Epithelial-thymocyte interactions in human thymus

SEMINARS IN T-CELL IMMUNOBIOLOGY

Epithelial-Thymocyte Interactions in Human Thymus Kay H. Singer and Barton F. Haynes ABBREVIATIONS SCC subcapsuiarcortex MHC major histocompatibili~ complex SRBC sheep red blood cells LFA lymphocyte function-associated antigen Ti T-cell antigen receptor CD cluster of differentiation "rE thymic epithelial ILl interleukin 1

IL2 PHA HPLC ETAF CTL TEM ACD MoAb

interleukin 2 phytohemaggludnin high performance liquid chromatography epiderm~l-derived thymocyte activatingfactor cytotoxic T lymphocytes mitomycinC treated 'rE cells accessorycell depleted monoclonalantibody

INTRODUCTION During fetal and early postnatal development, the thymus is critical for establishment of functionally mature, antigen specific T lymphocytes. Direct contact of developing thymocytes with nonlymphoid components of the thymus is important for normal T-cell maturation to occur [1-3]. Thymic hormones have been postulated to influence T-cell maturation within the thymus [4-6]; however, in spite of detailed biochemical characterization of those hormones, little is known regarding their role in vivo in T-cell maturation. Adoptive transfer studies in routine systems have been used to study functional maturation, and have shown that major histocompatibility complex restriction and self-tolerance are developed under the influence of the thymus [7,8]. Further studies in the mouse have been directed at understanding the role of thymic stromal elements on the development of self-tolerance and MHC restriction (reviewed in [9]). While the development of self-tolerance appears to be directed by bone marrow derived components [10-12], the control of MHC restriction remains controversial. Longo and his colleagues [13,14] have reported that thymic macrophages and dendritic cells dictate MHC restriction, while Lo and Sprent [15] have reported that thymic epithelial cells may be capable of dictating MHC restriction. Moreover, the role that extrathymic or post-thymic events may play in the development ofT-cell functional competence is not yet clear [3,16]. In addition to studies of T-lymphocyte function, phenotypic analysis of thymocytes, coupled with adoptive

From the Department of Medicine, Divison of Rheumatology and Immunology, and the Department of Microbiologyand Immunology, Division of Immunology, Duke University, Durham, North Carolina. Address reprint requests to Kay Singer, Dept. ol Medicine, Division of Rheumatd/lmmundogy, Duke University Medical Center, Durham, NC 27710. ReceivedMarch 4, 1987: acceptedJune 21, 1987.

HumanImmunology20, 127-144(1987) ©ElsevierSciencePublishingCo.,Inc., 1987 52 Vanderbil¢Ave.,NewYork,NY 10017

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K.H. Singer and B. F. Haynes transfer of thymocyte subsets as well as cell migration studies [9,17,18], have provided valuable information about pathways of intrathymic T-cell maturation in murine systems and have suggested multiple intrathymic T-cell lineages. In the human thymus, current models ofT-lymphocyte maturation are derived largely from studies in which thymocyte subsets have been isolated and their phenotype correlated with functional capabilities in vitro [ 19-21 ]. More recently, ontogeny studies of human fetal thymic development have suggested early pathways of T-cell maturation involving primarily CD7 +, CD2 + T-cell precursors [22]. However, the specific roles the nonlymphoid thymic microenvironment plays in promotion ofT-cell activation and maturation remain poorly understood. The cellular complexity of the thymus, coupled with the lack of methods to isolate and study individual components, have been the primary obstacles in addressing those questions. To approach these problems, during the past 3 yr we have produced a panel ofmonoclonal antibodies (MoAbs) reactive with human thymic epithelial (TE) ceils and developed in vitro culture systems for the longterm propagation and functional characterization of human TE cells with regard to 'rE cell interactions with thymocytes. This article reviews our recent work on the phenotypic characterization and in vitro growth of human TE ceils and functional studies of TE-thymocyte interaction.

P H E N O T Y P I C C H A R A C T E R i Z A T i O N OF T H Y M I C EPITHELIAL CELLS Recently, we developed a number of MoAbs that define components of the human and rodent thymic microenvironment (Figure 1) (reviewed in [23]). Two antibodies, TE-4 and TE-7, defined distinct and mutually exclusive thymic micr¢anvironment components. Antibody TE-4 (and a second antibody, A2BS) defined human endocrine thymic epithelium in the subcapsular cortex (SCC) and medulla of the thymus [24]. These cells also contained keratin, thymopoietin, and thymosin otl [25]. Antibody TE-7 defined nonkeratinized, mesodermal-derived thymic fibrous stroma and bound to cultured ¢human fibroblasts [24]. A

A2B5 and TE-3. (A) postnatal thymus. (B) Reactivity of antibody A2B5 with subcapsular cortical ~3CC) epithelium and medulla. (C) Reactivity of antibody TE-3 with cortical but not medullary epithelium. C: capsule ( x 400) (from {23]).

