Immunology 1991 73 151-158

Thymic nurse cells (TNC) in spontaneous thymoma BUF/Mna ratsas a model to study their roles in T-cell development

T. EZAKI, K. MATSUNO & M. KOTANI* 2nd Department of Anatomy, Kumamoto University Medical School,Honjo, Kumamoto, Japan

Acceptedfor publication 27 February 1991

SUMMARY

In order to elucidate possible roles of thymic nurse cells (TNC) we isolated them as lympho-epithelialcell complexes from spontaneous thymomas of BUF/Mna rats and characterized them usingimmuno- and enzyme-histochemical techniques. A remarkable increase in the number ofTNC wasseen at 8 months of age, immediately before or in accordance with the onset of thymomas. Nostructural abnormality in the TNC was detected by light-microscopy compared with those fromnormal control thymi. Phenotypically, the TNC per se were positive for major histocompatibilitycomplex (MHC) class I, class II, cytokeratin and thymulin, but lacked macrophage markers,indicating their epithelial origin. They also expressed some of the markers for non-epithelialcomponents, such as Thy- 1, SI 00 and peanut agglutinin (PNA). The majority ofintra-TNC cells wereMHC class 1+, Thy-I+, CD5+, CD4+CD8+ (double positive) and PNA+, but with someheterogeneity in their phenotype. The intra-TNC cells from thymomas revealed higher proliferationindices than those from control thymi, as assessed by 5-bromo-2'-deoxyuridine (BrdU)-uptake. Itwas also demonstrated for the first time that, not only in thymoma rats but also in normal controlrats, about 15-30% of TNC enclosed macrophage populations within them. These results maysuggest that the TNC in BUF/Mna thymomas represent typical TNC populations, but they arehyperactive particularly in their number and nursing capacity, resulting in the unusual increment ofthymocytes in the thymomas. This animal model lends itself greatly to studies on the regulatory rolesof TNC in T-cell development.

INTRODUCTION

Thymic nurse cells (TNC) are the specialized epithelial orstromal cells that form unique multi-cellular complexes byenclosing many thymocytes within them. ' They are known to beone of the most important components of the thymic microen-vironment which may induce and select T-cell repertoires in thisorgan.2 In vitro studies have revealed the possible roles ofTNCin T-cell development, such as presentation of self antigens3 andnon-self antigens4 or triggering leukaemogenesis.5 However,their real status and functions being manifested in situ still

* Present address: Kyoto College of Medical Technology, 1-3,Imakita, Koyama-Higashi-machi, Sonobe-cho, Funai-gun, Kyoto 622,Japan.

Abbreviations: ALP, alkaline phosphatase; BrdU, 5-bromo-2'-deoxyuridine; FCS, foetal calf serum; FTS, facteur thymique serique (orthymulin); HRP, horseradish peroxidase; mAb, monoclonal antibody;PBS, phosphate-buffered saline; PNA, peanut agglutinin; TNC, thymicnurse cell(s).

Correspondence: Dr T. Ezaki, Dept. of Anatomy, KumamotoUniversity Medical School, 2-2-1, Honjo, Kumamoto 860, Japan.

remain controversial.6 9 This may be due to the facts that TNCcan be obtained only in very young animals and that theirabsolute number found in a thymus is too few to analyse in situor in vivo.2"i0

Very recently, we have found numerous TNC increasing inthe cortical areas ofthymomas in Buffalo/Mna (BUF/Mna) ratsand found clear evidence of their existence in situ." The ratsdevelop spontaneous benign thymomas of epithelial origin at 9-15 months of age'2 in an autosomal dominant manner."' Theunusual increment ofTNC and their availability should enableus to analyse their characteristics in detail and to elucidate theirbiological roles in the T-cell development and the pathogenesisof the thymoma.

The present study deals with both TNC per se and intra-TNC cells by isolating the cellular complexes from thymiafter enzymatic digestion. This brings systematic characteriza-tion of TNC with regard to phenotypic and functional markersusing immuno- and enzyme-histochemical techniques. Thisanimal model of TNC has also provided novel evidence thatmacrophage populations may take part in forming the specialmicroenvironment within normal TNC.

