thymic epithelial cell culture

Thymic Epithelial Cell CultureCARSTEN ROPKEInstitute of Medical Anatomy, University of Copenhagen, The Panum Institute, DK-2200 Copenhagen N, Denmark

KEY WORDS serum-free; murine; human; cytokines; adhesion molecules; T-lymphocyte devel-opment

ABSTRACT Culture of epithelial cells from the thymus of children and laboratory animals hasbeen used for more than two decades to evaluate both the nature of these cells and their importancein the selection andmaturation of functional T cells. Especially by the use of serum-free cultures andby establishment of cell lines from cultured thymic epithelial cells (TEC), it has been possible toobtain basic information on morphology of subpopulations of TEC, including surface determinantsof importance for interactions with T-cell precursors, and on the repertoire of cytokines secreted bydifferent types of TEC. The available information, obtained by co-culture of pre-T cells and TEC, onthe effects of TEC on the fate of pre-T cells suggests that cultured TEC/TEC lines are able both tosecrete needed cytokines for T-cell development, and to deliver signals needed for T-cell selection. Invivo results showing cross-talk between TEC and T cells indicate that more careful evaluation ofinteractions between well-defined subtypes of cultured TEC and co-cultured subpopulations of pre-Tcells (as well as macrophages/dendritic cells) will be of importance in evaluation of the function ofthe thymus.Microsc. Res. Tech. 38:276–286, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONThe development of functionallymature T cells within

the thymus has been a major area of interest inimmunology during the last decades. It has long beenknown that a more or less constant influx of stem cellsfrom fetal liver and, thereafter, from the bone marrowinto the thymus, is needed for maintenance of T-celldevelopment. However, the exact nature and potency ofthese cells are still not clarified (surveys: Godfrey andZlotnik, 1993; Kruisbeek, 1993). Likewise, it is gener-ally agreed that T cells are functionally mature andrestricted to either the helper (CD4) or cytotoxic/suppressor (CD8) T-cell subpopulations when leavingthe thymus (surveys: Boyd and Hugo, 1991). But onlyduring the last few years has it been possible to get areasonable insight into the critical events within thethymus that lead to full maturation of immunocompe-tent, self-tolerant T cells, via T-cell receptor (TcR) generearrangement and positive as well as negative selec-tion. In these clarifications of the steps leading from atriple negative (CD3-4-8-) T-cell precursor entering thethymus, with no or only b-gene rearrangement of theTcR, to single positive (CD4182 or CD4281), ab-TcRrearranged T cellsmigrating from the thymus,monoclo-nal antibodies, flow cytometry, and transgenic micehave been important tools. These techniques haveelucidated the successive steps—characterized by phe-notype—and the control points, the T-cell precursor hasto go through to mature functionally. However, thisquite detailed picture of the developing T cell in thethymus has not been accompanied by a similar clearunderstanding of the importance of the thymicmicroen-vironment. Although the thymus has been recognizedas the central organ for T-cell development for morethan three decades, the importance of the variousstromal cells in T-cell development is still debated.Thus, until recently, it was generally accepted that

cortical epithelial cells were essential for positive selec-tion, whereas macrophages and dendritic cells at thecortical-medullary junction and in the medulla werenecessary for negative selection of T-cell precursors,although it was suggested that medullary epithelialcells were able to induce anergy (surveys: Blackman etal., 1990; Boyd and Hugo, 1991). However, within thelast few years this picture has been blurred. Reportsfrom several laboratories have indicated that T-cellselection was connected more to affinity between sur-face molecules on the T cell and on the stromal cell (andon avidity) than to the type of stromal cell interactingwith the T cell (e.g., Pircher et al., 1993), and evenintrathymically injected fibroblasts have been shown tobe able to induce positive selection (Hugo et al., 1993;Pawlowski et al., 1993). The latter finding, however,does not imply that thymus is not essential for matura-tion and selection of at least the majority of theperipheral ab-TcR1 T cells. Thus, the thymic microen-vironments are necessary for T-cell development,whereas the type of stromal cell involved in a given stepof T-cell maturation may be dependent on the locationof this cell type in the thymus, and, in addition, on thedevelopmental stage of the T-cell precursor present inthis given microenvironment. Recent studies indicatethat both surface determinants found on stromal cellsand determinants characteristic for a given T-cell pre-cursor stage, as well as cytokines secreted by bothstromal cells and certain T-cell precursors, are essentialfor the differentiation of a T-cell precursor from one stepto the next on the ladder of T-cell maturation. Thisrecognition of the importance of cross-talk between

*Correspondence to: Carsten Ropke, Institute of Medical Anatomy, Section A,University of Copenhagen, The Panum Institute, Blegdamsvej 3, DK-2200Copenhagen N, Denmark.Accepted in revised form 24 January 1995

MICROSCOPY RESEARCH AND TECHNIQUE 38:276–286 (1997)

r 1997 WILEY-LISS, INC.

