Human Thymocytes Bind to Autologous and Allogeneic Thymic Epithelial Cells in Vitro (Thymus/T-Celi Activation/Cell-Cell Binding) KAY H

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6588-6592, September 1986 Immunology

Human thymocytes bind to autologous and allogeneic thymic epithelial cells in vitro (thymus/T-celi activation/cell-cell binding) KAY H. SINGER*tt, LEANNE S. WOLFt, DAVID F. LOBACH*, STEPHEN M. DENNING§, DEBBI T. TUCK*, ANNETTE L. ROBERTSON*, AND BARTON F. HAYNES*t *Division of Rheumatology and Immunology, and §Division of Cardiology, Department of Medicine; and tDivision of Immunology, Department of Microbiology and Immunology, Duke University, Durham, NC 27710 Communicated by D. Bernard Amos, May 9, 1986

ABSTRACT The thymus plays a critical role in the gen- MATERIALS AND METHODS eration of immunocompetent T lymphocytes. In the thymus, this Cells. Thymus tissue was obtained at the time of corrective lymphocytes are in close contact with epithelial cells, and 1 to 16 contact is necessary for T-cell maturation. Using cultured cardiovascular surgery from 15 patients aged day years. Thymocyte single-cell suspensions were prepared as human thymic epithelial (TE) cells, we have found that human described (16). Human peripheral blood lymphocytes and thymocytes bind to human TE cells in vitro. Thymocytes bound tonsillar lymphocytes were prepared as described (16), and T to both allogeneic and autologous TE cells and to the lymphocytes were isolated by filtration over nylon wool. TE epidermoid carcinoma cell line A431 but did not bind to cell cultures were initiated, propagated, and subcultured as epidermal keratinocytes or to thymic fibroblasts. Thymocyte described (11). Cytocentrifuge preparations of cultured TE binding to TE cells was trypsin- and cytochalasin B-sensitive. cells were evaluated in indirect immunofluorescence assays Indirect immunofluorescence assays showed that both mature using a panel of monoclonal antibodies including AE-1 (T6-, T3+) and immature (T6+, T3-) thymocytes bound TE (anti-keratin) (17), Mo-1 (anti-monocyte, macrophage) (18), cells. In our system, TE-thymocyte binding was not inhibited Leu-M3 (anti-monocyte, macrophage) (19), TE-7 (anti-fibro- by antibodies to class I or class II major histocompatibility blast) (13), and L243 (anti-MHC class II antigen) (20). TE antigens. In vitro binding of thymocytes to TE cells may cultures were 86 ± 1.6% AE-1 (keratin)-positive, 2.3 ± 0.6% represent a correlate of in vivo TE-thymocyte interactions and L243-positive (all the L243-positive cells contained keratin), provides a model system for the study of human intrathymic and 13 ± 1.2% TE-7 (fibroblast)-positive; none ofthe TE cells T-lymphocyte maturation and activation. were positive for Mo-1 or Leu-M3. Thymic fibroblasts were isolated from TE cell cultures by treatment with 0.02% EDTA. The epidermoid carcinoma cell line A431 was pro- Microscopists have long appreciated the extensive physical vided by J. DeLarco (Otsuka Pharmaceuticals, Gaithersburg, contact between the and elements of lymphoid nonlymphoid MD) and was propagated in Dulbecco's modified Eagle's the thymus (1, 2). Direct contact of lymphoid and nonlymph- medium with 10%o fetal bovine serum (21). Neonatal human oid elements within the thymus is necessary for generation of foreskin epidermal keratinocytes were cultured as described functionally mature, antigen-specific, major histocompatibil- (22). ity complex (MHC)-restricted T lymphocytes (3-6). Al- Slide Technique for TE-Thymocyte Binding. TE cells were though the precise role played by the nonlymphoid compo- subcultured onto two-well Lab-Tek chamber slides (Miles nent of the thymus is poorly understood, a number of Laboratories, Naperville, IL) at 2-5 x 104 cells per chamber interactions of developing thymocytes with nonlymphoid and grown to 50-75% confluence. TE cells were overlaid with elements have been identified, including the formation of thymocytes, and chamber slides were then incubated 40 min lymphoepithelial cell complexes in