportionof CD45RA+ thymocytes withinthe CD4SP was observed in non-MG-thymoma, whereas thelowest proportion of CD45RA+

thymocytes within the CD8SPwas found in MG-thymoma, al-though the difference did notreach statistical significance.MG-thy MG-thymoma; non-MG-thy non-MG-thymoma; MG-NNT MG non-neoplastic thymus.Bars denote mean value.

J Clin Immunol (2008) 28:194–206 197197

CD45RA+ thymocytes was marginal in MG-thymoma andprominent in non-MG-thymoma, while the reduction ofCD8SPCD3+CD45RA+ thymocytes was prominent in MG-thymoma and marginal in non-MG-thymomas. Thus, itappeared that terminal thymopoiesis was preferentiallyskewed towards the CD4+ lineage in MG-thymoma andthe CD8+ lineage in non-MG-thymoma.

An increased frequency of CD4SP thymocytes lackingCD3 is a known feature of thymoma [26, 27] and representsan exaggerated expansion of a small thymocyte subsetpresent also in normal thymus. This feature seems to berelated to an inefficient or slowed down DP to immatureCD4+ transition consequent to a reduced expression of majorhistocompatibility complex (MHC) class II molecules inthymomas [26, 28]. In a recent review, Germain [29]highlighted a small transitional CD4+CD8lowTCRint/high thy-mocyte subset preceding the acquisition of the fully matureCD4SP phenotype in normal thymus. Thus, we speculatedthat the same subset might be enlarged in thymomas. Asshown in a representative MG-thymoma sample, thispopulation was indeed expanded (Fig. 2a). Notably, therewas no difference between MG- and non-MG-thymoma (notshown) indicating that the mechanisms leading to thedefective CD4SP thymocyte maturation are the same.Consistent with a normal thymopoiesis in MG not associatedwith thymoma [2], CD4+CD8lowCD3- frequency in MG-NNT was as low as in normal thymus [25] (not shown).

To analyze T cell receptor Vβ repertoire development,thymocytes from three MG-, five non-MG-thymoma, andthree MG-NNTwere costained with mAbs to Vβ families andCD4, CD8, and CD3. For comparison, two adult normalthymuses were also studied. We assessed the concordance ofthe Vβ repertoire usage among DP, CD4SP, and CD8SPsubsets. The correlation coefficient was calculated (r2=1 beinga perfect correlation and r2=0 being no correlation), asdescribed earlier by others [30] and ourselves [31]. As shownin Fig. 3a, in normal thymuses, there was a significantconcordance in the TCRVβ repertoire usage between CD4SPand DP thymocytes as well as between CD8SP and DPthymocytes. MG-NNT, MG-, and non-MG-thymomasshowed a significant correspondence in the TCRVβ reper-toire usage between CD4SP and DP thymocytes, whereas theconcordance in the TCRVβ repertoire usage between CD8SPand DP thymocytes was looser, being present in a minority ofpatients (1/3 MG-NNT, 2/3 MG-thymomas and 3/5 non-MG-thymomas). These data are shown in Fig. 3b–d.

Thymocyte Susceptibility to Steroids

The effect of steroid treatment on thymocyte subsetdistribution was investigated in 24 MG-thymomas and 18MG-NNT patients who had received prednisone treatmentbefore thymectomy and were under treatment at the time of

surgery. The frequency of DP thymocytes was significantlyreduced both in MG-thymoma and MG-NNT with asignificant increase of more mature subsets includingCD4SP and CD8SP (Fig. 4a), and CD3+ thymocytes withinCD4SP subset (Fig. 4b). Concomitantly, steroid therapyincreased the proportion of CD3+ thymocytes withinCD4SPCD8low subset (Fig. 2b), consistent with the viewthat these cells are precursors of fully mature CD4SPthymocytes. At variance with the generalized increase ofmore mature subsets, the proportion of mature CD3+

thymocytes within DP subset did not change, either inMG-thymoma or MG-NNT (Fig. 4b). In MG-thymomas,steroid therapy increased the frequency of matureCD45RA+ thymocytes within both CD4SPCD3+ andCD8SPCD3+ subsets, although a significant differencewas attained for CD8SPCD3+ subset only (Fig. 4c).Conversely, steroid treatment did not modulate CD45RAexpression in MG-NNT, either in CD4SPCD3+ orCD8SPCD3+ thymocytes (Fig. 4c).

The effect of steroid therapy on TCRVβ developmentare summarized in Table III. Steroid administration mar-ginally affected the concordance between TCRVβ usage inCD4SP and DP in both MG-thymoma and MG-NNT,whereas the concordance between TCRVβ usage in CD8SPand DP subset was significantly reduced in MG-thymomas.