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T. Ezaki, K. Matsuno & M. Kotani

MATERIALS AND METHODS

A nimialsInbred BUF/Mna rats and DA rats were bred in our LaboratoryAnimal Centre for Experimental Research. DA rats were usedas normal non-thymoma controls. Unless otherwise stated,male rats, more than three animals in each group, were used forthe following analyses.

Antibodies and other reagents for phenot-'pingWe used mouse monoclonal antibodies (mAb) against ratlymphoid cells as follows: OX 19 (anti-CD5), W3/25 (anti-CD4),OX8 (anti-CD8) and OX7 (anti-Thy-I) (purchased from Sera-Lab, Crawley Down, Sussex, U.K.); HAM2 (anti-MHC class I)and OX6 (anti-MHC class II or la) (kind gifts of Professor T.Fukumoto, Yamaguchi University Medical School, Ube,Japan); and EDI, ED2 and ED3 (anti-rat macrophage subpo-pulations) (kind gifts of Dr C. D. Dijkstra, Vrije University,Amsterdam, The Netherlands). A rabbit anti-FTS (facteurthymique serique or thymulin) antibody (a kind gift of ProfessorK. Seiki. Tokai University Medical School, Kanagawa, Japan),a rabbit anti-S 100 antibody (Advance Co. Ltd, Tokyo, Japan), arabbit anti-keratin antibody (Dako Corp., Santa Barbara, CA)and a biotin-conjugated peanut agglutinin (PNA; VectorLaboratories Inc., Burlingame, CA) were also obtained. Assecondary reagents, a horseradish peroxidase (HRP) conju-gated rabbit anti-mouse immunoglobulin (Ig) antibody (Dako),an alkaline phosphatase (ALP)-conjugated rabbit anti-mouseIg antibody (Dako), an HRP-conjugated goat anti-rabbit Igantibody (Bio-Rad Lab., Richmond, CA) and an HRP-conju-gated streptavidin (KPL Lab. Inc., Gaithersburg, MD) wereused in the indirect enzyme-immunostaining. A thymidineanalogue, 5-bromo-2'-deoxyuridine (BrdU) (Sigma, St Louis,MO) and a mouse mAb against BrdU (Becton-DickinsonImmunocytometry Systems, Mountain View, CA) were used todetect proliferating cells. BrdU (4 mg/100 g body weight) wasinjected into rats intraperitoneally 2 hr before the assay.

Thivmus dissociation and isolation of TNCRats were killed by cervical dislocation under ether anaesthesiaand thymi removed and put in calcium- and magnesium-freephosphate-buffered saline (PBS). Pericapsular connective tissueand parathymic lymph nodes were carefully trimmed off thethymi. The cleaned organs were minced into small pieces withscissors. Thymus fragments were placed into a dissociationbottle containing 20 ml of 0-25%, trypsin (Wako Pure ChemicalIndustries, Osaka, Japan) in PBS with I mg/ml collagenase(Wako) and 25 ,ug/ml deoxyribonuclease I (Sigma). The disso-ciation was achieved by low-speed stirring (about 200 r.p.m.) for20 min at room temperature. This enzymatic digestion did notaffect the efficiency of immunostaining in the subsequentphenotype analyses. The dispersed cell suspensions were cooledin an ice-bath to terminate the enzyme digestion, filteredthrough a stainless mesh to remove all debris and clots, thentransferred into 50-ml centrifuge tubes. Heat-inactivated foetalcalf serum (FCS) was carefully underlaid at the bottom and thetubes were centrifuged at g for 7 min at 4'. The cell pellets wereresuspended in 5 ml of PBS and the same volume of FCS wasunderlaid at the bottom of the conical centrifuge tube. TNCwere isolated by four or five runs of sedimentation at 1 gaccording to previous methods." 5 Each step yielded a top

supernatant layer and a bottom FCS layer with cell pellets. Thislatter fraction was submitted to repeated runs of sedimentationuntil a sufficient enrichment with TNC was obtained.