vivo, called thymic nurse at 4°C on a rotating platform (Tekpro, Evanston, IL) at 80 cells (7), and the binding of thymocytes to macrophages and rpm (23). Nonadherent cells were removed, and the remain- dendritic cells (8, 9). Farr et al. (10) recently observed a ing cells were fixed with 1% glutaraldehyde (Sigma) in subpopulation of thymocytes within the thymic cortex ex- Dulbecco's phosphate-buffered saline without Ca2' and pressing low levels of surface T-lymphocyte antigen recep- Mg2+, pH 7.4 for 15 min on the rotator. After washing, the tors (Ti). Where these thymocytes were found in contact with plastic chambers were removed, the slides were dipped in epithelial cell processes, Ti molecules were localized in the water, and the cells were stained with hematoxylin and eosin. region of thymocyte-epithelial contact. Recent development Scanning and transmission electron microscopy was per- of methods for the long-term culture of human thymic-stro- formed as described (11, 24). mal elements (11), as well as development of monoclonal Rosette Assay for TE-Thymocyte Binding. TE cell suspen- reagents specific for nonlymphoid components of human sions were prepared by trypsin treatment ofcultured TE cells thymus (12-15), have made it possible to investigate thymo- (0.5 mg of trypsin per ml in phosphate-buffered saline with cyte-stromal interactions in vitro. In this report we present 0.02% EDTA, 1-3 min) and kept at 4°C. Autologous or allogeneic thymocytes, peripheral blood T cells, or tonsillar evidence that human thymocytes bind to both autologous and lymphocytes were thawed, centrifuged through Ficoll-Hy- allogeneic thymic epithelial (TE) cells in vitro. Binding of paque, washed three times with RPMI 1640 medium contain- thymocytes to TE cells in vitro likely represents a correlate ing 5% fetal bovine serum (RPMI/FBS), and kept at 4°C. of in vivo TE-thymocyte interactions that are important for Thymocytes or other lymphoid cells (2 x 106 cells in 0.1 ml triggering sequential stages of thymocyte maturation. of RPMI/FBS) were combined with TE cells (2.5 x 105 cells

The publication costs of this article were defrayed in part by page charge Abbreviations: TE cell, thymic epithelial cell; MHC, major histo- payment. This article must therefore be hereby marked "advertisement" compatibility complex. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

6588 Downloaded by guest on October 2, 2021 Immunology: Singer et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6589 in 0.1 ml of RPMI/FBS) and centrifuged (380 x g, 4TC, 5 CD-2, LFA-2, T1l) (28), TS1/8.1.1 (anti-CD-2, LFA-2, T11) min). TE/thymocyte pellets were gently resuspended by (29), T3/RW2-8C8 (anti-CD-3, T3) (30), T4/19-THY5D7 rotation of the tube as for an erythrocyte (E)-rosette assay (anti-CD-4, T4) (31), 3A1 (anti-CD-7, 3A1) (32), T8/2T8-1B5 (25). Tubes were coded, and cell suspensions examined at (anti-CD-8, T8) (33), A2B5 (anti-GQ ganglioside) (34), TE-3 x 150 by light microscopy. TE cells binding >3 thymocytes (anti-cortical TE cell) (13), and AE-1 (anti-keratin) (17). were scored as positive for rosette formation. At least 200 TE cells were counted for each tube, and each determination was RESULTS performed in duplicate or triplicate. Data were analyzed by Student's t test. Binding of Thymocytes to TE Cells in Vitro. Graded num- Phenotypic Characterization of Cells in TE-Thymocyte bers ofthymocytes were added to TE cells growing adherent Rosette Assay. The TE-thymocyte rosette assay was per- to glass slides. Maximal binding of thymocytes to TE cells formed as described above, and the percentage of TE cells was achieved with 107 thymocytes per chamber well. forming rosettes with thymocytes was determined. TE/ Thymocytes bound to TE cells with varying morphologies, thymocyte suspensions were centrifuged onto glass slides including small, polygonal TE cells (Fig. LA), TE cells with using a cytocentrifuge (Shandon Southern Instruments, extensive processes (Fig. 1B), and large TE cells with clear Sewickley, PA) and processed for