In MG patients, steroids were originally shown to inducedepletion of cortical thymocytes [32, 33], but this changeshowed a considerable variability [33, 34]. Cortical thymo-cytes largely correspond to DP thymocytes. Thus, weanalyzed the individual responses to steroid treatment interms of DP reduction. In 58% of steroid-treated MG-NNTand 21% of steroid-treated MG-thymomas (thereafterreferred to as steroid resistant), the percentage of DPthymocytes remained within the range observed in untreat-ed patients (Fig. 4a, upper left panel). The observation thatMG-thymoma was considerably less resistant than MG-NNT suggested that thymic epithelial cells could mediatesteroid susceptibility. To test this hypothesis, purifiedthymocyte suspensions from steroid-resistant and -sensitivethymuses were exposed to dexamethasone in vitro. Thisanalysis included 12 MG-NNT (4 steroid treated andresistant and 8 untreated), 9 MG-thymomas (5 steroidtreated and resistant and 4 untreated), and 1 normal thymus.As shown in Fig. 5, dexamethasone induced a comparableloss of DP thymocytes in all samples.

Contribution of MG- and non-MG-Thymoma to PeripheralT Cell Pool

We analyzed, before thymectomy, the number of newlygenerated T lymphocytes (recent thymic emigrants, RTE) inPB samples from 23 patients (10 MG-, 5 non-MG-thymomas,and 8 MG-NNT, all untreated). Twenty-two age-matched

198 J Clin Immunol (2008) 28:194–206

healthy individuals were included as controls. Measurementof RTE in the context of MG was performed in earlier studiesby flow-cytometric characterization of CD4+ and CD8+

lymphocytes with a naïve phenotype, either CD45RA+ orCD45RA+CD62L+ [9–11, 35]. More recently, RTE wasmeasured through the assessment of T cell receptor (TCR)-αexcision circles (TREC) generated during TCR-α gene rear-rangement [36]. Each method has its own pitfalls [37, 38], and

there is no conclusive demonstration of one’s superiority incomparison to the other. In fact, it has been recognized thatmost, if not all, TREC activity is contained within thephenotypically identified naïve T lymphocytes [33], andconsistently, time-course measurements of RTE frequency afterthymectomy showed that the molecular and phenotypicalapproach produced comparable results [39]. Lastly, it has beenshown that TREC levels in adults were correlated with naïve T

Fig. 2 CD3 expression in thy-moma. Representative dual-colordot plot displaying the coexpres-sion of CD4 and CD8 in a anuntreated MG-thymoma patientand b a MG-thymoma patient onsteroids. Shown are the electronicregions used to measure therelative proportions of CD3+

thymocytes in CD4SP subset andin the transitional CD4SPCD8low

stage. In the untreated patient,15% of CD4SPCD8low thymo-cytes and 44% of CD4SP coex-pressed CD3 (a, upper and lowerinset, respectively) indicating anoverall impaired CD3 expressionpathway. In steroid treated pa-tient, 62% of CD4SPCD8low

thymocytes and 97% of CD4SPcoexpressed CD3 (b, upper andlower inset, respectively), con-sistent with a CD3 inducingactivity on developing thymo-cytes (see also Fig. 4b).

J Clin Immunol (2008) 28:194–206 199199

0 1 2 3 4 0 1 2 3 4

0 1 2 3 4 5 0 1 2 3 4 50

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r2 0.516

p<0.01r2 0.491

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TCRVβ usage in DP cells

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200 J Clin Immunol (2008) 28:194–206

lymphocytes identified as CD45RA+CD45RO− rather thanCD45RA+CD62L+ [40]. Therefore, in this study, we decidedto assess RTE phenotypically as CD4+ and CD8+ CD45RA+

CD45RO− lymphocytes. The number of circulating naïveCD4+ lymphocytes did not significantly differ between MG-,non-MG-thymoma, and age-matched healthy subjects(Table IV). In contrast, in non-MG-thymoma patients, naïveCD8+ lymphocytes outnumbered naïve CD4+ lymphocytes;their number was significantly higher than in MG-thymomaand tended to be higher than in healthy subjects (Table IV). Wealso measured RTE level in MG-NNT patients. As the meanage in this patient group was lower than in thymoma patients,we included an appropriate age-matched healthy control group.There was no difference in the number of naïve CD4+ and

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Fig. 4 Phenotypic analysis of thymocyte subsets of steroid treatedpatients with MG-thymoma and MG-NNT. a Steroid administrationsignificantly reduced DP thymocyte frequency and concomitantlyenhanced CD4SP and CD8SP thymocyte frequency in MG-thymoma and MG-NNT. b CD3+ cell frequency in DP subset wasnot modified by steroid that, conversely, significantly increasedCD3+ cell frequency in CD4SP subset of MG-thymoma and MG-

NNT. c Steroid therapy increased CD45RA+ thymocyte frequencywithin both CD4SPCD3+and CD8SPCD3+subsets in MG-thymoma,although a statistical significance was attained for CD8SPCD3+sub-set only. No effect was seen in MG-NNT. Bars denote mean values.Shaded area identifies the range assessed in untreated correspon-dent patients. MG-thy MG-thymoma; MG-NNT MG-non-neoplasticthymus.