Immunohistochenistr pThymi were freshly frozen in liquid nitrogen and 6-pm thickcryosections were made, air-dried, and then fixed in acetone for10 min at room temperature. Smears of the isolated TNC werealso made and fixed in the same way. For staining, the indirectimmunoenzymatic method was employed as described pre-viously.'4 Finally, the samples were lightly counterstained withhaematoxylin. The positive staining for various markers wasestimated as either the marginal pattern of TNC or thehoneycomb-like pattern of intra-TNC cells by bringing themindividually into focus under a light-microscope. All negativecontrols stained either with unrelated mouse mAb or in theabsence of the first specific reagents showed no or only negligiblelevel of background stain.

EnZ mehistochemistrJ'For acid phosphatase staining, sections or smears were fixed inacetone for 5 min at room temperature before the enzymereaction (at pH 5 0) by the method of Goldberg & Barka.'5 Fornon-specific esterase staining, the sections were fixed in 1%glutaraldehyde in PBS for 10 seconds and washed in PBS. Thereaction (at pH 6 1) was carried out according to Yam et al.'6

Statistical anali'sisAt least three individual samples in each assay were analysed.Statistical significance of differences in mean values was assessedusing Student's t-test. P values less than 0 05 were considered tobe statistically significant.

RESULTS

Changes in the number of TNC with age

TNC were isolated from thymi ofBUF/Mna rats at various agesand compared with those from non-thymoma control DA rats.Light-microscopically, most TNC from thymomas usuallyenclosed 10-100 viable lymphoid cells and no structural differ-ence was found in comparison with those from normal controlthymi. In BUF/Mna rats, the absolute numbers of TNC perwhole thymus were higher than those of control DA rats at allages examined (Table 1). TNC per IO thymocytes and TNC perg thymus were, however, almost similar to those of DA rats tillabout 2 months of age. All three values began to increasesuddenly, explosively at 8 months of age, when the size of thymiwas still in the normal range. After this critical period, the totalnumber ofTNC kept increasing as the weight of thymus and thenumber of thymocytes increased, while the other two valuesremained at almost the same level.

Phenotypes of TNC in BUF/Mna thymoma rats

Smears of the isolated TNC from 14-16-month-old thymomaswere examined in their phenotypes by immunohistochemistryand enzymehistochemistry. The results are summarized in Table2. Most of the TNC were strongly positive for both MHC class I(Fig. Ia) and class II (Fig. Ib), cytokeratin (Fig. Ic), Thy-I (Fig.

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TNC in thymoma BUFIMna rats

Table 1. Changes in the number of TNC with age

Thymus Thymocyte TNC TNC per TNC perAge weight number number 108 thymocytes g thymus

Strain (month) (g) (X 108) (X 103) (X 103) (X 103) n*

DA 1 0-19 5-0 5 0 1-0 26-3 42 0-30 9-1 5-1 0-6 16-5 92-5 0-24 6-3 3 0 0-5 12-5 37 0-12 0-5 1-6 3 9 13-8 5

BUF/Mna 1 0-39 13-2 14-7 1-1 36-8 41-5 0-79 22-8 12-3 0-5 15-7 32 1-35 58-7 23-4 0-4 17-4 36 1 05 31-7 34-9 1-3 37-4 48 1 10 20-1 216-7 10-8 197-0 316 15-86 508 6 3872-6 7-7 255-3 422 25 03 834-1 6024 3 7-4 240-1 3

* n: number of animals examined.

Table 2. Phenotype ofTNC in thymomas

Phenotype % positive* Intensityt % negative

MHC class I 92 + + + 8MHC class II 99 + -~mostly+ + IThy-i 94 + + 6PNA 99 + + IEDI <1 >99ED2 < 1 >99ED3 0 100Keratin 95 + . + + 5S100 76 + 24FTS 92 + - + 8

* More than 500 TNC from 14-16-month-old thymoma ratswere assessed in each assay.

t The staining intensity was expressed as: -, negative; +weakly positive; +, positive; + +, strongly positive.