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third antibody, TE-3, selectively defined a population of epithe|ial cells in the inner thym[c cortex that did not conta/n thymosin c~l and did not react with antibody T E d [26]. Study of the tissue distribution of the T]/~, A2B5, TE-7, and TE-3 antigens as well as a det~filed ontogeny study during fetal development of the thymic microenvironment, reve~ed that antibody T~-3 likely defines ~he endodertual derived thytuic epithelial component, while antibodies TE-~ and A2B5 define the ectodermal-derived thymic epithelial compc~nent ([22,2~,27], reviewed in [23]). IN V I T R O G R O W T H OF H U M A N TF~ C ~ , S In order to study the roles that antigenically defined components of the thymi¢ tuicroenvironment may play in T-cell maturation, it was first necessary to grow individual thymic microenvironment components in in vitro cukure for phenotypic and functional characterization. Using an exphnt technique, we established long-term c~!~re~ ofTE ce~s from norm~! hutuan thymus [28]. TE ce~s were cultivated under conditions previously established for epidermal keratinocytes [29]. An enriched culture medium conta/ning epidermal growth factor, cholera toy/n, insu~n, hydrocortisone, fet~ c~f serum (FCS), and adenine was used along with a feeder layer of mitomycln C treated mouse 3T3 fibrob|asts. Most importantly, steps were taken to minimize growth of human fibroblasts. Initially, these steps included selective remov~ of fibroblasts using ethylenediamine tetracetic acid and subculture onto mitomycir.~ C treated mouse fibrobl~t feeder layers that inhibit the growth of human fibroblasts. More recently, we have produced a MoAb (IB10) that selectively binds to the surface of human fibrobhsts and is cytotox/c for hu.~an fibrobhsts in the presence of rabbit complement. This antibody has been successfu~y used to remove fibroblasts from mixed cultures of fibrobiasts and epithelial cells (KH Singer, RM Scearce, DT Tuck, BF Haynes, unpublished observations) and thus facilitate TE cell propagation in pure cultures in vitro. Indirect immunofluorescence assays using MoAbs showed that TE cells propagated in vitro were 100% AE-1 + (anti-keratin) (Figure 2), 60-80% T~-3 + , 20-40% TE-4+, and nonreactive with antibodies MO-I and Leu M3 (anti-

FIGURE 2 (A) Transm/ssion electron m/crograph of two ~jacent ~nlred k ~ ~y~ epithelial ceils, hi indicates nucleus, arrowheads indicate tonofihments, arrows indic~ desmosomes. Insert shows detai! of desmosome. (B) Indirect hnmnnofluorescence on cultured human thymic epithefial cells using anti-keratin MoAb AE-1 (400 x ) (from [28]).

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K.H. Singer ,~,d B. F. Hayn_es monocyte/macrophage). TE cells could be subcultured repeatedly over a 6 - 8 month period, although for a finite number of population doublings (20-40) and could be retrieved from a frozen state in liquid nitrogen. Ultrastrucmral analysis of cultured human TE cells demonstrated the presence of tonofilaments and desmosomes, hallmarks of epithelial ultrastructure (Figure 2). Once we had established techniques for cultivating TE cells, it was then possible to use these cells in functional studies.