cells in the thymus has prompted many researchers tostudy the nature of thymic stromal cells more closely,their localization, their phenotypes, and their function,including cytokine secretion (surveys: Ritter and Boyd,1993; van Ewijk et al., 1994).In the present survey, the intention is to report

results obtained by culture of thymic epithelial cells(TEC), results with relevance for our understanding oftheir role in T-cell development. From the above it isobvious that investigation of TEC brought into cultureis hampered by changes induced by transferring thecells from in vivo to in vitro environment, as well as theabsence of other cell types, the most obvious being theT-cell precursors (thymocytes), the latter being able tochange the biology of TEC (e.g., van Ewijk et al., 1994).However, to dissect themicroenvironments and to learnabout the functional capabilities of the TEC per se, theold ‘‘divide et impera’’ notion is still valid, and resultsreported from co-culture of TEC with thymocytes willdiminish some of these problems. Thus, the main areasin TEC culture research, namely characterization ofthe biology of TEC (morphology, growth, surface deter-minants), measurements of cytokine production, estab-lishment of cell lines, and evaluation of functionalimplications from coculture with thymocytes, will bepresented. Since areas, basic to the understanding ofthe significance of findings in TEC cultures—develop-ment and topography of TEC and their subsets invivo—are covered by other surveys in this issue, thesetopics will not be included in the present review.

DISCUSSIONEstablishment and Maintenance

of Human and Murine TEC CulturesThe basis for bringing TEC to culture is an effort to

understand the role of TEC in the development of theT-cell repertoire, the nature of the direct interactionbetween T-cell precursors and TEC, and the importanceof possible secreted factors (hormones/peptides). Forthis, it seems essential to studywell-defined cell popula-tions. Numerous methods have been used to cultureTEC from humans and rodents (Farr et al., 1986;Gershwin et al., 1978; Jordan and Crouse, 1979; Loor,1979; Mizuno et al., 1978; Nieburgs et al., 1985; Pa-piernik and Nabarra, 1981; Potworowski et al., 1986;Schuurman et al., 1986; Singer et al., 1985; Small et al.,1984; Sun et al., 1984; Taubenberger and Haar, 1987).In all these reports, fetal calf serum (FCS) or humanserum has been used as a necessary constituent of theculture medium. However, it is well known that theaddition of serum to the medium has several disadvan-tages (see also Barnes and Sato, 1980a,b), such as theaddition of unknown factors (hormones, endotoxins,viruses, and other macromolecules), which may varyfrom batch to batch. This not only creates problems ofinfection and lack of reproducibility, but also makes itdifficult to define growth requirements for the cells, andto isolate and interpret the significance of biologicalactivities executed by molecules released by the cellsinto the culture medium.The culture of TEC in serum-containing (SC) me-

dium is further hampered by the ability of the mediumto support the growth of macrophages, fat cells, andespecially fibroblasts, the latter often rapidly overgrow-ing the epithelial cells. To overcome especially the

growth of fibroblasts in SC cultures, several methodshave been used. Subculture of cells on mitomycinC-treated mouse 3T3 fibroblast feeder layers, andwashes of cultures with EDTA, diminish the number offibroblasts (Farr et al., 1986; Galy et al., 1989; Singer etal., 1985; Sun et al., 1984), as does the use of D-valineinstead of L-valine in the medium (Munoz-Blay et al.,1987; Nieburgs et al., 1985). However, with thesemethods, other side-effects of SC medium are not dealtwith. Rimm et al. (1984) succeeded in obtaining highpercentages of TEC in cultures initiated with serum,but later maintained in serum-free (SF) medium. Sinceeven a very short presence of serum in the culturesystem may influence cultured cells for a long time(Barnes and Sato, 1980a), it is desirable to avoid serumcompletely in these cultures if the above-mentioneddisadvantages of serum are to be removed. Relativelyclean populations of TEC (80%) have been obtained bythe use of commercially available SF media supple-mented with growth factors (Christensson et al., 1989).However, cultures of TEC, essentially free of ‘‘contami-nating’’ cells, can bemade by seeding thymic fragments/cells on a collagen matrix in a defined SF medium(Eshel et al., 1990a,b; Ropke et al., 1990, 1994; Ropkeand Elbroend, 1992; Schreiber et al., 1991), as alsoindicated by Figure 1.TEC cultures are usually initiated by cutting the

thymic tissue into about 1–2 mm3 large pieces, whichthen are digested with DNAase and collagenase ortrypsin, washed, and plated out in the culture cham-bers. (Adescription of these initial steps for preparationof cultures from newborn and fetal mouse, as well ashuman thymus, can be found in Ropke et al., 1994).Culture chambers may be used without coating in SC