Fig. 3 Correlation analysis of TCRVβ expression in CD4SPCD3+

and CD8SPCD3+ vs DP CD3+ thymocytes. Expression of the variousTCRVβ families was determined in four-color flow cytometry withinDPCD3+and CD4SPCD3+or CD8SPCD3+ thymocytes. Concordantusage of TCRVβ repertoire between CD4SPCD3+ and DPCD3+

thymocytes in a normal adult thymus, b MG-NNT, c MG-thymomas,and d non-MG-thymomas. The analogous analysis of TCRVβrepertoire usage by CD8SPCD3+ and DPCD3+ thymocytes indicatesa looser correlation in all pathological thymuses.

R

J Clin Immunol (2008) 28:194–206 201201

CD8+ lymphocytes between patients and controls (Table V),indicating that thymus contribution to the periphery in MG-NNT patients and normal individuals is similar, consistent withthe essentially normal thymic functionality in nonparaneo-plastic MG.

Intrathymic Treg Frequency, TCRVβ Usage, and Effectof Steroid Therapy

Current evidence indicates that, in the thymus, mostCD4SPCD3+CD25+ cells have a regulatory capacity [41–43].In keeping with this notion, we found that both in MG-NNTand thymomas, most CD4SPCD3+CD25+ thymocytesexpressed Foxp3 (Fig. 6). Of note, CD4SPCD3- thymocytesdid not stain for Foxp3 and CD25, indicating that only matureCD4SP thymocyte can differentiate into Treg cells (notshown). Table VI shows that Treg level in MG-NNT (n=10),MG-thymoma (n=6), and non-MG-thymoma (n=6) wassimilar and close also to normal thymus (12.8±7, n=3, andreference [19]). Treg level was enhanced by corticosteroid(CS) therapy in both MG-thymoma (n=24) and MG-NNT

(n=11), although the difference was not significant owing to alarge inter-individual variability (Table VI).

The TCRVβ repertoire is first determined by negativeand positive selection in the thymus [44]. In an earlierreport [31], we assessed the concordance in TCRVβ usageamong the Treg and not Treg CD4+ cells as an indicator ofthe capacity of the two subsets to recognize similar sets ofantigens. Using this approach, we simultaneously tested forthe expression of 12 TCRVβ families in CD4SPCD25- andCD4SPCD25+, i.e., Treg. As a measure of concordance ofthe TCRVβ repertoire between two given samples, thecorrelation coefficient (Pearson’s correlation analysis) wascalculated (r2=1 being a perfect correlation and r2=0 beingno correlation), as described [31]. We first tested twonormal thymuses and found that both showed a significantconcordance (r2=0.65, p<0.01 and r2=0.92, p<0.005),implying a nonrandom TCRVβ usage between the twopopulations. Then, we tested 12 MG-NNT (8 not treatedand 4 CS treated), 6 MG-thymomas (2 not treated and 4 CStreated), and 4 non-MG-thymomas. We found a significantcorrelation between the TCRVβ repertoire of CD4SPCD25-

and Treg thymocytes in 6 out of 8 not treated MG-NNT andin half of both MG-and non-MG-thymomas (Fig. 7).Steroid treatment increased the concordance both in MG-NNT and MG-thymomas (Fig. 7).

Table III Steroid-induced Modifications of the Concordant Usage of TCRVβ Repertoire Between CD4SP and DP and CD8SP and DPThymocytes

MG-thymoma (%) MG-NNT (%)

CD4SP CD8SP CD4SP CD8SP

Not treated 100 (3/3) 66 (2/3) 100 (3/3) 33 (1/3)Steroid-treated 78 (7/9) 11 (1/9)* 83 (5/6) 50 (3/6)

Figures indicate the percentage and (numbers) of thymuses exhibiting a significant concordance between CD4SP and DP and CD8SP and DPthymocytes (see Fig. 3).*Different from not treated by one-sided χ2 (p<0.05)

Normal

thym

us

Untreate

d

Sensit

ive

Resist

ant

MG-NNT MG-thy

Untreate

d

Sensit

ive

Resist

ant

Rec

ov

ered

DP

th

ym

ocy

te

nu

mb

er (

%)

0

25

50

75

Fig. 5 In vitro response of isolated thymocytes to steroid. Thymusspecimens were cultured for 18 h in medium or in the presence of 10−6 Mdexamethasone and the number of DP thymocytes assessed as describedin the “Methods”. Steroid induced an evident loss of DP thymocytes inall samples, irrespective of prior in vivo treatment and thymocyte origin,i.e., MG-thymoma, MG-NNT, and normal adult thymus.