Id) and PNA, but negative for ED I (Fig. 3a), ED2 (Fig. 3b) andED3 macrophage markers. TNC lacked reactivity for typicallysosomal enzymes, such as acid phosphatase (Fig. 3c,d) andnon-specific esterase (data not shown). As for the other T-cellmarkers, such as CD5, CD4 and CD8, the majority of TNCwere negative. Occasionally, however, some TNC were partiallystained for CD4 or CD8 (Fig. le). About 76% of TNC wereweakly positive for S100. The ability of TNC to produce athymic hormone, FTS (or thymulin), was also examined byimmunostaining ofTNC with an antibody to FTS (Fig. I fg). Inthymomas, more than 90% of TNC were positive for FTS(Table 2). There was no significant difference in the percentageand staining intensity of the FTS-positive TNC betweenthymomas and thymi from I 5-2-month-old DA and BUF/Mnarats (data not shown).

Phenotypes of intra-TNC cells in BUF/Mna thymoma rats

The smears of TNC were also examined for reactivity of intra-TNC cells with PNA and various mAb against rat lymphoid

cells. Most staining patterns of cells within a single TNC wereeither almost all positive or almost all negative for the markers,as shown in Fig. Id or Fig. le, but with occasional exceptions ofmixed patterns (i.e. some cells were positive but the others werenegative). Therefore, the phenotypes of intra-TNC cells wereestimated by measuring the percentages of TNC of these threestaining patterns. The result is summarized in Table 3. Ingeneral, most of the cells were positive for MHC class I, CD5and PNA. The staining pattern of intra-TNC cells for Thy-lshowed the greatest variety amongst all markers tested (Table 3and Fig. Id). Approximately 73% and 85% ofTNC containedCD4+ and CD8+ cells, respectively, indicating that the majorityof the intra-TNC cells were double positive (CD4+CD8+).However, there was a significant difference (P=0-013) in thepercentages of CD8+ cell-containing TNC (85+4%o) (Fig. Ig)and CD4+ cell-containing ones (73 + 4%). When the TNC werestained with a cocktail of W3/25 (anti-CD4) and OX8 (anti-CD8) mAb, the percentage of TNC that contained CD4+ and/or CD8+ cells was 87%. This was almost equivalent to thepercentage of those stained with OX8 alone (85%), indicatingthat there were substantial numbers (about 10%) ofTNC whichcontained only CD8-single positive thymocytes. Most of theintra-TNC cells were negative for MHC class II (Ia) antigen, buta few Ia+ cells were occasionally found inside TNC (Fig. Ib).

Proliferative capability of whole thymocytes and intra-TNC cells

There was no significant difference in the proportion of BrdU +cells among 2-month-old DA rats (16 + 5%0), 2-month-old BUF/Mna rats (12+2%) and 13-month-old BUF/Mna rats(14+ 1%/), as measured in whole thymocyte suspensions. Thiswas confirmed in the individual thymic tissue sections by theBrdU-immunostaining (Fig. 2a-c).

On the other hand, in order to examine the capability ofTNC to support intra-TNC cells to proliferate, the proportionof isolated TNC that enclosed BrdU+ proliferating cells withinthem was measured (Fig. 2d, Table 4). In 13- and 22-month-oldthymoma rats, more than 35% of TNC enclosed BrdU+ cells(3610/ and 351J/o, respectively), whereas TNC from 1 5-2-month-old DA and BUF/Mna rats showed only 16-8% and

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i

Figure 1. Immunoperoxidase (a-e) and immunoalkaline phosphatase (fand g) stainings ofTNC isolated from thymomas of 14-month-old (a-e) and 23-month-old (f and g) BUF/Mna rats. The smears of TNC were stained for the following antigens by the indirectimmunostaining method. (a) MHC class I antigen stained with HAM2 mAb. Intra-TNC cells were also stained, though the photo wasnot focused on them (563 x ). (b)MHC class II (Ia) antigen stained with OX6 mAb. Note that IaI cells were occasionally found inside ofTNC (arrow) (563 x ). (c) Cytokeratin stained with a rabbit anti-bovine keratin antibody (450 x ). (d) Thy- I antigen stained with OX7mAb (450 x). (e) CD8 antigen stained with OX8 mAb. Note that some parts of the TNC were occasionally stained (arrowheads)(563 x ). (fand g) FTS stained with a rabbit anti-porcine FTS antibody. The photographs were taken either by light microscopy (f ) or byfluorescence (490 nm) microscopy (g) (225 x ).