H U M A N T H Y M I C EPITHELIAL CELLS PRODUCE I N T E R L E U K I N 1 Antigen presentation to mature T lymphocytes requires triggering of the T cells through the T-cell receptor (T3/Ti) complex and also requires the presence of the cytokine interleukin 1 (ILl). ILl induces T-cell interleukin 2 (IL2) receptor expression and induces the synthesis and secretion of IL2 itself [30-33]. The interaction of IL2 with its receptor is thought to be an obligatory step in the proliferation of mature T lymphocytes [32,34]. More importantly, ILl has been suggested to be an important cytokine that synergizes with other cytokines such as interleukin 3 and colony stimulating factors for activation of pleuripotent bone marrow cells [35]. Because other epithelial cells can serve as a source of ILl [36-38], it seemed likely that TE cells may produce an interleukin 1-11ke molecule and that 'rE cellderived ILl may play a role in thymocyte proliferation in vivo. Supernatants from cultures of human TE cells were screened for ILl activity in the C3H/HeJ mouse thymocyte assay [34]. We found that both postnatal and fetal human thymic epithelial cells produced an ILl-like factor that augmented proliferation of C3H/HeJ mouse thymocytes to phytohemaggludnin (PHA) [39]. ILl activity (20-200 units/ml) was detected in ammonium sulfate fractionated supernatants of TE cultures from all individuals (2-13-yr old) tested. ILl activity was also detected in supernatants of TE cultures from a 17-week fetus but not from those of a 10week fetus and was shown to be present in vivo at 17 weeks but not 10 weeks by indirect immunofluorescence assay using anti-ILl on fetal thymus sections [39]. Production of TE-IL1 was dependent on TE cell density and time in culture, with optimal ILl activity observed with l06 TE cells/ml after 48-72 hr of culture [39]. Moreover, TE-IL1 was produced by thymic epithelial cells themselves and not by other contaminating cell types such as macrophages or fibroblasts [39]. The cornitogenic activity of TE-IL1 was immunologically related to human monocyte ILl. A polyclonal rabbit antiserum to human monocyte ILl neutralized the TE-IL1 activity in the mouse thymocyte assay. Human monocyte ILl has been well characterized with regard to molecular weight and isoelectric points. To compare further TE-IL1 and monocyte-derived ILl, the relative molecular mass (M,) and isoelectric point (pI) of TE-IL1 were determined and compared to that of monocyte ILl. "rE cell supernatants were concentrated by 65% ammonium sulfate fractionation and chromatographed on high performance liquid chromatograph (HPLC) gel filtration columns. The thymocyte comitogenic activity ofTE-IL1 eluted in a single peak of Mr 18,000-20,000 (Figure 3A). When the same material was electrophoresed under reducing conditions in a 12.5% acrylamide gel containing 1% SDS, the comitogenic activity was found in a 15,000-17,000 Mr band. Additional ILl activity was found at 23,000-29,000 Mr [39] These values are similar to those reported for human monocyte ILl [34]. To determine the pI of TE-IL1, the HPLC fraction (18,000-20,000 M~°) was concentrated and electrofocused in a 4% acrylamide gel with 2% ampholine~. The majority (85%) of TE-IL1 activity was associated with pIs of 5.7-5.8 and 6.9-7,0 (Figure 3B) [39]. Similar charge heterogeneity has been reported for human monocyte ILl and human epidermal-derived thymocyte activating factor

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F I G U R E 3 (A) Thymocyte comitogenic activity of TE cell supernatant separated by HPLC gel filtration. A 2.0-ml ~ q u o t of an ammonium sulfate fracdonated TE cell cukure supernatant was chromatographed on TSK 3000-TSK 2000 gel filtration columns con-nected ~n series. All fractions were tested for TE-IL1 activity at a 1:4 dilution. Dam represent the means of triplicate determinations. Background 3H-TdR incorporation with PHA alone was 2320 -+ 190 cpm. (B) Thymocyte comitogenic activity of HPLC purified TE-IL1 separated by isoelectricfocuaing. HPLC purified TE-IL1 was subjected to isoelectricfocusing in a 4% ac~lamide gel containing 9.2 M urea, 2% NP-40, and 2% ampholines ....',pH 5 - 6 and pH 3.5-10). The gel was sliced into 2-mm sections, TEdL1 eluted and tested at 1:4 dilution for comitogenic activity in the mouse thymocyte assay. Background 3H-TdR incorporation with PHA alone was 2132 - 462 cpm. A pH calibration curve was constructed using a mixture of protein markers of known pls (3.5-9.3) (from [39]).

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K.H. Singer and B. F. Haynes (ETAF) [40,41]. Like monocyte ILl and ETAF, TE-IL1 was insensitive to sodium dodecyl sulfate, stable at 56°C, and at pHs between 2 and 11 (PT Le, BF Haynes, KH Singer, unpublished observations). The indistinguishable biochemical characteristics of monocyte-derived ILl, ETAF, and TE-IL1 suggested that they are similar polypeptides produced by different cell types. Further comparison of these molecules awaits studies at the amino acid, mRNA, and DNA levels. In order to confirm the location of TE ILl producing "rE cells in situ, we performed double immunofluorescence assays using a polyclonal rabbit anti-ILl antiserum and a monoclonal antikeratin antibody [34]. The majority of cells staining positive with the anti-ILl antibody were also positive with the anti-keratin antibody confirming the reactivity of anti-ILl in situ with TE cells. Macrophages that were positive for ILl but negative for keratin were also observed in the thymic medulla [39].