Table IV Number Per Microliter of Naïve CD4+ and CD8+ Cells inUntreated MG- and Non-MG-thymoma Patients and Age-matchedHealthy Subjects

MG-Thymoma(n=10)

Non-MG-Thymoma(n=5)

HS(n=8)

Naïve CD4+ 291±223a 118±108 352±223Naïve CD8+ 277±197 678±90* 380±222Age range(mean)

38–69 (53) 33–79 (61) 33–79 (54)

aMean±SD*Significantly different (p=0.049) from MG-Thymoma by ANOVAand Tukey’s test

202 J Clin Immunol (2008) 28:194–206

Discussion

The role of thymoma in the pathogenesis of MG has beenrelated to a flawed T lymphocyte generation and export [8–13].In particular, an increase of circulating CD4+RTE in MG-thymoma patients has been taken as evidence for acontinuous contribution of autoreactive T lymphocytes bythe neoplastic thymus [10, 11]. In this paper, we confirm thatterminal thymopoiesis is skewed towards the CD4+ compart-ment in MG-thymoma, but we show that the intratumorouspreferential production of CD4+ lymphocytes is not associ-ated with a concomitant increase of CD4+ RTE in PB. Thisdiscrepancy may be owing to differences in flow cytometrytechnique leading to the incorrect inclusion of transitionalCD45RAdim/CD45ROdim circulating not naïve T lympho-cytes in RTE counting. Additionally, we investigated changes

occurring in absolute RTE numbers rather than percentages,thereby providing a more correct picture of the distribution oflymphocyte subsets in the periphery. Thus, our data suggestthat a flawed intratumorous CD4 thymopoiesis does exist inMG-thymoma but is likely to be less relevant in MGpathogenesis than commonly thought. Accordingly, we foundthat a further indicator of an altered CD4+ thymocytedevelopment, i.e., the abnormal accumulation of CD4SPthymocytes lacking CD3 expression, equally characterizedMG and non-MG-thymomas. Present observations suggestthat, although thymopoietic activity remains a prerequisite forMG development in thymoma [8], once thymoma-derivedintolerant T lymphocytes have been exported to the extra-tumorous immune system, e.g., lymph nodes, the autoim-mune process proceeds independently [7]. This hypothesis isalso in line with the observation that thymoma removal

Table V Number Per Microliter of Naïve CD4+ and CD8+ Cells inUntreated MG-NNT Patients and Age-matched Healthy Subjects

MG-NNT (n=8) HS (n=14)

Naïve CD4+ 277±232a 359±180Naïve CD8+ 293±205 379±187Age range (mean) 17–50 (40) 16–50 (33)

aMean±SD

Fig. 6 CD25 and Foxp3 expression on CD4SP thymocytes. Thymussample (MG-NNT) was stained with mAb to CD4, CD8, CD25, andFoxp3. An electronic gate was established on CD4SP thymocytes toexplore CD25 and Foxp3 expression (not shown). Quadrants aredrawn based on fluorescence signal generated by a sample stainedwith isotype-matched control antibody for PE-CD25 and an eightfoldmolar excess of unlabeled anti-Foxp3 mAb followed by FITC-conjugated anti-Foxp3 mAb for FITC-Foxp3. In this representativesample, 2.4% of cells are CD25+Foxp3−, 9.1% coexpress CD25 andFoxp3, and 5.7% are CD25−Foxp3+. The latter population is anartifact due to the reduction of CD25 staining intensity induced by thefixation/permeabilization procedure required for Foxp3 detection.Thus, a strict correlation exists between CD25 and Foxp3 expressionin CD4SP thymocytes.

Table VI Percentage of Treg Within CD4SPCD3+ Thymocytes inMG-NNT, and MG- and Non-MG-thymoma, and Effect of CSTherapy

Treg

NT MG-NNT 12.9±6.2NT MG-thymoma 13.0±12.1Non-MG-thymoma 10.8±5.7CS MG-NNT 20.1±14.8CS MG-thymoma 16.8±11.8

0.00

0.25

0.50

0.75

1.00

Not

trea

ted

MG

-thy

Non

MG

-thy

CS M

G-t

hy

Not

trea

ted

MG

-NN

T

CS M

G-N

NT

Co

rrel

atio

n c

oef

fici

ent

(r2)

Fig. 7 Correlation analysis of TCRVβ expression in Treg vs non-Treg CD4SP thymocytes. The expression of the various TCRVβfamilies was determined in four-color flow cytometry within theCD4SP subset. Pearson’s correlation coefficient (r2) for each thymussample is shown on y-axis. A significantly concordant (p<0.05) andnot concordant usage of TCRVβ repertoire between Treg and non-Treg CD4SP thymocytes is represented as filled and empty circles,respectively. A significantly concordant usage is prevalent in not-treated MG-NNT compared to both non-MG and not-treated MG-thymoma. The analogous analysis made after CS therapy shows anincrease in the concordant usage of TCRVβ repertoire in MG-NNTand MG-thymoma. Bars represent median value.