22-7% positivities, respectively. Furthermore, the TNC fromthymoma rats had a tendency to contain more BrdU+ cellsinside them than those from non-thymoma young control rats,because the percentages ofTNC enclosing more than 5 BrdU+cells were greater in the thymoma rats than in the controls.

Demonstration of macrophage populations within TNC

TNC per se lacked macrophage markers (Table 2). However,some of them did enclose cells bearing ED1, ED2 markers oracid phosphatase (Fig. 3a-c). In order to exclude the possibilitythat they were artificially enveloped during the process ofTNC-

isolation, not only the ED I + or ED2 + cells (data not shown) butalso the acid phosphatase-positive cells in situ (Fig. 3d) weredemonstrated within TNC in tissue sections of thymi. Theproportion of TNC that enclosed EDI+ or ED2+ cells wasmeasured and compared between thymoma rats and non-thymoma young rats of both DA and BUF/Mna strains (Table5). In thymomas, the frequencies of TNC with EDI + cells andED2+ cells were 18 3% and 28-6%, respectively, but nearlycomparable values were also obtained in control DA thymi(14-5% and 204%, respectively) and 1 5-month-old youngBUF/Mna rats (23-8% and 27-2%, respectively). In general,however, TNC of BUF/Mna rats contained more ED2+ cellswithin them than those of DA rats.

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155TNC in thymoma BUF/Mna rats

Table 3. Phenotype patterns of intra-TNC cells in thymomas

Stained for % all positive* Intensityt % all negative % mixed patterns

MHC class I 93 + 6 1CD5 92 + 4 4CD4 73 + ~+ 20 7CD8 85 + 10 5CD4+CD8 87 ±-.-+ 7 6Thy-l 68 +++ 23 9PNA 94 + 6 0

* The percentages were calculated by measuring the number ofTNC of differentstaining patterns of intra-TNC cells. Total 500-2000 TNC from 14-month-oldthymoma rats were assessed in each assay.

t The staining intensity was expressed as: -, negative; ±, weakly positive; +,positive; + +, strongly positive.

Table 4. Proportion of TNC that contain BrdU-positive cells

Figure 2. Immunoperoxidase staining of BrdU+ cells. BrdU+ cells incryosections of thymi from a 2-month-old DA rat (a), from a 2-month-old BUF/Mna rat (b), and from a 13-month-old thymoma BUF/Mnarat (c). C, cortex; M, medulla (143 x ). (d) BrdU+ cells (arrows) in a

TNC isolated from the 13-month-old thymoma rat (870 x).

Strain Age (month) % * ofTNC that contain BrdU I cells

DA 1-5-2 16-8+4-Ot (0)$

BUF/Mna 1-5-2 22-7+5 2 (2-5)13 36-1+3-2§ (4-0)22 35-1+6-8§ (48)

* The percentages were calculated by measuring the number ofTNCthat contained BrdU + cells. Total 100-500 TNC were assessed in eachassay.

t Mean + SD of four to six individual assays.t Among the positive TNC, the percentage of TNC that contained

more than five BrdU+ cells was further estimated.§When compared with those of 1-5-2-month-old BUF/Mna and

DA rats, the differences in the values are statistically significant(0-001 < P< 0-014). Differences in the other measured values are notstatistically significant.

DISCUSSION

Previously, we have reported that the altered thymic microenvir-onment in the thymoma of BUF/Mna rats may regulate theunique T-cell differentiation pathway without causing anyserious immune dysfunction.' As suggested in human thymo-mas,'7"8 the difference in phenotypic patterns of each cellularcomponent of thymomas may be simply due to the imbalancedpopulation sizes of the cells at a given normal differentiationstage. Taking this into consideration, we have characterized theTNC increasing in BUF/Mna rats based on the phenotypes ofboth TNC per se and intra-TNC cells to elucidate theirimmunological significance in the T-cell development.