Wright's stain or acetone- cytoplasm (Fig. 1C). Indirect immunofluorescence assays on fixed for indirect immunofluorescence assay using anti-T cell companion slides, using an anti-keratin antibody (AE-1), and anti-epithelial monoclonal antibodies followed by fluo- demonstrated that all the morphological cell types that bound rescein isothiocyanate-conjugated goat anti-mouse immuno- thymocytes contained keratin (results not shown). globulin as described (26). The following monoclonal re- To quantitate TE-thymocyte binding, we developed a agents were used: NA1/34 (anti-CD-i, T6) (27), 35.1 (anti- TE-thymocyte rosette technique. Representative samples of rosetting and nonrosetting TE cells are shown in Fig. 2. Small and large thymocytes bound to both large and small TE cells L: (Fig. 2 A and B). TE cells that did not bind any thymocytes were frequently observed (Fig. 2C). Occasionally TE cells 0 were observed that appeared to contain thymocytes within 0 their cytoplasm (Fig. 2D). When the ratio of thymocytes to .0. TE cells was varied over a range of 1:1 to 32:1, maximal binding was achieved between 8:1 and 16:1 (data not shown). i. In experiments below, unless otherwise indicated, a thymocyte/TE cell ratio of 8:1 was used. In a comparison ofT cells from other sources, we found that tonsillar and peripheral blood T cells bound to TE equally as well as thymocytes (data not shown). Peripheral blood B cells did not bind to TE cells S.. E (data not shown). Treatment ofthymocytes and TE cells with trypsin indicated that thymocyte surface molecules involved .d-fw * * in binding were trypsin-sensitive, whereas TE cell surface :e:::. molecules were not (data not shown). Cytochalasin B (10 ,g/ml) totally inhibited thymocyte-TE binding (data not shown). Electron Microscopic Analysis of TE-Thymocyte Binding. Binding of thymocytes to TE cells was evaluated using scanning and transmission electron microscopy. TE cells binding thymocytes included compact TE cells as well as TE cells with extensive cell processes. Many TE cells exhibited a polar distribution of bound thymocytes (Fig. 3). By means of transmission electron microscopy, the interface between A B I: ;* .1*. II

000 Ae00 0 7 D 0~~~~~ _

e.>.I' *0 *. *, A.* '.V I S 50 *0 0 a On*@ _ . . ...F W.:1 0 *

FIG. 1. Binding of thymocytes to adherent TE cells grown on FIG. 2. TE-thymocyte rosettes formed in suspension. (A) Large glass slides. Thymocytes bound to adherent TE cells of various (solid arrows) and small (arrowheads) thymocytes bound to TE cells morphologies, including polygonal cells (A), cells with elongated (open arrow). (B) Thymocytes bound to a large TE cell. (C) TE cell processes (B), and large cells with clear cytoplasm (C). (Hematox- (open arrow) that did not bind thymocytes. (D) TE cell containing ylin/eosin stain; x570.) thymocytes. (Hematoxylin/eosin stain; x320.) Downloaded by guest on October 2, 2021 6590 Immunology: Singer et al. Proc. Natl. Acad. Sci. USA 83 (1986)

CD) ii 0

0 E a c e FIG. 3. Scanning electron micrograph of thymocytes bound to .r-6 B autologous adherent TE cells. (x 1500.) 3- 80r 0C) TE cells and could be characterized as a close 0 thymocytes 0 70 _ association of plasma membranes devoid of desmosomes 0)2 (data not shown). .cmC Binding of Thymocytes to Autologous and Aliogeneic TE E 60 _ Cells and Other Cell Types. The ability of thymocytes to bind F-1- to TE cells in autologous combinations (n = 4) and allogeneic 0 combinations (n = 12) was compared, and no difference in 50 _ binding was observed [45.5 ± 6.5% of TE cells bound to 0 autologous thymocytes vs. 45.7 ± 3.8% of TE cells bound to 0- 40 F17 allogeneic thymocytes (P > 0.1)] (Fig. 4A). In addition, we determined the ability of allogeneic thymocytes to bind to a .