cultures, but it is necessary to coat with an extracellu-lar matrix to obtain adhesion of thymic fragments andcells to the bottom, when SF medium is used. Type-Icollagen (Vitrogen-100, Flow Laboratories, McLean,VA; Ropke et al., 1990) or extracts from cultures ofbovine cortical endothelium (Eshel et al., 1990a), haveproved to be effective. The basic medium for both SCand SF cultures is usually RPMI-1640, Dulbecco’smodified Eagle’s medium (DMEM), or Ham’s F12. Inour hands a 1:1 mixture of DMEM and F12 works verywell. By omitting Ca21 from the DMEM, keratinization,which is a problem in murine cultures, is avoided(Ropke et al., 1990).A variety of growth factors have been used in SC

culture media with positive results for growth andsurvival of TEC. However, the various compositions ofthe added serum and the unknown amounts of hor-mones and endotoxins in serum make it difficult toevaluate the importance of the different factors. Aclearer picture is obtained by the use of SF medium.Previous experiences from culture of other epitheliasuggested the following factors as valuable additives inmurine and/or human TEC cultures: epidermal growthfactor, hydrocortisone, high-density lipoproteins, insu-lin, transferrin, cholera toxin, selenenium, and triiodo-thyronine (Eshel et al., 1990a; Ropke et al., 1990, 1994;Ropke and Elbroend, 1992; Schreiber et al., 1991). Byvarying the amounts of these factors or by deleting oneof these at a time, it appeared that insulin, hydrocorti-sone, cholera toxin, and epidermal growth factor wereessential for growth as measured by 3H-TdR incorpora-

277THYMIC EPITHELIAL CELL CULTURE

Fig. 1. A: Phase contrast micrograph of a primary culture ofmurine thymic epithelial cells (TEC). Note predominance of rathersmall cells with typical cobblestone appearance (3140). B: Unstainedmicrograph of an autoradiographic preparation of part of an expand-ing 10-day-old cell islet from a primary culture of murine TEC.[3H]-thymidine was added to the culture for the last 24 h. It is seenthat DNA-synthesizing cells are present all over the islet, and thatlarger cells are most numerous in the periphery. The remains of thetissue fragment are found in the lower right corner (340). C: Light

micrograph of a primary human TEC culture incubated with anantibody against cytokeratins (keratins 6 and 18) and stained by thePAP method (3130). D: Light micrograph of a secondary human TECculture, incubated and stained by the same procedure (3250).E: Lightmicrograph of a secondary human TEC culture incubated with anantibody against medullary TEC and stained by the PAPmethod. Note‘‘Hassall’s body-like’’ accumulations of TEC (3130). F: Fluorescencephotomicrograph of cultured TEC from an SCID mouse. Cells werelabeled with an FITC-labeled antibody againstmedullary TEC (3250).

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tion and areameasurement of cell islets, but—as shownin Figure 2—transferrin could be deleted in murinecultureswithout any effects (Ropke et al., 1990), whereasboth transferrin and cholera toxin decreased the growthrate in human cultures (Ropke and Elbroend, 1992;Andersen et al., 1993). However, by cell counts, epider-mal growth factor, insulin, and hydrocortisone werefound to be essential for murine TEC culture, andselenium, transferrin, and insulin were important forgrowth of human TEC, as reported in other studies(Eshel et al., 1990a; Schreiber et al., 1991). Thesefindings indicate that the definitive composition of anoptimal medium has not been found and further thatTEC cultures from different species have differentrequirements. Generally, the SF media will sustaingrowth of TEC for a prolonged period (months) and/orfor several transfers, although a decline in growth ratemay be seen after several transfers (Petersen et al.,1994).In optimizing a medium, growth rate is not the only

thing to consider. Cell morphologymay change consider-ably in media with varying factor compositions(Schreiber et al., 1991), and omission of hydrocortisonefrom the medium has been shown to increase theamount of cytokines, of importance for T-cell function,in culture supernatants (Andersen et al., 1993).

Morphology and Nature of Cultured TECIn primary cultures, epithelial cells spread out from

the tissue fragments as a carpet mainly consisting offlattened cells. It is usual to see both small (10–20 µm)and large (20–100 µm) cells from the start of cultures,and these types are also found in later cultures invarying numbers (Fig. 1). We found about equal, rela-tively high DNA synthesis rates (4–6%/h) in large andsmall cells within the first 4 weeks of culture (Ropke etal., 1990). However, Nieburgs et al. (1985) observedfaster proliferation of large TEC than of small. Inconfluent cultures, TEC may be organized both as‘‘cobblestones’’ or as more widespread cells connectedvia long cytoplasmic processes. Reversible morphologi-cal changes from cobblestone-shaped to elongated fusi-form cells resembling fibroblasts (but remaining cyto-keratin positive) after addition of IL-1 were seen byGaly et al. (1989), besides increase in TEC prolifera-tion; the latter finding, however, could not be repeatedby us (Ropke and Elbroend, 1992).Ultrastructural studies confirm the epithelial nature