In general, no structural abnormality, as was reported in thecase ofAKR thymic lymphomas,'9 was detected in the TNC ofthymomas by light-microscopy. Phenotypically, the TNC inthymomas were MHC class 1+, class II+, cytokeratin+ andFTS+, but lacked any of the markers for macrophages (ED1,ED2, ED3, acid phosphatase and non-specific esterase), indicat-ing their epithelial origin but not phagocytic origin. It has beenreported that TNC have some phenotypic heterogeneity withvarious characteristics as an epithelial component.20 Interest-

T. Ezaki, K. MAatsun) & M. Kotani

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Figure 3. Demonstration of mnacrophage populations (arrows) within TNC. Immunoperoxidase staining of EDI + (a) and ED2± (b)

cells within isolated TNC from 14-month-old thymomas (698 x ). (c) Acid phosphatase-positive cells found in a smear of the isolatedTNC (563 x ). (d) Acid phosphatase-positive cells found within a TNC in situ of a thymus in an 8-month-old BUF/Mna rat. Arrowheadindicates the nucleus of the TNC (698 x ). Note that TNC pet se are negative for adl the macrophage markers.

Table 5. Proportion ofTNC that contain ED I - and ED2-positive cells

%Y..* of TNC that contain positivecells for

Strain Age (month) EDI ED2

DA 2 14-5+3-9t (21 2)4 20-4±4-3 (0)

BUF Mna 1-5 23-8+6-8 (30-8) 27-2+7-3 (27-1)14-16 183+70 (17-0) 28-6+4-0§ (16-5)

* The percentages were calculated by measuring the number ofTNCthat contained EDI I or ED2+ cells. Total 100- 500 TNC were assessedin each assay.

t Mean + SD of three to five individual assays.+ Among the positive TNC, the percentage of TNC that contained

more than one EDI ' or ED2+ cells was further estimated.§ Difference in the value from that of 2-month-old DA rats is

statistically significant (P=0-035). Differences in the other measuredvalues are not statistically significant.

ingly, several non-epithelial markers were also shown to beexpressed by the TNC of thymomas in this study. First, Thy-iwas strongly expressed on the TNC as well as intra-TNCthymocytes, though there seemed to be a species differencebetween human TNC6 and mouse TNC. I' Since Thy- I has beenreported to function as a signal transduction molecule throughthe cell membrane, '' the expression of Thy-i may play somerole in the cellular interactions both inside and outside of TNC.

Second, SlOO was detected in more than 75%, of TNC in thethymoma. Besides in nervous tissues, SI 00 has been shown to beexpressed in human thymic interdigitating cells (IDC) in themedulla and skin Langerhans' cells, but this is still controversialin the rat thymus.22'23 This protein, one of the calcium-bindingproteins related to calmodulin or troponin C,24 might be relatedto the signal transduction like Thy-i molecules. Third, PNAreceptors were also expressed on TNC, though PNA was

originally reported to be negative on mouse TNC. ' Finally, CD4and CD8 molecules were usually negative on most of the TNC,but occasionally these molecules were partially detected on

TNC. Recently, Tucek & Boyd25 reported that co-expression or

sharing of some T-cell markers, namely CD4 and Thy-I,between thymocytes and thymic stromal cells may have impor-tant implications for intercellular communications within thethymus. The present results not only support their idea but alsofurther extend the range of the shared markers between TNCand other thymic components. Since these shared markers seem

to be closely related to various biological functions, this may

also indicate the heterogeneity of TNC depending on thevarious differentiation stages of intra-TNC thymocytes.

It has been suggested that the TNC are, at least in part,responsible for the remarkable increment of lymphocytes in

thymomas, though we have not estimated the exact turnover

and traffic of the thymocytes in this study. This is because an

enormous increase in the number of TNC occurred at 8 monthsof age, which was immediately before or in good accordancewith the onset of thymomas.'' In addition, TNC in the thymomarats seem to have a higher capability of supporting cellproliferation within them, as assessed by BrdU-uptake, while

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TNC in thi'inoma BUF/Mna rats 157

the porportion of BrdU + cells to whole thymocytes revealed nosignificant difference between thymomas and normal and youngcontrol thymi. Furthermore, this report has demonstrated forthe first time that quite a large proportion of TNC containdetectable levels of FTS. Some types of epithelial cells have beenreported to be positive for FTS;26 however, there has been nodirect evidence to show that TNC can actually produce such athymic hormone. Although the proportion and staining inten-sity of FTS+ TNC in thymomas showed no difference fromthose of non-thymoma controls, the absolute amount of FTS inthe thymoma rats must be much larger than the controls becausethe total numbers of TNC in thymomas were approximately200-4000 times more than the controls. It has been reported thathyperplastic epithelial cells in BUF/Mna rats are stronglypositive for thymosins and epithelial growth factor (EGF).9 Inthis report, we have not studied whether these factors are alsorelated to the TNC in thymomas.