THYMOCYTES BIND T O THYMIC EPITHELIAL CELLS A number of physical interactions of developing thymocytes with nonlymphoid elements of the thymus have been identified, including the formatic,n of lymphoepithelial cell complexes in vitro called thymic nurse cells [42,43~, and the binding of thymocytes to macrophages and dendritic cells [44,45]. Farret al. [46] observed a subpopulation of thymocytes within the mouse thymic cortex expressing low levels of surface T-lymphocyte antigen receptors (Ti). Where these thymocytes were found in contact with epithelial cell processes, Ti molecules were localized in the region of thymocyte-epithelial contact. Thus it was of particular interest, once large numbers of TE cells were available from in vitro culture, to determine if human thymocytes could direcdy bind to "rE cells, and if so, to determine the molecules involved in TE-thymocyte interactions. To establish techniques to evaluate the ability of thymocytes to bind to 'rE cells in suspension, we used an adaptation of the E-rosette binding assay for Tcell sheep cell binding [47], and for binding of thymocytes to adherent TE cells, we used an adaptation of the Woodruff lymphocyte-high endothelial cell binding assay [48]. In the latter assay, graded numbers of thymocytes were added to TE cells growing adherent to glass slides. Nonadherent cells were removed by washing and cells were fixed with glutaraldehyde and stained with hematoxylin and eosin (H&E). As shown in Figure 4A, thymocytes bound to TE cells, but did not bind to human foreskin fibroblasts [49]. In order to quantitate TE-thymocyte binding, a suspension binding assay was developed similar to that used for T-cell E-rosette formation [49]. Under these conditions, 30-60% of TE cells bound ~3 thymocytes (Figure 4B). When the ratio of thymocytes to 'rE cells was varied over a range of 1:1 to 32:1, maximal TE-thymocyte binding was achieved with thymocyte:TE ratios of between 8:1 and 16:1. Peripheral blood T cells and tonsillar T cells also bound "rE cells; however, peripheral blood B cells did not bind to TE cells. No quantitative difference was seen in the binding of thymocytes to TE in autologous or allogeneic combinations. Under the conditions of binding in the suspension assay, thymocytes did not bind to thymic fibroblasts, to the rat thymic epithelial cell line IT26R21, or to cultured epidermal keratinocytes. However, human thymocytes did bind to the epithelial cell line A431 [49]. Thymocytes that bound to TE cells included both the immature CD1 + and mature CD3 + thymocyte phenotypes. In addition, purified populations of immature double negative (CD7 +, CD2 +, C D 4 - , C D 8 - ) thymocytes bound to TE cells as well [50]. We next used a large panel of anti-thymocyte and antiTE cell MoAbs to determine the molecules involved in TE-thymocyte binding. Contrary to our expectations, TE-thymocyte binding was not inhibited by an-

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FIGURE 4 (A) Binding of thymocytes to adherent TE cells grown on #ass slides (hematoxylin/eosin stain, 400 ×). (B) TE-thymocyte rosettes formed in suspension (Wr/ght's stain, 400 ×) (from [49]).

tbodies to MHC class I nor class I! antigens. However, TE cells cultured in vitro and used in these studies did not express class II MHC ant/gens [49] although in vivo thytoic epithelial ceils do express class II antigens [23,51]. Upon in vitro cultivation, TE cells cease to express class II antigens but can be induced to express class II antigens by treatment with ~/-intefferon [52] or by cocultvaton for 72 hr with thytoocytes [53]. To da~e, we have been unable to detect HLADR mediated binding of TE to thytoocytes using ~/-intefferon treated HLA-DR + TE cells. Interestingly, antibodies directed against the CD2 (Tll, LFA-2, E-rose~e receptor) toolecule and the lymphocyte functon-assochted antigen-3 (LFA-3) did inhibit TE-thytoocyte binding [54]. Ant-CD2 antibodies TS1/8, TS2/18, T11/3Pt2H9, T11 ROLD2-1H8, T11/TT47A9, and 35.1 blocked greater than 80% of TE-thymocyte binding. And-CD2 antibodies D66 and T56 partially