of the cultured cells by showing the cells to havedesmosomes and tonofilaments/intermediate filamentbundles. In addition—besides Golgi complexes, mito-chondria, ribosomes, and granular endoplasmatic retic-ulum—it is characteristic to see vacuoles in the largercells (Eshel et al., 1990a; Papiernik and Nabarra, 1981;Rimm et al., 1984; Ropke et al., 1990; Schreiber et al.,1991).By the use of anti-cytokeratin antibodies, heterogene-

ity of cultured TEC with respect to cytokeratins hasbeen detected (Nicolas et al., 1985), and such antibodiesas well as antibodies against surface molecules havebeen used to characterize TEC as either cortical ormedullary. From these studies it appears that bothcortical and medullary TEC can be cultured, but thenumbers of either of these subtypes in cultures varyconsiderably from laboratory to laboratory, dependent

on culture conditions, e.g., SC or SF medium, additionof factors such as IL-1, and age of donors. Thus, inmurine SC cultures, Piltch et al. (1990) find that TECwith cortical markers disappear quickly, leaving TECwith subcapsular/medullary markers in the cultures,whereas Small et al. (1984, 1989a,b) by the use ofmedium containing D-valine obtain spindle-shaped,singly situated cortical type TEC and confluent carpetsof medullary TEC. This latter type increased, by hydro-cortisone addition and by donor age, up to 100%,

Fig. 2. Example on evaluation of effects of added growth factors oncell proliferation in murine TEC cultures. A: Area measurements ofmurine TEC islets in primary cultures done by the aid of a Leitz TASSPLUS image analyzer. a.u.: arbitrary units. Averages of measure-ments from 4 to 12 islets are shown. B: Percentages of DNA-synthesizing cells in primary TEC cultures evaluated by 3H-thymidineincorporation and autoradiography. Composition of medium: 1, com-plete medium; 2, mediumwithout epidermal growth factor; 3, mediumwith 103 less epidermal growth factor than normal; 4, mediumwithout transferrin; 5, medium without cholera toxin; 6, mediumwithout hydrocortisone; 7, medium without insulin; 8, 3.33 normalinsulin concentration. Adapted from results presented in Ropke et al.(1990).


whereas cortical-type TEC were predominant whenyoung donors were used, and after Con A supernatantswere added to cultures. Eshel et al. (1990b) found aweak reaction for medullary TEC in SF murine cul-tures, and we found, also by the use of SF medium,predominance of medullary TEC in murine cultures,although some cortical TEC were always present (Fig.1; Ropke et al., 1990). The ratio medullary/cortical TECappeared stable irrespective of culture age, also incultures obtained from fetal mice. Interestingly, thisratio was also found in cultures obtained from SCIDmice (Ropke et al., unpublished data), irrespective ofthe fact that very fewmedullary TEC are seen in vivo inthese latter mice (van Ewijk et al., 1994).When the same SF medium was employed for human

cultures, cortical TEC were present in small numbers(below 20%) in all cultures, whereas medullary typeTEC increased steadily up to 80–95% in late cultures(Ropke and Elbroend, 1992). In addition, in thesecultures many cells were positive for antibodies againstHassall’s bodies (these bodies also being detected mor-phologically in cultures as shown in Fig. 1E), and mostcells were positive for an antibody against sialomucinstypical of secretory epithelia (Hilkens et al., 1989). Incontrast, by the use of a SCmedium, Singer et al. (1985)found the majority of cells to be positive for corticalmarkers (40–80%), whereas only 18% were positive fora marker for medullary/subcapsular cells. However, inthis latter study, two antibodies against Hassall’s bod-ies labeled 10–20 and 20–100%, respectively, indicatinghigh numbers of medullary TEC in some cultures.This heterogeneity in findings shows a surprising

plasticity in phenotypes of the cultured cells, dependenton the milieu of the cultures, and may indicate that themedullary and cortical phenotypes are not fixed types.This is also supported by findings in vivo by changingenvironment for TEC, e.g., the change in numbers ofmedullary TEC in SCID mice after BM cell injections(van Ewijk et al., 1994). Whether these changes are dueto the presence in cultures of common stem cells, topreferential survival of a given TEC type, or to changeof phenotype of individual cells, is not known.