-FI variety of other cell types (Fig. 4A). Allogeneic thymocytes 30 did not bind to thymic fibroblasts, to a rat thymic epithelial cell line (data not shown) (35, 36), or to cultured epidermal 20 keratinocytes. Interestingly, thymocytes bound well to cells of the epidermoid carcinoma cell line A431 (36.3 ± 3.1%). Comparison of Thymocyte Binding to TE Cells from Differ- 10 ent Individuals. To compare the ability of different TE cultures to bind thymocytes, binding was evaluated using thymocytes and TE cells from four different individuals. As 1 2 3 4 can be seen in Fig. 4B, binding ranged from 38 to 60%. Thymocytes from all four individuals bound significantly FIG. 4. Ability ofTE cells and A431 cells to bind thymocytes. (A) better to TE cells from patient 2 than to the other three TE Binding of thymocytes to autologous TE cells (bar a, n = 4), cell cultures (P < 0.005). allogeneic TE cells (bar b, n = 12), thymic fibroblasts (bar c, n = 3), Antibodies to Class I and Class II MHC Antigens Do not cultured epidermal keratinocytes (bar d, n = 3), and A431 epidermoid Affect Thymocyte Binding to TE Cells. To determine whether carcinoma (bar e, n = 4). Vertical bars represent mean ± SEM. (B) class I and class II MHC antigens are involved in binding of Binding of thymocytes to cultured TE cells from four individuals. thymocytes to cultured TE cells, antibodies 3F10 (anti-MHC Vertical bars represent mean ± SEM. class I; ref. 48) and L243 (anti-MHC class II) were added to the TE-thymocyte binding assay. To reduce the possibility of define T-cell antigens, we found among rosetting thymocytes bridging of cells by antibody molecules, either thymocytes, both T6+ (65 ± 9%) (Fig. 5C) and T3+ (48 ± 8%) (Fig. 5D) or TE cells, or both were pretreated with antibody, and thymocytes bound to primarily TE-3+ cortical TE cells excess antibody was removed by washing. TE cells and (Table 2). thymocytes were then combined to allow binding. Pretreat- ment of thymocytes, TE cells, or both with either antibody DISCUSSION 3F10 or L243 did not affect thymocyte-TE binding (Table 1). The specific roles played by the epithelial component of the Phenotypic Analysis of Thymocytes Bound to TE Cells. human thymus in T-lymphocyte maturation are controversial Following TE-thymocyte rosette formation, bound thymo- and in large part are inferred from histologic studies ofthymic cytes as well as TE cells were characterized in indirect sections. Although it is generally accepted that epithelial cells immunofluorescence assays using monoclonal antibodies are the source of thymic hormones (37-39), the effects of (Fig. 5; Table 2). In the TE cell suspension, 85% ofcells were these hormones as well as other influences of epithelial cells TE-3' (cortical TE cell marker) (13), while 15% were A2B5' on maturing T cells are not well understood (reviewed in ref. (medullary and subcapsular cortical TE cell marker) (12, 34). 12). In this study, using methods developed for long-term OfTE cells bound by thymocytes, >90% were ofthe cortical culture ofTE cells and assays for TE-thymocyte binding, we TE phenotype (TE-3+) (Fig. 5A). Rare (1-3%) A2B5+ TE have established that human thymocytes bind to TE cells. cells rosetting with thymocytes were also seen (not shown). Thymocytes did not bind to epidermal keratinocytes or to Thymocytes in suspension were 56% T6' (immature cor- thymic fibroblasts but did bind to cells of the epidermoid tical thymocyte), 37% p80+ (A1G3+) (mature thymocyte), carcinoma cell line A431. 75% T4+, 65% T8+, and 95% 3A1+ (pan thymocyte). Fig. SB Thymocytes bound to both allogeneic and autologous TE shows a TE cell surrounded by thymocytes expressing the cells, and TE-thymocyte binding was not inhibited by anti- pan-thymocyte antigen 3A1. Using a panel of reagents that bodies to class I and class II MHC antigens. However, TE Downloaded by guest on October 2, 2021 Immunology: Singer et A Proc. Natl. Acad. Sci. USA 83 (1986) 6591 Table 1. Monoclonal antibodies to MHC antigens do not affect TE-thymocyte binding Percent TE cells Cells pretreated forming rosettes Exp. Antibody with antibody (mean ± SEM) 1 3F10 Thymocytes 69.2 ± 4.6 TE cells 72.8 ± 5.3 Both 74.5 + 4.9 Neither 68.0 ± 3.5 2 L243 Thymocytes 62.5 + 1.0 TE cells 56.0 ± 2.7 Both 57.5 ± 1.2 Neither 58.0 + 3.0

