Expression of antigen-specific, major histocompatibility complex-restricted receptors by cortical and medullary thymocytes in situ

Cell, Vol. 43, 543-550,

December

1985 (Part

l), Copyright

0 1985 by MIT

0092-8674/85/l

20543-08

$02.0010

Expression of Antigen-Specific, Major Histocompatibility Complex-Restricted Receptors by Cortical and Medullary Thymocytes In Situ Andrew G. Farr: Susan K. Anderson: Philippa Marrack,t and John Kapplert * Department of Biological Structure SM-20 University of Washington Seattle, Washington 98195 t Department of Medicine National Jewish Hospital and Research Center Denver, Colorado 80206

Summary We have examined the distribution of the antigenspecific, major histocompatibility complex-restricted receptor on mouse thymocytes in situ, using immunohistochemical techniques and the monoclonal antibody KJ16-133. This antibody reacts with the beta chain of the receptors on about 20% of peripheral murine T cells. Of the cortical thymocytes reacting with KJ16-133, cells with only cytoplasmic staining were most frequently observed. Such cytoplasmic staining was not observed in the medulla. Occasional cortical cells had low levels of surface expression, which was almost invariably patched in the region of contact with epithelial cell processes. KJ16-133+ medullary thymocytes had high levels of uniform surface labeling. These results suggest that thymic selection of MHC restriction and/or tolerance may occur in the cortex, where the receptors on maturing thymocytes interact with MHC proteins on epithelial cells. Introduction The T cell antigen receptor has many features similar to the B cell antigen receptor (immunoglobulin). Structurally, the T cell antigen receptor is composed of two glycosylated polypeptide chains (termed alpha and beta) linked by disulfide bonds (Allison et al., 1982; Haskins et al., 1983; Kappler et al., 1983; McIntyre and Allison, 1983; Meuer et al., 1983). At the gene level, the beta chain gene resembles that of immunoglobulin heavy chain, in that it results from rearrangement of variable, diversity, joining, and constant gene segments (Yanagi et al., 1984; Hedrick et al., 1984; Siu et al., 1984; Kavaler et al., 1984; Gascoigne et al., 1984; Malissen et al., 1984). Accumulating data indicate that the alpha gene has a similar organization (Chien et al., 1984; Saito et al., 1984; Sim et al., 1984). Although there are similarities between the T cell antigen receptor and immunoglobulin in terms of gene organization and protein structure, there is at least one important feature of the T cell antigen receptor that is significantly different from immunoglobulin. lmmunoglobulin can bind free antigen, but antigen recognition by T cells occurs only in the context of major histocompatibility complex (MHC) gene products (Zinkernagel and Doherty, 1975; Bevan, 1975). In the case of helper/amplifier T cells, antigen recognition requires the participation of accessory cells bearing syngeneic or “self” Class II antigens (in the mouse, la

antigens) which serve an antigen presentation function (Kappler and Marrack, 1976; Sprent, 1978; Waldmann et al., 1978). Acquisition of this MHC-linked recognition of antigen by T cells has been thought to occur during the thymic phase of T cell differentiation, since studies with bone marrow chimeras (lethally irradiated mice reconstituted with semisyngeneic bone marrow) showed that the thymus determines which major histocompatibility antigens will be recognized as “self” (Bevan, 1977; Zinkernagel et al., 1978). The cellular process whereby restricted MHC antigen recognition is generated within the thymus is not understood. Recognition of MHC antigens by thymocytes has been thought to play an important role in the thymic phase of T cell differentiation, serving as a proliferative stimulus to expand clonally thymocytes with receptors for the expressed MHC antigens (Jerne, 1971; Zinkernagel et al., 1978). Consistent with this hypothesis is the observation that both epithelial cells and nonlymphoid bone-marrowderived cells within the thymus express la antigens (Hoffman-Fezer et al., 1978; Rouse et al., 1979; van Ewijk et al., 1980; Farr and Nakane, 1983). Rock and Benacerraf (1984a, 1984b) have shown that syngeneic bone-marrowderived accessory cells bearing la antigens can stimulate proliferation of both cortical and medullary thymocytes in vitro. Attempts to define the capacity of thymic epithelial cells to similarly stimulate thymocyte proliferation in vitro have been frustrated in part by difficulty in generating rigorously defined thymic epithelial cultures (Jones and St. Pierre, 1981; Papiernik and Nabarra, 1981) and the observation that thymic epithelium cells maintained in culture no longer express la antigens (Sun et al., 1984). In models of T cell differentiation that invoke receptor-ligand interaction as a proliferation/differentiation signal, expression of receptor structures by immature thymocytes would be a necessary event. Consistent with this prediction, Roehm et al. (1984) have utilized a monoclonal antibody (KJ16-133) that recognizes a determinant on the beta chain of the T cell antigen receptor (Epstein et al., 1985; Roehm et al., 1985) to identify a population of immature thymocytes expressing cell surface antigen receptors. Here we have used the same monoclonal antibody in immunohistochemical studies at the light and electron microscopic levels to identify thymocytes within the thymus that express antigen receptors, to characterize the expression of antigen receptors by these cells, and to define the spatial relationship of receptor-bearing thymocytes with stromal cells within the thymus. Emphasis was placed on the cortical thymic environment because this area contains the majority of immature T cells, is the site of extensive thymocyte proliferation, and may be the site where thymic selection of T cell repertoire occurs. Results Distribution of Cells Bearing KJlG-133~Reactive Antigen Receptor Molecules The distribution of cells bearing antigen receptors