Surface Molecules on Cultured TECBecause of the importance of TEC in thymocyte

selection, it is of interest to map surface molecules oncultured TEC, molecules of relevance in thymic selec-tion. It is likely that the selection steps mediatedthroughMHCClass I or II and TcR 1 CD8 or 4 requiresextensive cell-to-cell contact in which adhesion mol-ecules such as LFA-3, B7/BB1, Thy-1, and ICAM-1 areof importance, if present on TEC.MHC Class I molecules are relatively strongly ex-

pressed on both human and murine TEC in culture(Eshel et al., 1990a; Fernandez et al., 1994; Ropke etal., 1990; Ropke and Elbroend, 1992) whereas MHCclass II is weakly expressed or absent (Berrih et al.,1985; Christensson et al., 1989; Denning et al., 1987b;Eshel et al., 1990a; Farr et al., 1986; Nonoyama et al.,1989; Rimm et al., 1984; Ropke et al., 1990; Ropke andElbroend, 1992). However, by adding gIFN or thymo-cytes to the cultures, class II is expressed on TECwithin 24–48 h (Berrih et al., 1985; Nonoyama et al.,1989; Ropke, unpublished data), as also shown inFigure 3. Of the adhesion molecules, LFA-3 is constitu-

tively expressed on both human and murine TEC inculture, whereas ICAM-1 is weakly expressed, but it isupregulated after addition of gIFN (Fig. 4; Nonoyamaet al., 1989; Ropke and Elbroend, 1992), and it has beenshown that cultured TEC bind thymocytes via CD2-LFA-3 and LFA-1-ICAM-1 interactions (Nonoyama etal., 1989; Ropke and Elbroend, 1992; Singer et al., 1990;Vollger et al., 1987).Several recent reports point to the importance of

Class I/II-TcR 1 CD8/4 interactions in positive andnegative selection (survey: Kruisbeek, 1993), whereasthe importance of the adhesion molecules is less clear.Adhesion (rosette formation) has been shown betweenTEC and immature—low CD31—thymocytes, whichseem to be in GO/1 phase (Nonoyama et al., 1989),whereas both early and late thymocyte subsets bind toTEC, when the former cells are actively proliferating(Ropke and Elbroend, 1992; Singer et al., 1990). It hasbeen shown that ICAM-1-LFA-1 and LFA-3-CD2 inter-actions rather than Class I/II-TcR 1 CD8/4 interactionsare essential for activation of CD4-8-thymocytes (Den-ning et al., 1988), and it has recently been shown byPircher et al. (1993) that the level of expression ofICAM-I, but not of Class I, influences the effectivenessof deletion of CD4181 cells in vitro. PHA inducedactivation of moremature thymocytes is also dependenton interaction between these adhesion molecules (Den-ning et al., 1987a). However, inhibition of thymocytemitogenic responses by TEC, as well as increase, hasbeen shown in murine cultures (e.g., Munoz-Blay et al.,1989) whereas supernatants from TEC cultures in-crease Con A and PHA responses (Ropke et al., 1990;Schreiber et al., 1991). By the use of several types ofthymocytes CD4-82, CD4181, CD4182, and CD4-81,it was shown that cultured human TEC affect theIL-2-dependent proliferation of these cells reversely,namely by depressing proliferation in the absence ofmitogen, whereas the PHA response of the differentthymocyte types was augmented (Ropke and Elbroend,1992). These reverse effects seem to indicate thatseveral receptors are involved in the interactions be-tween TEC and thymocytes.The presence of high levels of CD28 on thymocytes

(Gross et al., 1992), as well as documented expression ofB7/BB1 in the thymus (Turka et al., 1991) indicate thatCD28/B7 interaction may be of importance in thymo-cyte selection. This is supported by the results ofDegermann et al. (1994), suggesting that the B7-positive medullary TEC cause intrathymic deletion ofCD41 VB51 T cells, dependent on a costimulatorypathway associated with CD28/B7 interaction. On theother hand, Reiser and Schneeberger (1994) find noeffects on thymocyte development when murine fetalorgan cultures are treated with anti-B7-antibody. It isnot known whether cultured TEC express B7 constitu-tively, but Nelson et al. (1993) have shown B7 to bepresent on several murine TEC lines of medullaryorigin. However, results obtained by Vukmanovic et al.(1994) by the use of a TEC line indicate that B7—although of importance in antigen-presentation—is notessential in positive selection. These partly conflictingresults may indicate that B7 acts in concert with othersurface molecules and/or that the function of thismolecule is dependent on a given microenvironment.Apparent normal thymic phenotype distribution in

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CD28 deficient mice (Shahinian et al., 1993) and in B7deficient mice (Freeman et al., 1993) in conjunctionwith functional abnormalities in immune responses ofthese mice, suggest a need for further insight into thecomplex B7-CD28 receptor/counter-receptor system, forevaluation of the importance of this system in thymicselection.Thy-1 is a glycoprotein expressed abundantly on

cortical thymocytes and to a lesser degree on medullarythymocytes. The significance of Thy-1 has for a longtime been unknown, but it has now been shown thatThy-1 is of importance in thymocyte-TEC adhesion (Heet al., 1991), pointing to a role for Thy-1 in corticalthymocyte maturation. Since Thy-1 has also been dem-onstrated on TEC (Raedler et al., 1978), this moleculemay prove to be of bidirectional importance. However,the ligand for Thy-1 has not been established yet.CD40 is an activation molecule for B cells, and this

molecule has been shown on both cortical and medul-lary TEC using immunohistochemistry, as on culturedTEC (Galy and Spits, 1992). These authors showedfurther that CD40 expression was regulated by cyto-