cells cultured in vitro and used in this study did not express 11 class II MHC antigens (11). In vivo, both cortical and medullary TE cells express class II antigens (12, 40-42). FIG. 5. Indirect immunofluorescence ofTE-thymocyte rosettes. Upon in vitro cultivation, TE cells cease expression of class (A) TE cell reactive with antibody TE-3. The TE cell is surrounded II antigens but can be induced to express TE cell surface class by thymocytes (arrows), which did not bind TE-3 antibody. (B) 3A1+ II antigens by treatment with y-interferon (ref. 43; Singer, thymocytes (arrow) bound to TE cells. (C) T6+ (NA1/34) thymo- K. H. et al. unpublished observations) or by cocultivation for cytes bound to TE cells. (D) T3+ (T3/RW2-8C8) thymocytes bound to TE cells. (x200.) 72 hr with autologous or allogeneic thymocytes (44). The effect of MHC class II antibodies on thymocyte binding to y-interferon-treated TE cells has yet to be determined. In our thymocyte activation. Thus, it is likely that at least one present system, binding of TE cells to thymocytes is not function (thymocyte activation) of the epithelial component mediated by class I or class II antigens. In this regard, we of thymus is mediated by direct thymocyte-TE cell contact. have recently established that TE-thymocyte binding is Since TE-thymocyte binding is not mediated via MHC inhibited >90% by antibodies to the lymphocyte function- molecules (at least in our present system), it is possible that associated LFA-3 and LFA-2 (CD2, Til) antigens (45). With MHC restriction is not mediated via cortical (TE-3+) TE regard to the specificity of TE-thymocyte binding, cells. Alternatively, other epithelial cell types (such as TE-4+ thymocytes, peripheral blood T cells, and tonsillar T cells medullary epithelial cells) or thymic macrophages may be bound equally well to TE cells. Thus, it is likely that the important in this regard. TE-thymocyte binding we report here is not related to We have shown that keratin-containing TE cells bind homing of T-cell precursors to the thymus but rather repre- human thymocytes, and we have described an in vitro system sents an early step in thymocyte activation. In support ofthis whereby the molecular and cellular events that are sequelae notion, we have recently shown that following binding to of TE-thymocyte binding (i.e., activation and progressive thymocytes, human TE cells act as accessory cells for T-cell maturation) can be studied in vitro. This assay system, phytohemagglutinin-induced thymocyte activation (44), and coupled with the ability to characterize phenotypically and to that TE cells produce interleukin 1 (46). separate subsets ofboth T cells and TE cells, should allow the Of the thymocytes bound to TE cells in vitro, 48% were dissection of the roles TE cells play in promotion of various T3+ and 65% were T6+ (Table 1). These data suggest that at stages of human thymocyte maturation. least 13% ofthymocytes that bound TE cells coexpressed T3 We thank Drs. R. F. Todd III, T.-T. Sun, E. L. Reinherz, J. A. and T6 antigens and likely represent recently matured Hansen, P. J. Martin, and T. A. Springer for monoclonal reagents thymocytes located in situ in the thymic cortex (47). and Ms. Joyce Lowery and Ms. Kim McClammy for expert secre- In contrast to the nonspecificity of the type of T cells that tarial assistance. This work was supported by Grants AM34808, bind TE cells, epithelial cells from peripheral microenviron- CA28936, K0400695, and T32 CA09058 from the National Institutes ments such as skin and cornea (data not shown) did not bind of Health. K.H.S. is a Scholar of the Leukemia Society of America. thymocytes or peripheral blood T cells. These observations 1. Mandel, T. (1970) Z. Zellforsch. Mikrosk. Anat. 106, 498-515. suggest that T cells that pass through the thymic microenvi- 2. Hwang, W. S., Ho, T. Y., Luk, S. G. & Simmon, G. T. (1974) ronment are exposed to receptors specifically present on TE Lab. Invest. 31, 473-487. cells that, upon binding to thymocyte CD-2 antigens, result in 3. Cantor, H. & Weissman, I. (1976) Prog. Allergy 20, 1-64.