J Clin Immunol (2008) 28:194–206 203203

neither improves MG symptoms nor is associated with asignificant decrease of serum antibody level [1, 3].

In non-MG-thymoma, thymopoiesis tended to be skewedtowards the CD8+ compartment, and there was a concom-itant increase of circulating CD8+ RTE, indicating that inthese patients, thymoma contributes to peripheral CD8+

lymphocyte homeostasis. It has been hypothesized that theCD8+ T lymphocytes could be involved in a very earlyphase of auto-sensitization in MG, producing a muscletissue damage long before the clinical onset of the disease[7, 14, 35]. This would explain the occasional occurrence ofMG after thymoma removal [45, 46].

A number of reports emphasized the role of an intra-thymic and/or peripheral TCRVβ repertoire skewed to-wards the expression of certain TCRVβ families in MG[47–50]. Rather than looking for preferential TCRVβfamily usage, we focused on intrathymic TCRVβ develop-ment during the differentiation of DP thymocytes intomature CD4SP and CD8SP thymocytes, crucial for shapingTCR repertoire. We first assessed the intrathymic pattern ofTCRVβ usage in normal adult thymus and found asignificant concordance in TCRVβ usage between CD4SPand DP thymocytes and CD8SP and DP thymocytes. Then,we explored TCRVβ usage in MG- and non-MG-thymo-mas and MG-NNT. A concordance was present betweenCD4SP and DP thymocytes in all thymuses, less frequentlybetween CD8SP and DP thymocytes. This result wassurprising. In fact, we expected a distorted TCR develop-ment in CD4 rather than CD8 compartment in thymomas,owing to the reduced MHC class II expression on thymomaepithelial cells [9, 26, 28]. The significance of thesefindings and especially of the altered TCRVβ usage inCD8 thymocytes in some patients will require furtherinvestigation.

Next, we approached the issue of thymic Treg alterationin the context of paraneoplastic and nonparaneoplastic MG.Treg frequency in MG-NNT did not differ from that innormal thymus and was also similar to MG- and non-MG-thymoma, suggesting that a numerical Treg defect is notinvolved in MG. This conclusion contrasts with an earlierhypothesis based on the finding of a reduced Foxp3message in whole-tissue extracts of MG- and non-MG-thymoma [17]. The most likely reason for the discrepancyresides in the modality of Treg assessment. In this paper,we measured Treg as CD4SPCD25+Foxp3+ cells in purifiedthymocyte preparations avoiding the interference of cellsother than thymocytes. We found that Treg TCRVβdevelopment was moderately altered in MG-NNT, while itwas more obviously distorted in thymomas. The data werenonparametrically distributed, and the number of patientswas small, preventing statistical significance to be attained.However, the lack of difference between MG- and non-MG-thymoma tempers the relevance of this finding in the

context of paraneoplastic MG. We hypothesize that ratherthan a gross thymic Treg cell deficiency being the cause ofdisease precipitation, there may be subtle maturativedefects. For example, it has been shown that the thymicTreg in MG-NNT are less functional but are present withinthe same range of percentages as control thymus [19].

In the last part of the study, we asked how steroids,frequently administered for the treatment of MG, influencethymopoiesis in these patients. Steroids were originallyshown to induce immunohistochemical changes indicativeof a selective cortical thymocyte depletion [32–34] thoughtto be due to a preferential killing of immature thymocytes.Present data confirm those findings and provide also aquantitative evaluation of thymocyte subsets after steroidtreatment. However, we disagree with the simplisticconclusion that steroid-driven modifications are fullyexplained by depletion of less mature thymocyte subsets.The persistence of immature CD3− thymocytes within theDP subset concomitant with their depletion within theCD4SP subset and the interference with TCRVβ develop-ment point to a more complex activity on developingthymocytes, and we speculate that microenvironment isimportant on the ground that (a) the effect on terminalthymopoiesis produced by steroids in MG was much moreevident in thymoma than in MG-NNT, and (b) steroidresistance was much more frequent when thymocytes wereassociated with non-neoplastic thymic epithelial cells, inkeeping with previous observations [32–34]. Support to thishypothesis comes from earlier studies showing that steroidtreatment regulates terminal differentiation of thymicepithelial cells [51] and enhances the production of IL-7,a cytokine that favors terminal thymopoiesis and modulatesCD3/TCRαβ rearrangement [52, 53]. Additionally, humor-al factors released from thymic epithelial cells rescuethymocytes from steroid-induced killing [54], and steroidadministration induces profound morphological changes inthymoma epithelial cells [55].