Fig. 4. Histograms obtained by FACScan analysis of secondaryhuman TEC cultures by the use of fluorescence labeled antibodiesagainst adhesionmolecules. LFA3 (CD58) is present on TEC in normalmedium (LFA3), whereas ICAM-1 (CD54) expression is weak or absentin normal medium (not shown), but is detected after addition ofg-interferon (G-IF) to the medium (ICAM-1 1 G-IF). Non-shadedareas indicate fluorescence of cells labeled with isotype controls.Y-axis: number of cells; X-axis: fluorescence intensity in arbitraryunits.

Fig. 3. Histograms obtained by FACScan analysis of secondaryhuman TEC cultures by the use of fluorescence labeled antibodiesagainst MHC class I, MHC class II, and MAM-6. It is seen that class Iis present on cultured TEC (CLASS-I 2 G-IF), but that the presence ofg-interferon (G-IF) in the medium increases the number of class Imolecules on the cells (CLASS I 1 G-IF). Class II antigens are notdetected in normal medium (CLASS II 2 G-IF), but are detected afteraddition of G-IF (CLASS II 1 G-IF). The majority of the cultured TEC

are positive for sialomucins typical for secretory epithelia, detected bythe antibody MAM-6, when cultured in normal medium in thepresence of usual growth factors (1ALL), but the amounts of sialomu-cins decrease significantly when hydrocortisone (HC) is withdrawnfrom the medium (2HC), concomitant with an increase in cytokinsecretion (see text). Non-shaded areas indicate fluorescence of cellslabeled with isotype controls. Y-axis: number of cells; X-axis: fluores-cence intensity in arbitrary units.


kines on cultured cells, and that CD40 can act as acoactivation molecule for TEC in cytokine production.This demonstration of interplay between a surfacemolecule and cytokines stresses the importance of awell-defined milieu in the evaluation of TEC biology.It is likely that the extracellular matrix in the

thymus plays an important role in T-cell maturation.Cultured TEC and TEC in situ have been shown toproduce basement membrane proteins such as fibronec-tin, laminin, and Type IV collagen; and VLA6, presenton both TEC and thymocytes, has been shown to be alaminin receptor on these cells. Thus, laminin mightlink thymocytes to TEC, or TEC to TEC (surveyed bySavino et al., 1993). Furthermore, it has recently beenshown that a stromal thymic cell clone bears fibronectinon its surface, and that immature (mainly DN) thymo-cytes, expressing large amounts of VLA-4, adhere to thestromal cells, whereasmoremature thymocytes express-ing lower levels of VLA-4 are less adherent (Sawada etal., 1992). In addition, this adhesion seems to be aprerequisite for differentiation of T-cell precursors(Utsumi et al., 1991).Finally, an adhesionmolecule, H-CAM (CD44), which

is involved in lymphocyte homing and recognizes hyal-uronic acid and collagen has been shown to be ex-pressed on cultured TEC and TEC lines (Fernandez etal., 1994). Although the functional role of this moleculeis unknown, it seems likely that it participates incell-to-cell or cell-matrix interactions within the thymicmicroenvironment.

Cytokine Production by Cultured TECIt has recently been established that several cyto-

kines are important in intrathymic T-cell differentia-tion concomitant with cell-to-cell interaction betweenT-cell precursors and stromal cells (Haynes, 1990; Le etal., 1991; van Ewijk, 1991). Cytokines are producedboth by thymocyte subpopulations and by stromal cells,such as macrophages, dendritic cells, and epithelialcells (Kendall, 1991; Wolf and Cohen, 1992). Thus,evaluation of the roles played by individual cell typeswithin the thymic microenvironment is dependent onestablishment of clean populations of these cell types.Numerous studies using TEC cultures in SC mediumhave established that cultured TEC secrete cytokines/lymphokines of known importance in development ofstem cells, prethymic T-cell precursors, subsets of thy-mocytes, and mature T cells (Aime et al., 1991; Cohen-Kaminsky et al., 1991; Dalloul et al., 1991; Denning etal., 1988; Galy et al., 1989; Le et al., 1987, 1988, 1990,1991; Singer et al., 1990). This includes IL-1a and b,IL3, IL6, IL7, G-CSF, M-CSF, and GM-CSF, and severalof these cytokines have also been detected in SFcultures (Andersen et al., 1993; Eshel et al., 1990b;Petersen et al., 1994; Ropke and Elbroend, 1992), aswell as in supernatants from cultured TEC lines (e.g.,Faas et al., 1993; Fernandez et al., 1994). Interactionsbetween T-cell precursors and TEC may, in addition tointeractions between surface molecules, include cross-talk between surface molecules and cytokines, leadingto both upregulation and downregulation of surfacemolecule expression on both cell types. Further, thisinterplay may regulate the cytokine repertoire and thecytokine production by both cell types in a givenmicromilieu (Fig. 5). Thus, it seems essential to investi-

gate the importance of production of the cytokines incocultures between defined thymocyte subpopulationsand defined subtypes of TEC to unravel the apparentcomplexity.