Table 2. Phenotypic characterization of thymocytes bound to human TE cells Percent of rosetted Thymocyte surface antigen and Monoclonal thymocytes positive distribution in thymus antibody (mean ± SEM)* CD-7 (3A1), pan-thymocyte 3A1 97 ± 1 CD-4 (T4), inner cortical thymocytes and subset of mature medullary thymocytes T4/19- THYSD7 93 ± 2 CD-8 (T8), inner cortical thymocytes and subset of mature medullary thymocytes T8/2T8-1B5 79 ± 7 CD-1 (T6), inner cortical thymocytes NA1/34 65 ± 9 CD-3 (T3), mature medullary thymocytes and foci of inner cortical thymocytes T3/RW2-8C8 48 ± 8 CD-2 (T11, LFA-2), pan-thymocyte 35.1, TS1/8.1.1 96 ± 1 *From 2-5 experiments. Downloaded by guest on October 2, 2021 6592 Immunology: Singer et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4. Stutman, 0. (1978) Immunol. Rev. 42, 138-184. 29. Krensky, A. M., Sanchez-Madrid, F., Robbins, E., Nagy, 5. Zinkernagel, R. M., Callahan, A., Althage, A., Cooper, S., J. A., Springer, T. A. & Burakoff, S. J. (1983) J. Immunol. Klein, P. & Klein, J. (1978) J. Exp. Med. 147, 882-8%. 131, 611-616. 6. Fink, P. J. & Bevan, M. J. (1979) J. Exp. Med. 148, 766-775. 30. Reinherz, E. L., Meuer, S. C., Fitzgerald, K. A., Hussey, 7. Wekerle, H. & Ketelson, V. P. (1980) Nature (London) 283, R. E., Levine, H. & Schlossman, S. F. (1982) Cell 30, 402-404. 735-743. 8. Kyewski, B. A., Rouse, R. B. & Kaplan, H. S. (1982) Proc. 31. Reinherz, E. L., Morimoto, C., Fitzgerald, K. A., Hussey, Natl. Acad. Sci. USA 79, 5646-5650. R. E., Daley, J. A. & Schlossman, S. F. (1982) J. Immunol. 9. El Rouby, S., Praz, F., Halbwachs-Mecarelli, L. & Papiernik, 128, 463-468. M. (1985) J. Immunol. 134, 3625-3631. 32. Haynes, B. F., Mann, D. L., Hemler, M. E., Schroer, J. A., 10. Farr, A. G., Anderson, S. K., Marrack, P. & Kappler, J. Shelhamer, J. A., Eisenbarth, G. S., Thomas, C. A., (1985) Cell 43, 543-550. Mostowski, H. S., Strominger, J. L. & Fauci, A. S. (1980) 11. Singer, K. H., Harden, E. A., Robertson, A. L., Lobach, Proc. Natl. Acad. Sci. USA 77, 2914-2918. D. F. & Haynes, B. F. (1985) Hum. Immunol. 13, 161-176. 33. Haynes, B. F. (1986) in Leukocyte Typing I1, ed. Reinherz, 12. Haynes, B. F. (1984) Adv. Immunol. 36, 87-142. E. L., Haynes, B. F., Nadler, L. M. & Bernstein, I. D. 13. Haynes, B. F., Scearce, R. M., Lobach, D. F. & Hensley, (Springer, New York), Vol. I, pp. 3-30. L. L. (1984) J. Exp. Med. 159, 1149-1168. 34. Haynes, B. F., Shimizu, K. & Eisenbarth, G. (1983) J. Clin. 14. McFarland, E. J., Scearce, R. M. & Haynes, B. F. (1984) J. Invest. 71, 9-14. Immunol. 133, 1241-1249. 35. Itoh, T. (1979) Am. J. Anat. 156, 99-104. 15. Lobach, D. F., Scearce, R. M. & Haynes, B. F. (1985) J. 36. Lobach, D. F., Itoh, T., Singer, K. H. & Haynes, B. F. (1986) Immunol. 134, 250-257. Clin. Res. 34, 500A (abstr.). 