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blocked TE-thymocyte binding (59 and 44%, respectively) [55]. D66 and T56 have been shown to bind only to a subset of CD2 positive peripheral blood T cells and to bind to a distinct epitope on the CD2 molecule [56,57]. No other CD group of antibodies in 4°C binding assays inhibited TE-thymocyte binding to the degree that anti-CD2 reagents inhibited [55]. Specifically, anti-CD18 (LFAl) antibodies (TS1/22, TS1/18) and anti-CD8 antibodies Ehat are known to inhibit binding of cytotoxic T lymphocytes (CTL) to target cells [58,59] and anti-CD3 antibodies that inhibit CTL-mediated killing at a postconjugation step [60], did not block TE-thymocyte binding. To determine the site of inhibition of TE-thymocyte binding by anti-CD2 and anti-LFA-3 antibodies, experiments were performed using either TE cells, thymocytes, or both, that had been preincubated with antibody, washed, and then tested for binding in the TE-thymocyte binding assay (Figure 5). Inhibition of TE-thymocyte binding occurred when TE cells were pretreated with LFA-3 (94% inhibition) or when thymocytes were preincubated with anti-CD2 antibody (94% inhibition). Indirect immunofluorescence assays confirmed that < 10% of resting thymocytes were LFA-3 + while greater than 95% were CD2 +. Cultured TE cells were >-98% LFA-3 + and C D 2 - [54]. Double immunofluorescence assays on frozen sections of human thymus using anti-keratin antibody AE-1 and LFA-3 antibody confirmed that TE cells indeed expressed the LFA-3 antigen in vivo [54]. Thus, we showed that human thymocytes bind to autologous and allogeneic TE cells via LFA-3 molecules on TE cells and CD2 (Tll, LFA-2, E-rosette receptor) molecules on thymocytes. The simplest and most attractive interpre-

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tation of these data was that thymocyte-TE binding involves a receptor-ligand interaction between CD2 and LFA-3. Plunkett et al. have recendy shown th~ the CD2 antigen binds directly to the LFA-3 molecule on human er~throcytes [61]. Recently, we have shown that affinity purified LFA-3 antigen i~duces the agglutination of CD2 + thymocytes and this agglutination is specifically inhibited by anti-CD2 or anti-LFA-3 antibodies (LW Vollger, SM Denning, BF Haynes, KH Singer, unpublished observations). Taken together, these data ,trongly suggest that the LFA-3 antigen on TE cells can bind to the CD2 antigen on thymocytes. In the TE-thymocyte binding assay, it was interesting that CD2 + thymocytes did not bind to LFA-3 + cultured epidermal cells [54] or corneal epithelial cells (L~t Vollger, BF Haynes, KH Singer, unpublished observations). However, LFA3 exhibits molecular heterogeneity on different cell types [62] and ;t is possible that glycosylation of LFA-3 on cultured epidermal or corneal cells is inappropriate for binding to thymocytes. Likewise, the surface charge or other properties of epidermal or corneal cells may differ from thymic epithelL~ cells and hinder thymocyte binding. Alternatively, LFA-3 molecules on epit},elial cells may be necessary but not sufficient for binding to CD2 on thymocy~es. Hunig [63,64] has recendy isolated a 42,000 Mr glyco~rotein from sheep erythrocytes (SRBC) as well as sheep leukocytes which binds ~o CD2 and blocks E-rosette formation. LFA-3 antibody does not bind to sheep erythrocytes; however, it is likely that the 42,000 Mr glycoprotein from SRBC is sinfilar to the human LFA-3 molecule but lacks the epitope detected bg anti-LFA-3 antibody. T H Y M I C EPITHELIAL CELLS ARE P O T E N T ACCESS~'JRY CELLS FOR M A T U R E T H Y M O C Y T E P R O L I F E R A T I O N In order to study sequelae of thymocyte-TE binding, ~e determined the influence of TE cells on thymocyte proliferation in the presence and absence of mitogen. In the presence of subopthn',d concentrations of phytohemag#udnin (PHA), thymic epithelial ceils function as potent accessory ceils enhancing thymocyte proliferation as measured by 3H-thymidine incorporation, blast transformation, and 3H-leucine incorporation into protein [53]. Thymocytes alone or thymocytes that have been rigorously depleted of monocyte/macrophage accessory cells (accessory cell depleted or ACD-thymocytes) by passage over nylon wool, responded poorly to PHA at 1 /zg/ml (Figure 6). Addition of TE cells pretreated with mitomycin C (TEM) to autologous or allogeneic ACD-thymocytes in the presence of 1 ~g/ml PHA, markedly enhanced thymocyte proliferation measured by 3Hthymidine incorporation (Figure 6). Thus, human TE cells can serve as potent accessory cells for PHA-induced thymocyte activation in vitro. TE accessory cell function for PHA-induced thymocyte activation was present at all PHA concentrations tested but was most pronounced at suboptimal concentrations. Kinetic experiments showed that peak PHA-induced mitogenesis in the presence of TE cells occurred at day 3. TE cell induced proliferation of thymocytes in the presence of PHA was not due to macrophage contamination of'rE cell suspension because pretreat~ent of the TE cell prepare*ion with anti-HLA-DR mltibody (L243) plus complement did not abolish the accessory cell function. Likewise, TE accessory cell function tbr thymocytes could not be attributed to contaminating fibroblasts because addition of thymic fibroblasts to ACD-thymocytes in the presence of 1/~g/ml PHA did not induce thymocyte proliferation [53]. "rE accessory cell function was maximal when 2 × 104 TE ceils were added to 1 x 105 ACD-thymocyte (TE:thymocyte = 1:15); however, "rE accessory cell function was detected when TE:thymocyte ratios as low as 1:100 were used. The