Effects of Cultured TEC and of Cell LinesDerived from TEC, on T-cell DifferentiationA main reason for growing TEC in culture is to

establish the role of TEC, and their subsets, in T cellmaturation. However, despite much work applied to thecharacterization of SC and SF TEC cultures, the pre-cise knowledge obtained on T-cell differentiation andfunction induced by cultured TEC is relatively sparse.This is in part due to lack of well-defined cell popula-tions in cultures, and to the missing feedback fromother cell types. (As previously mentioned, even in SFcultures the cell composition may vary from predomi-nantly cortical-to predominantly medullary TEC. Fig-ure 6 shows morphological differences between non-selected cultures and cell lines). In an effort to overcomethe former problem, a number of laboratories haveestablished cell lines from cultured TEC to obtain cleansubpopulations of TEC, and to elucidate the function ofsubcapsular, cortical, and medullary TEC. These lineshave probably given the clearest information, and workusing cell lines from which information on thymocytedifferentiation and functional maturation can be ob-tained is included in the following.The above-noted specific adhesion of T-cell precursors

to, and secretion of cytokines by, cultured TEC, pointsto an essential role for TEC in T-cell maturation, butonly a few publications show direct specific effects ofnon-transformed cultured TEC on admixed thymo-cytes. However, by short-term culture of thymocytes oncultured thymic stromal cells (presumably of TECnature), Sen-Majumdar et al. (1992) have shown thatvarious subsets of immature murine thymocytes candifferentiate into all the mature phenotypes of thymo-cytes, and Claesson and Ropke (1990) showed thatcultured murine TEC of medullary origin were able toinduce energy (by the so-called veto activity) in cyto-

Fig. 5. Example of interaction between cytokines and TEC. Hu-man TEC was grown for 3 days in serum-free medium, either innormal medium (control), in medium containing IL-1b (IL-1), inmedium containing TNFa (TNF), or in medium containing LPS (LPS).Changes in IL-6 secretion into the supernatant are shown.

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toxic T-cell precursors directed against the MHC class Itype of the TEC, indicating a possible role for TEC innegative selection.Further, Ropke et al. (1993) demonstrated, by the use

of TcR transgenic mice, that both cultured female andmale TEC from these mice were able to induce in-creased proliferation of thymocyte subpopulations,whereas male TEC were unable to delete female cyto-toxic cells directed against male H-Y antigens. Bytransforming thymic stromal cells with SV40, Glimcheret al. (1983) developed TEC lines that secreted a factorcapable of inducing functional maturation of thymo-cytes to cytotoxic effector cells, possibly indicatingpositive selection or post-selection maturation. By theuse of TcR transgenic mice expressing a receptor for adefined viral peptide antigen presented by MHC class Imolecules, Pircher et al. (1993) showed that a corticalTEC line was able to induce antigen-specific deletion ofCD4181 thymocytes. Further, results obtained byVukmanovic et al. (1992) and Hugo et al. (1992), afterintrathymic injection of TEC lines, showed that thesecell lines were able to induce positive selection ofthymocytes. As mentioned in the introduction, injectedfibroblasts have also been shown to induce positiveselection (Hugo et al., 1993; Pawlowski et al., 1993).These results, obtained by injection of cell lines, arehowever inconclusive. The results show that positiveselection is possible, but do not define the factors(besides MHC) necessary for selection, factors presentin the thymic microenvironments but not outside the

thymus. In parallel, Nishimura et al. (1990) showedthat mainly CD4181 and CD4-82 immature thymo-cytes interacted with a thymic stromal cell line ofpresumably epithelial nature, and that this cell linewas able to induce growth and differentiation ofCD4181 cells into CD4-81 T cells. In these latterpapers, the origin—cortical or medullary—of the TEClines was not established, and thus it is not possible toestablish specific abilities in selection by the two sub-sets of TEC, i.e., whether the view of cortical TEC aspositive selectors and medullary TEC as possible nega-tive selectors and ‘‘maturators’’ is correct, or whetherone TEC type is capable of doing the whole job.Previously, Palacios et al. (1989) compared the effects