Treg were also susceptible of modulation followingsteroid administration: their frequency increased, andTCRVβ development was normalized in both MG-NNTand MG-thymoma. As the acquisition of regulatory activityrepresents a further differentiative step of CD4SP thymo-cytes, these findings are consistent with the view that CSexerts a generalized maturative effect on the thymus.However, whether these changes are somehow related tothe clinical effects of steroids in MG patients remainsspeculative.

In summary, our findings question the relevance of acontinuous CD4+ T cell export from MG-thymoma in thedisease pathogenesis and support the view of an enhancedgeneration and export of CD8+ naïve T lymphocytes innon-MG-thymoma. We also show that that gross alterationsin Treg development are not evident in either paraneoplastic

204 J Clin Immunol (2008) 28:194–206

and nonparaneoplastic MG. Lastly, we suggest that thymo-cyte response to steroid administration is largely dependenton thymic microenvironment.

Acknowledgments Supported by Catholic University grant (LineaD1) to A. E.

References

1. Vincent A. Unraveling the pathogenesis of myasthenia gravis. NatRev Immunol 2002;2:797–804.

2. Haynes BF, Hale LP. The human thymus. A chimeric organcomprised of central and peripheral lymphoid components.Immunol Res 1998;18:175–92.

3. Willcox N. Myasthenia gravis. Curr Opin Immunol 1993;5:910–7.4. Shiono H, Roxanis I, Zhang W, Sims GP, Meager A, Jacobson

LW, et al. Scenarios for autoimmunization of T and B cells inmyasthenia gravis. Ann N Y Acad Sci 2003;998:237–56.

5. Katzberg HD, Aziz T, Oger J. In myasthenia gravis, clinical andimmunological improvement post-thymectomy segregate withresults of in vitro antibody secretion by immunocytes. J NeurolSci 2002;202:77–83.

6. Okumura M, Ohta M, Takeuchi Y, Shiono H, Inoue M, FukuharaK, et al. The immunologic role of thymectomy in the treatment ofmyasthenia gravis: implication of thymus associated B-lympho-cyte subset in reduction of the anti-acetylcholine receptor antibodytiter. J Thorac Cardiovasc Surg 2003;126:1922–8.

7. Vincent A, Willcox N. The role of T-cells in the initiation ofautoantibody responses in thymoma patients. Pathol Res Pract1999;195:535–40.

8. Müller-Hermelink HK, Marx A. Thymoma. Curr Opin Oncol2000;12:426–33.

9. Nenninger R, Schultz A, Hoffacker V, Helmreich M, Wilisch A,Vandekerckhove B, et al. Abnormal thymocyte development andgeneration of autoreactive T cells in mixed and cortical thymomas.Lab Invest 1998;78:743–53.

10. Ströbel P, Helmreich M, Menioudakis G, Lewin SR, Rüdiger T,Bauer A, et al. Paraneoplastic myasthenia gravis correlates withgeneration of mature naive CD4+ T cells in thymomas. Blood2002;100:159–66.

11. Ströbel P, Preisshofen T, Helmreich M, Müller-Hermelink HK,Marx A. Pathomechanisms of paraneoplastic myasthenia gravis.Clin Dev Immunol 2003;10:7–12.

12. Evoli A, Minicuci GM, Vitaliani R, Battaglia A, Della Marca G,Fattorossi A. Paraneoplastic diseases associated with thymoma. JNeurol 2007;254:756–62.

13. Buckley C, Douek D, Newsom-Davis J, Vincent A, Willcox N.Mature, long-lived CD4+ and CD8+ T cells are generated by thethymoma in myasthenia gravis. Ann Neurol 2001;50:64–72.

14. Machens A, Loliger C, Pichlmeier U, Emskotter T, Busch C,Izbicki JR. Correlation of thymic pathology with HLA inmyasthenia gravis. Clin Immunol 1999;91:296–301.

15. Sun Y, Qiao J, Lu CZ, Zhao CB, Zhu XM, Xiao BG. Increase ofcirculating CD4+CD25+ T cells in myasthenia gravis patients withstability and thymectomy. Clin Immunol 2004;112:284–9.