16. Haynes, B. F., Harden, E. A., Telen, M. J., Hemler, M. E., 37. Dardenne, M., Savino, W., Gastinel, L. & Bach, J. F. (1984) in Strominger, J. L., Palker, T. J., Scearce, R. M. & Eisenbarth, Thymic Hormones and Lymphokines, ed. Goldstein, A. L. G. S. (1983) J. Immunol. 131, 1195-1200. (Plenum, New York), pp. 37-42. 17. 'Tseng, S. C. G., Jarvinen, M. J., Nelson, W. G., Huang, 38. Low, T. K. & Goldstein, A. L. (1984) in Thymic Hormones J.-W., Woodcock-Mitchell, J. & Sun, T.-T. (1982) Cell 30, and Lymphokines, ed. Goldstein, A. L. (Plenum, New York), 361-372. pp. 21-35. 18. Todd, R. F., Nadler, L. M. & Schlossman, S. F. (1981) J. 39. Goldstein, G. & Audhya, T. K. (1985) Surv. Immunol. Res. 4, Immunol. 126, 1435-1442. Suppl. 1, 1-10. 19. Dimitriu-Bona, A., Burmester, G. R., Waters, S. J. & Win- 40. Bhan, A. K., Reinherz, E. L., Poppema, S., McCluskey, chester, R. J. (1983) J. Immunol. 130, 145-152. R. T. & Schlossman, S. F. (1980) J. Exp. Med. 152, 771-782. 20. Lampson, L. A. & Levy, R. (1980) J. Immunol. 125, 293-299. 41. Scollay, R., Jacobs, S., Jeraber, L., Butcher, E. & Weissman, 21. Fabricant, R. N., DeLarco, J. E. & Todaro, G. J. (1977) Proc. I. (1980) J. Immunol. 124, 2845-2853. Natl. Acad. Sci. USA 74, 565-569. 42. Janossy, G., Thomas, J., Bollum, F., Granger, S., Pizzalo, G., 22. Hashimoto, K., Shafran, K. M., Webber, P. S., Lazarus, Bradstock, K., Wong, L., McMichael, A., Ganeshaguru, K. & G. S. & Singer, K. H. (1983) J. Exp. Med. 157, 259-272. Hofbrane, A. V. (1980) J. Immunol. 125, 202-212. 23. Butcher, E. C., Scollay, R. G. & Weissman, I. L. (1979) J. 43. Berrih, S., Arenzana-Seisdedos, F., Cohen, S., Devos, R., Immunol. 123, 1996-2003. Charron, D. & Virelizler, J.-L. (1985) J. Immunol. 135, 24. Miller, S. E. (1985) in Electron Microscopy, ed. Jones, B. R. 1165-1171. (Library Research Association, Monroe, NY), pp. 293-316. 44. Denning, S. M., Tuck, D. T., Singer, K. H. & Haynes, B. F. 25. Jondal, M., Holm, G. & Wigzell, H. (1972) J. Exp. Med. 136, (1986) Clin. Res. 34, 669A (abstr.). 207-215. 45. Wolf, L. S., Tuck, D. T., Springer, T. A., Haynes, B. F. & 26. Haynes, B. F., Hensley, L. L. & Jegasothy, B. V. (1982) J. Singer, K. H. (1986) Clin. Res. 34, 674A (abstr.). Invest. Dermatol. 78, 323-326. 46. Le, P. T., Tuck, D. T., Dinarello, C. A., Haynes, B. F. & 27. McMichael, A. J., Pilch, J. R., Galbre, G., Mason, D. Y., Singer, K. H. (1986) Clin. Res. 34, 499A (abstr.). Fabre, J. W. & Milstein, C. (1979) Eur. J. Immunol. 9, 47. Umiel, T., Daley, J. F., Bhan, A. K., Levy, R. H., Schloss- 205-210. man, S. F. & Reinherz, E. L. (1982) J. Immunol. 129, 28. Martin, P. J., Longton, G., Ledbetter, J. A., Newman, W., 1054-1062. Braun, M. P., Beatty, P. A. & Hansen, J. A. (1983) J. Immu- 48. Haynes, B. F., Reisner, E. G., Hemler, M. E., Strominger, nol. 131, 180-185. J. L. & Eisenbarth, G. S. (1982) Human Immunol. 4, 273-285. Downloaded by guest on October 2, 2021