of a cloned murine TEC line with heterogeneous TECcultures on pro-T clones and bone marrow cells, andfound that the cloned line only induced an increase ofCD41 cells, whereas the heterogeneous TEC popula-tion in addition gave rise to CD81 and CD4181 cells.Later, Gutierrez and Palacios (1991) established sev-eral nontransformed TEC lines. Lines which expressedmarkers of cortical TEC produced IL7 and supported invivo generation of CD41 cells or both CD81 abTcR1cells, but not of CD4181 cells. A medullary TEC lineproduced IL1a but did not support T-lymphocyte differ-entiation. Thus, these findings support the idea offunctional heterogeneity of TEC based on surface mol-ecules. However, a correlation to the above papers,using TEC injections and measuring antigen-specificresponses, is needed. The finding of greater potential ofheterogeneous TEC as compared with a TEC line maybe significant for the understanding of the successivesteps in T-cell maturation.In this, TEC lines, as the ones developed by Faas et

al. (1993) from SV40-T transgenic mice, are of interest.These lines were characterized as subcapsular/nursecells, cortical, and medullary cells. All 3 types secretedGM-CSF, subcapsular cells secreted IL-5 and gIFN,cortical cells secreted IL-7, and both cortical andmedul-lary types secreted IL-6. Preliminary results from thispaper indicate different effects of the subtypes of TECon pre-thymocyte development. Knowledge of the func-tional capabilities of subtypes of human TEC is sparse,but Fernandez et al. (1994) developed non-transformedTEC lines of cortical nature, and showed that theselines produced IL-1a, IL-6, and IL-7.When the above results, derived from cultures of TEC

and of TEC lines, are considered, it appears that finalproof—of the ability of TEC alone to bring thymocyteprecursors through all maturation steps in the thy-mus—is missing. This may be due to culture conditions,e.g., absence of microenvironmental factors such ascytokines and extracellular matrix present in the thy-mus, or changes in gene expression. However, theelegant results obtained by Anderson et al. (1993) bythe use of reaggregation of fetal murine mesenchymaland epithelial cells, point to the fact that mesenchymalcells are needed for the initial maturation steps in thethymus, whereas TEC alone are able to support differ-entiation of CD4181 cells to single-positive CD41 andCD81 cells.Although macrophages at the cortico-medullary

boundary have been regarded as wastebaskets fornon-selected and negatively selected dying/apoptoticthymocytes, TEC may also be involved in these pro-

Fig. 6. 3D view of the morphology of murine TEC in primaryculture (C3H TEC), of murinemedullary and cortical TEC lines, and ofhuman TEC in tertiary culture, as obtained by FACScan analysis.FSC: forward scatter, equivalent to cell size; SSC: side scatter,equivalent to ‘‘granularity,’’ or maturity, of cells. It is seen that smallcells predominate in the primary murine culture and in the corticalcell line, whereas a broader spectrum of cell sizes is seen in themedullary cell line. The human cells show a great variety of sizes andgranularity, as it is typical for late cultures (tertiary and quartenary),in which cytokine secretion usually is greater than in primary andsecondary cultures (see Petersen et al., 1994). The latter cultures arebeing dominated by smaller, non-granulated cells.


cesses. A murine TEC line capable of binding—andforming aggregates resembling nurse cells with—especially immature thymocytes, was shown to containapoptotic cells (Hiramine et al., 1990), and selectiveelimination of CD4181 and CD32 or low thymocytes—not CD4-82 and not mature thymocytes—by integra-tion into a cloned cortical TEC line in culture, points toa possible function of TEC in removal of non-selectedthymocytes (Nakashima et al., 1990). Parallel findingsbyAguilar et al. (1994), showing that thymic nurse cellscontain a large number of apoptotic cells after treat-ment of mice with antiCD3e, which induces apoptosis inimmature thymocytes, suggest that nurse cells mayparticipate in clearance of nonfunctional, nonselected,apoptotic thymocytes. However, by the use of a newtechnique for demonstration of apoptotic cells in tissuesections, Suhr and Sprent (1994) have shown the vastmajority of apoptotic cells in the thymus in vivo to beengulfed in macrophages at the corticomedullary junc-tion. These latter results add to the insecurity concern-ing nurse cells: are they an in vivo or an in vitrophenomenon?

CONCLUSIONThe results obtained from cultured TEC, and from

cell lines derived from these cells, have given us areasonably clear picture of the biology of TEC, and theirpotential for surface expression of molecules and forcytokine production of importance to T-cell develop-ment. Considering the new information on the abilitiesof both TEC lines and, for example, fibroblasts forselection of T-cell precursors, after injection of the linesinto the thymus, it is apparent that the selectionprocess is not confined to TEC due to, for example, aspecialized antigen presentation, but rather due tospecialized microenvironments created in the thymusby TEC or subtypes of TEC in concert with other cells,most noteworthy subpopulations of pre-T cells. Thus,successful advancement of pre-T cells through thethymus will be dependent on the T-cell precursor beingin the right place, defined by surface receptors andproduced cytokines, at the right stage of its develop-ment. The challenge to future TEC culture work is theestablishment of well-defined nontransformed TECpopulations for coculture with selected pre-T cells, andsimultaneously the measurement of surface molecules,cytokines, and differentiation/selection of pre-T cellstowards functional T cells.

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