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ability of TF. cells to function as accessory cells did not require TE cell DNA synthesis. However, TE accessory cell function did require TI8 cell protein synthesis as treatment of TE cells with the irreversible prote:in synthesis inhibitor emetine completely inhibited TE cell induction of thymocyte response to MHC. T-cell activation by mitogen or antigen is dependent upon production of IL2 and driven by IL2 [65]. In our studies, thymocyte activation via TE cells was also IL2 dependent. Addition of a MoAb to the IL2 receptor anti-TAC [66] resulted in greater than 75% reduction in 3HoTdR incorporation by thymocytes in the presence of 1 ~g/ral PHA and cultured TE cells [53]. In order to determine the thymocyte subset responding in PHA-stimulated TE-thymocyte cocultures, the thymocytes were separated into CD14- (T6+), p S 0 - cells, (i.e.~ immature cells, predominantly found in the cortex) and CD1 -, p80 + cells (i.e., mature cells found in the medulla and in foci within the cortex) [53]. In an exten~sive series of separation experiments, the thymocytes that proliferated in the presence of PHA (1 /~g/ml) and TE cells were predominantly mature C D I - , p80+ thymocytes. Thus, one consequence of TE binding to mature thymocyt~:s is the provision of accessory signals necessary for thymocyte activation by the mitogen PHA. We have proposed that TE cells may have dual effects on mature thymocytes: (1) that of promoting activation toward thymocyte terminal maturation and/or clonal expansion and (2) facilitation of thymocyte response to antigens intrathymically [53,67,68]. To investigate the cell surface molecules involved in mediation of TE cell