16. Fattorossi A, Battaglia A, Buzzonetti A, Ciaraffa F, Scambia G,Evoli A. Circulating and thymic CD4 CD25 T regulatory cells inmyasthenia gravis: effect of immunosuppressive treatment. Im-munology 2005;116:134–41.

17. Strobel P, Rosenwald A, Beyersdorf N, Kerkau T, Elert O,Murumagi A, et al. Selective loss of regulatory T cells inthymomas. Ann Neurol 2004;56:901–4.

18. Evoli A, Minicuci GM, Vitaliani R, Battaglia A, Della Marca G,Lauriola L, et al. Paraneoplastic diseases associated withthymoma. J Neurol 2007;254:756–62.

19. Balandina A, Lécart S, Dartevelle F, Saoudi A, Berrih-Aknin S.Functional defect of regulatory CD4(+)CD25+ T cells in thethymus of patients with autoimmune myasthenia gravis. Blood2005;105:735–41.

20. Okumura M, Miyoshi S, Fujii Y, Takeuchi Y, Shiono H, Inoue M,et al. Clinical and functional significance of WHO classificationon human thymic epithelial neoplasms: a study of 146 consecutivetumors. Am J Surg Pathol 2001;25:103–10.

21. Evoli A, Tonali PA, Padua L, Monaco ML, Scuderi F, Batocchi AP,et al. Clinical correlates with anti-MuSK antibodies in generalizedseronegative myasthenia gravis. Brain 2003;126:2304–11.

22. Keesey JC. Clinical evaluation and management of myastheniagravis. Muscle Nerve 2004;29:484–505.

23. Pacifici R, Zuccaro P, Cozzi-Lepre A, di Carlo S, Bacosi A,Fattorossi A. Quantification of the variation due to lysingtechnique in immunophenotyping of healthy and HIV-infectedindividuals. Clin Biochem 1998;31:165–72.

24. Battaglia A, Buzzonetti A, Monego G, Peri L, Ferrandina G,Fanfani F, et al. (2007) Neuropilin-1 expression identifies a subsetof regulatory T cells in human lymph nodes that is modulated bypre-operative chemoradiation therapy in cervical cancer. Immu-nology (in press).

25. Bertho JM, Demarquay C, Moulian N, Van Der Meeren A, Berrih-Aknin S, Gourmelon P. Phenotypic and immunohistologicalanalyses of the human adult thymus: evidence for an activethymus during adult life. Cell Immunol 1997;179:30–40.

26. Takeuchi Y, Fujii Y, Okumura M, Inada K, Nakahara K, MatsudaH. Accumulation of immature CD3−CD4+CD8− single positivecells that lack CD69 in epithelial cell tumors of the humanthymus. Cell Immunol 1995;161:181–7.

27. Inoue M, Fujii Y, Okumura M, Takeuchi Y, Shiono H, Miyoshi S,et al. Mature CD4 single positive thymocytes in human thymoma:T cells may differentiate in the thymic epithelial cell tumor.Pathobiology 1997;65:216–22.

28. Kadota Y, Okumura M, Miyoshi S, Kitagawa-Sakakida S, Inoue M,Shiono H, et al. Altered T cell development in human thymoma isrelated to impairment of MHC class II transactivator expressioninduced by interferon-gamma (IFN-γ). Clin Exp Immunol2000;121:59–68.

29. Germain RN. T-cell development and the CD4-CD8 lineagedecision. Nat Rev Immunol 2002;2:309–22.

30. Reinhardt C, Melms A. Skewed TCR V beta repertoire in humanthymus persists after thymic emigration: influence of genomicimposition, thymic maturation and environmental challenge onhuman TCRV beta usage in vivo. Immunobiology 1998;199:74–86.

31. Battaglia A, Ferrandina G, Buzzonetti A, Malinconico P, Legge F,Salutari V, et al. Lymphocyte populations in human lymph nodes.Alterations in CD4+CD25+ T regulatory cell phenotype and T-cellreceptor Vbeta repertoire. Immunology 2003;110:304–12.

32. Berrih S, Safar D, Levasseur P, Gaud C, Bach JF. The in vivoeffects of corticosteroids on thymocyte subsets in myastheniagravis. J Clin Immunol 1984;4:92–7.

33. Willcox N. The thymus in myasthenia gravis patients, and the invivo effects of corticosteroids on its cellularity, histology andfunctions. In: Kendall MD, Ritter MA, editors. Thymus update.Harwood Academic Publ, Amsterdam 1989, p 105–24.

34. Willcox N, Schluep M, Sommer N, Campana D, Janossy G,Brown AN, et al. Variable corticosteroid sensitivity of thymiccortex and medullary peripheral-type lymphoid tissue in myas-thenia gravis patients: structural and functional effects. Q J Med1989;73:1071–87.