Concerning intra-TNC thymocytes in thymomas, the major-ity were Thy- I +, PNA +, double-positive (CD4 +CD8 +) imma-ture thymocytes, as reported by Kyewski et al.,'8 van Vliet eta/.27 and de Waal Malefijt et al.28 We analysed the stainingpatterns ofTNC as a whole instead of assessing individual intra-TNC cells; however, it was clear that there existed someheterogeneity in the phenotype ofintra-TNC cells. For example,there were small but substantial numbers of TNC whichcontained only CD8 single-positive thymocytes. In contrast, ithas also been reported that a mouse TNC clone established invitro introduces double-negative (CD4-CD8- ) foetal thymo-cytes into CD4+CD8- single positive cells.29 These data mayreflect that TNC can contain cells at various different stages ofthymocytes, because TNC may be responsible for a relativelylate step of intra-cortical T-cell development' and play some rolein active cell migration.30 It has yet to be established, however,which adhesion mechanisms3' are involved in the interactionsbetween TNC and intra-TNC cells.

One novel and intriguing finding about intra-TNC cells wasthat macrophage populations bearing EDI, ED2 markers oracid phosphatase activity were present within some TNC of notonly thymoma rats but also normal control rats. Since theincidence of the macrophage-containing TNC was not so high(about 15-300%), they may represent cells related to a certainstage of the T-cell differentiation. As for their immunologicalsignificance, there may be at least two possible roles. Firstly, likeordinary scavenger macrophages, they may clear off apoptoticcells after positive- or negative-selection within TNC.' In thiscase, TNC are the sites not only for nursing T cells but also fortheir destruction and disposal, as reported by Leene et al.32 andHiramine et al.33 These investigators, however, never have takenaccount of the possibility that the cell-depletion within TNC canbe mediated by the macrophage populations. Alternatively, theintra-TNC macrophagesper se may actively take part in nursingthymocytes within TNC, like ED2+ macrophages which forman immuno-proliferative microenvironment for B-cell differen-tiation in the spleen, as reported by Matsuno et al.'4 In thephenotypic study, we have occasionally found Ia+ cells insideTNC. These cells may also play an important role in the intra-TNC T-cell development. It has been reported that la+macrophages enhanced cell proliferation,34 whereas Ia macro-phages decreased the cell proliferation and viability.35 Thepresence or absence of Ia molecules on the intra-TNC macro-phages may thus become of great importance. In any case, our

findings revaluate TNC as a member of the thymic micro-environment for T-cell development, as the events happeningwithin TNC may be performed not only by two-cell interactionsbetween thymocytes and TNC but also by multi-cellularinteractions, at least among thymocytes, TNC and macro-phages. The exact roles and biological significance of the intra-TNC macrophages within TNC should be elucidated further.

In conclusion, considering the benign nature of the thymo-mas'2 and the absence of any abnormal features, it is speculatedthat, like other epithelial components,9 the TNC in BUF/Mnarats may simply be hyperactive, especially in their number andnursing capacity, resulting in the unusual increment of lympho-cytes in the thymomas. The results obtained from the thymomasshould thus provide useful information for understanding thenormal constitution of the thymic microenvironment and itsroles in T-cell development.

ACKNOWLEDGMENTS

The authors are grateful to Professor T. Fukumoto, Professor K. Seikiand Dr C. D. Dijkstra for their supply of antibodies; to Professor Y.Uehara (Kumamoto University Medical School, Japan) for his encour-agement and support; and to Mrs E. Kinoshita for her technicalassistance. This work was supported by a Grant-in-Aid for Encourage-ment of Young Scientists (No. 01770018) from the Japanese Ministry ofEducation and by a Yukyokai Grant for Encouragement of Scientificand Medical Research (Kumamoto University Medical School).

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