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dependent thymocyte activation, MoAbs were added to PHA-stimulated autolognus and allogeneic TE-ACD-thymocyte cocultures. In agreement with our data on inhibition of thymocyte-TE binding, antibodies to CD2 and LFA-3 antigens inhibited TE cell dependent, PHA-induced ACD-thymocyte activation in both autologous and allogeneic cultures [69]. Antibody 1.243 against MHC class II (HLA-DR) antigen did not inhibit thymocyte 3H-TdR incorporation in autolognus or allogeneic TE-thymocyte combinations. Antibody 3F10, agalns~ MHC class I antigen, did not significantly inhibit ACD-thymocyte PHA responses in autolognus TE-thymocyte cocultures, but did inhibit by 38% the TE dependent PHA response of ACD-thymocytes in allogeneic TE-thymocyte suspensions. Interestingly, antibody to the LFA-1 antigen did not inhibit TE-thymocyte binding in the rosette formation assays but did inhibit TE cell dependent thymocyte proliferation to PHA. Shaw has recently shown that adhesion of cytoto:dc T cells to their targets involves both LFA-3/CD2 mediated binding and binding via LFA-1 on the CTL to a structure on the target cell [70]. Dustin et al. have shown that LFA-1 binds to intercellular adhesion molecule-1 (ICAM-1) and that this binding is more efficient at 37°C ([71], M Dustin, TA Springer, personal communication). Thus, it is possible, that in the rosette forming assay we did not detect binding via LFA1/ICAM-1 due to the fact that the rosette assay was done at 4°C where conditions were not appropriate for measuring LFA-I mediated binding. In contrast, the accessory cell experiments were performed at 37°C, conditions under which LFA1 mediated binding would be expected to occur. Thus, it is likely that two pathways of interactions may be involved in TE-mature thymocyte interactions, one involving LFA-3 and CD2 molecules and the other thymocyte LFA-1 molecules. TE CELLS DIRECTLY ACTIVATE DOUBLE NEGATIVE (iMMATURE) T H Y M O C Y T E $ T O PROLIFERATE To investigate TE thymocyte interactions in the absence of mitogen, cocultures were established of autologous combinations of ACD-thymocytes and TEM cells and assessed for 3H-TdR incorporation on days 2-7 of coculture in the absence of mitogen [50]. Peak 3H-TdR incorporation occurred at day 3 with a two to fourfold increase in 3H-TdR incorporation when ACD-thymocytes were cultured with TEM as compared to ACD-thymocytes cultured alone. In order to determine which thymocytes proliferated in the presence of aa~o|ogous "gEM cells, thymocytes were fractionated using a panning technique into two subpopu!~tions. One of these subpopulations, the double negative thymocytes, consisted of thymocytes that did not express the CD4 or CD8 surface markers, and are thought to be the most immature cells found within the thymus [15,16,21]. While C D 4 - , C D 8 - , these double negative thymocytes were 5-15% CD3 +, and greater than 90% CD7 + and CD2 +. The CD4 +, CD8 + thymocyte subset was made up of ceils that either expressed both CD4 and CD8 antigens or reciprocally expressed these antigens. These two subpopulations of thymocytes (CD4 - , C D 8 - and CD4 + , CD8 + ) were cocultivated for 5 days with autologous TEM in the absence of mitogen. As can be seen in Figure 7, the thymocytes that proliferated in the presence of TEM were the C D 4 - , C D 8 - immature thymocytes. In addition, subsequent experiments showed that C D 3 - , CD7 + double negative thymocytes that did not express surface or cytoplasmic T-cell receptor ~ or fl chains (using antibody WT31) [72], similarly responded to TE cell activation signals [50]. It has been previously shown in humans that this :mbset of thymocytes includes the most

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immature T cells lacking expression of CD3 molecules and nonrearranged Ti a genes [73]. Thus, in the absence of mitogen, TE cells directly stimulated the most immature thymocytes to proliferate. Whereas purified doubie negative thymocytes did not respond to ILl, addition of TE cells to ILl-stimulated double negative thymocyte cultures augmented the immature thymocyte response to ILl [50]. At least two pathways have been described for activation of human T lymphocytes. The classical antigen dependent pathway involves triggering through the context of MHC antigens and antibodies co T3 or Ti can mimic antigen activation through this pathway. The alternative pathway of human T-cell activation is antigen independent and involves triggering via the CD2 molecule [74,75]. Combinations of antibodies to different epitopes of the CD2 molecule can promote activation via this pathway. Since binding of double negative thymocytes to TE cells was inhibited by anti-CD2 antibodies [69], we determined the ability of a combination of CD2 antibodies (9.6, 9-1) that is mitogenic for mature T cells [76], to activate double negative thymocytes. We found that the mitogenic effect of TE cells could be replaced by the incubation of double negative thymocytes with 9.6 and 9-1 anti-CD2 antibodies in the presence of either ILl or IL2 [50]. Further, we found double negative thymocytes proliferated in the presence of IL2 and this responsiveness to IL2 was augmented following thymocyte binding to TE cells [50]. Once activated, double negative human thymocytes proliferated in vitro in IL2 and remained double negative [50]. Taken together, these data demonstrate that one role of TE cells in vivo may be to ac:ivate immature thymocytes via the alternative or CD2 pathway of T-cell activation [75] toward clona! expansion, and in doing so, augment the double negative thymocy.*e response :o ILl and IL2 [50]. EFFECTS OF T E - T H Y M O C Y T E B I N D I N G O N TE CELLS We recently noted that cocultivation of autologous thymocytes and TE cells resulted in increased levels of'l~dL1 celeased into TI/culture supernatants ([771, PT Le, BF Haynes, KH Singer, unpublished observations). Because we had already shown that thymocytes and TE cells bind via CD2 molecules on thymocytes and LFA-3 molecules on TE cells, we reasoned that triggering TE cells through the LFA-3 molecule might provide a signal for TE cell release of ILl. We therefore assessed the ability of anti-LFA-3 antibody to trigger ILl release via binding to the LFA-3 molecule. Incubation of TE cells with anti-LFA-3 ~ntibody (1-10 ~g/ml) for 24--48 hr resulted in a two to fivefold increase in ILl activity in TE supernatants ([77], PT Le, BF Haynes, KH Singer, manuscript in preparation). When human peripheral blood monocytes were incubated with antiLBA-3 antibody (1-10 ~g/rul) for 24-48 hr, a 10-40-fold increase in ILl activity was detected in the supernatant. Thus, thymocytes may regulate their own activation by modulating the availability of ILl within the thymus by triggering TE cells (and monocytes/macrophages) to release ILl. Additionally, we have shown that HLA-DR negative TE cells were induced to express HLA-DR by incubation with y-interferon or by cocultlvation with autologous thymocytes [53]. Taken together, these data suggested that thymocytes are capable of regulating their mlcroenviroument with regard to TE cell expression of HLA-DR antigens as well as with regard to TE and monocyte production of ILl. SUMMARY Our data demonstrate that the epithelial component of the human thymic microenvironment is not an inert cell type, but rather is capable of being directly