35. Hoffacker V, Schultz A, Tiesinga JJ, Gold R, Schalke B, NixW, et al.Thymomas alter the T-cell subset composition in the blood: a

J Clin Immunol (2008) 28:194–206 205205

potential mechanism for thymoma-associated autoimmune disease.Blood 2000;96:3872–9.

36. Ye P, Kirschner DE. Measuring emigration of human thymocytesby T-cell receptor excision circles. Crit Rev Immunol 2002;22:483–97.

37. Hazenberg MD, Borghans JA, de Boer RJ, Miedema F. Thymicoutput: a bad TREC record. Nat Immunol 2003;4:97–9. (quiprobabilmente è 109).

38. Poulin JF, Viswanathan MN, Harris JM, Komanduri KV, Wieder E,Ringuette N, et al. Direct evidence for thymic function in adulthumans. J Exp Med 1999;190:479–86.

39. Sempowski G, Thomasch J, Gooding M, Hale L, Edwards L,Ciafaloni E, et al. Effect of thymectomy on human peripheralblood T cell pools in myasthenia gravis. J Immunol 2001;166:2808–17.

40. Steffens CM, Al-Harthi L, Shott S, Yogev R, Landay A.Evaluation of thymopoiesis using T cell receptor excision circles(TRECs): differential correlation between adult and pediatricTRECs and naïve phenotypes. Clin Immunol 2000;97:95–101.

41. Stephens LA, Mottet C, Mason D, Powrie F. Human CD4(+)CD25(+) thymocytes and peripheral T cells have immunesuppressive activity in vitro. Eur J Immunol 2001;31:1247–54.

42. Wing K, Ekmark A, Karlsson H, Rudin A, Suri-Payer E.Characterization of human CD25+CD4+ T cells in thymus, cordand adult blood. Immunology 2002;106:190–9.

43. Annunziato F, Cosmi L, Liotta F, Lazzeri E, Manetti R, Vanini V,et al. Phenotype, localization, and mechanism of suppression ofCD4+CD25+ human thymocytes. J Exp Med 2002;196:379–87.

44. Davis MM, Bjorkman PJ. T-cell antigen receptor genes and T-cellrecognition. Nature 1988;334:395–402.

45. Wakata N, Fukuya H, Niizuma M, Ishida T, Kinoshita M.Myasthenia gravis developing after discovery of thymoma. ClinNeurol Neurosurg 1992;94:303–6.

46. Mineo TC, Biancari F, D’Andrea V. Late onset of myastheniagravis after total resection of thymoma: report of two cases. JCardiovasc Surg 1996;37:531–3.

47. Grunewald J, Ahlberg R, Lefvert AK, DerSimonian H, Wigzell H,Janson CH. Abnormal T-cell expansion and V-gene usage inmyasthenia gravis patients. Scand J Immunol 1991;34:161–8.

48. Truffault F, Cohen-Kaminsky S, Khalil I, Levasseur P, Berrih-Aknin S. Altered intrathymic T-cell repertoire in human myasthe-nia gravis. Ann Neurol 1997;41:731–41.

49. Navaneetham D, Penn AS, Howard JF Jr, Conti-Fine BM. TCR-Vbeta usage in the thymus and blood of myasthenia gravispatients. J Autoimmun 1998;11:621–33.

50. Tackenberg B, Kruth J, Bartholomaeus JE, Schlegel K, OertelWH, Willcox N, et al. Clonal expansions of CD4+ B helper Tcells in autoimmune myasthenia gravis. Eur J Immunol 2007;37:849–63.

51. Hale LP, Markert ML. Corticosteroids regulate epithelial celldifferentiation and Hassal body formation in the human thymus. JImmunol 2004;172:617–24.

52. Zubkova I, Mostowski H, Zaitseva M. Up-regulation of IL-7,stromal derived factor-1 alpha, thymus-expressed chemokine, andsecondary lymphoid tissue chemokine gene expression in thestromal cells in response to thymocyte depletion: implication forthymus reconstitution. J Immunol 2005;175:2321–30.

53. Okamoto Y, Douek DC, McFarland RD, Koup RA. Effects ofexogenous interleukin-7 on human thymus function. Blood 2002;99:2851–58.

54. Gao Y, Kinoshita Y, Hato F, Tominaga K, Tsuji Y. Suppression ofglucocorticoid-induced thymocyte apoptosis by co-culture withthymic epithelial cells. Cell Mol Biol 1996;42:227–34.

55. Tateyama H, Takahashi E, Saito Y, Fukai I, Fujii Y, Niwa H, et al.Histopathologic changes of thymoma preoperatively treated withcorticosteroids. Virchows Arch 2001;438:238–47.

206 J Clin Immunol (2008) 28:194–206