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K.H. Singer and B. F. Haynes involved in the promotion of both early and late stages ofT-cell maturation. Data from our laboratory [54,69], together with the work of Plunkett et al. [61] and Shaw et al. [70] suggest that an endogenous ligand for the CD2 molecule in humans is the LFA-3 molecule. Using an SV40 transformed human thymic epithelial cell line of subcapsular cortical origin, Mizutani et al. have confirmed that thymic epithelial cells bind thymocytes via a CD2/LFA-3 interaction [78]. The data reviewed in this paper suggest that within the thymus one endogenous ligand for the alternative pathway of thymocyte activation via the CD2 molecule is the LFA-3 molecule on TE cells. Following thymocyte binding to 'rE cells, immature thymocytes are directly activated to proliferate, and their response to both ILl and IL2 is augmented. Also, following TE-thymocyte binding, TE-IL1 secretion is augmented and TE cell MHC class II antigen expression is induced, Moreover, while undergoing activation, thymocytes appear to be able to modulate their microenvironment milieu of MHC antigens and ILl. Further analysis of the sequelae of TE-thymocyte interactions using phenotypic characterization of thymocytes with anti-T-cell MoAbs, coupled with molecular analysis of thymocyte T-cell receptor genes, should allow for the determination of the precise sequential stages that immature T cells undergo enroute to functional maturity. Understanding these steps in T-cell maturation will be critical to our understanding of the events that transpire in the genesis of autoimmune, lymphoproliferative, and immunodeficiency diseases.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grants AR34808, CA28936, and T32CA09058 and a Basic Research Grant from the National March of Dimes Foundation. KHS is a Scholarof the LeukemiaSocietyof America. We gratefullyacknowledge the contributions of our colleagues Stephen M. Denning, David F. Lobach,Phong T. Le, Joanne Kurtzberg, Leanne W. Vollger, Debbi T. Tuck, Bobbi P. Crumment, and Richard M. Scearce. T.A. Springer and C.A. Dinarello provided antibodiesand critical discussion. We thank Joyce Lowery and Kim McClammyfor expert secretarial assistance. REFERENCES 1. MillerJFAP,Osaba D: Current concepts of the immunologicalfunctionof the thymus. Physiol Rev 47:257, 1967. 2. Weissman IL: Thymus cell maturatloa. Studies on the origin of cortisone-resistant thymic lymphocytes.J Exp Meal 137:504, 1973. 3. Stutman O: Intrathymic and extrathymic T-cell maturation, lmmunol Rev 42:138, 1978. 4. Dardenne MD~ SavinoW, Gastinel L, BachJF: Thymulin: new biochemicalaspects. In: AL Goldstein, Ed. Thymi¢hormones and lymphokines.New York, Plenum, 1984, p. 37. 5. Low TK, Goldstein AL: Thymosin: isolation, structural studies, and biological activities. In: AL Goldstein, Ed. Thymic hormones and lymphokines.New York, Plenum, 1984, p. 21. 6. Goldstein G, AudhuaTK: Thymopoietin to thymopentin:experimentalstudies. Surv Immunol Res 4 (Suppl. 1):1, 1985. 7. ZinkernagelRM: Thymusand lymphohemopoieticcells: their role in T cell maturation in selectiono f t cells H-2 restrictionspecificityand H-2 linkedgene control. Immunol Rev 42:224, 1978.

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