in thy-

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Figure

1. Distribution

of Thymocytes

Bearing

KJ16-133’

Antigen

*

.

Receptors

(a) Cross-section of a C3H thymus demonstrating KJ16-133’ cells in both the cortex and medulla. The pericapsular staining in the lower right corner of the micrograph (arrow) is due to avidin-peroxidase binding to associated fat cells (Farr and Anderson, 1985). C, cortex; M, medulla. (b) KJ16-133 cells in the thymic medulla. Note the clusters of some of the reactive cells (arrows). Endogenous peroxidase activity of eosinophils is indicated by arrowhead. (c) KJ16-133’ cells in the thymic cortex. Clusters of cells are less numerous than in the medulla and contain fewer cells (arrows). C. capsule. (d) Section of SWR thymus treated identically to C3H thymus shown in (a). Reaction product is associated with eosinophils in the medulla and faint background staining throughout the tissue. (e) C3H thymus tissue reacted with avidin-peroxidase conjugates without exposure to biotinylated KJ16-133. As in (d) faint background staining and peroxidase activity of eosinophils are observed. Bar = 100 pm (a, d, e); 10 ,um (b and c).

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mus tissue of a 4 week old C3HIHeJ mouse is shown in Figure la. Cells reacting with the antibody were located in both cortical and medullary areas. In the medullary area, the majority of the positive cells reacted strongly, while in cortical areas the labeling was more heterogeneous and contained both lightly and heavily labeled cells. In the medulla, clusters of KJ16-133’ cells were quite apparent and contained up to seven or eight cells (Figure lb), while in the cortex clusters were usually restricted to two or three cells (Figure lc). Thymus tissue from some of the other strains of mice that express the antigenic determinant recognized by KJ16-133 (CBA/J, BALB/c, and C57Bl16J) displayed a staining pattern indistinguishable from that described here for C3HIHeJ mice (data not shown). This was contrasted by thymus tissue from SWR mice, which does not express the KJ16-133 reactive determinant (Haskins et al., 1984; Roehm et al., 1985) and showed only faint background staining in the tissue (Figure Id) comparable to the staining observed when avidinperoxidase was applied to the tissue without prior treatment with biotinylated KJ16-133 (Figure le). Also present in these control sections was the endogenous peroxidase activity associated with eosinophils located in the thymus medulla of all thymuses examined (data not shown; see Farr and Nakane, 1983). Ultrastructural Localization of KJlG-133~Reactive Antigen Receptor Molecules Although light microscopic immunohistochemistry could characterize the distribution of cells bearing antigen receptors recognized by KJ16-133, this approach lacked sufficient resolution to define the cellular distribution of these molecules. Consequently, we employed immunoperoxidase techniques to define the distribution of KJ16-133 labeling at the ultrastructural level. In the cortex the majority of cells were not labeled with KJ16-133 antibodies. Those that did react exhibited three patterns of staining: cytoplasmic only, simultaneous cytoplasmic and surface, and surface only. In cells expressing cytoplasmic staining only, the intracellular reaction product was restricted to the cisternae of rough endoplasmic reticulum surrounding the nucleus (referred to hereafter as the perinuclear envelope; Figure 2a). The extent of perinuclear staining was variable. In some instances, the entire perinuclear envelope contained reaction product, while in others the staining was limited to a few areas. Although profiles of solitary cells with perinuclear staining were observed, it was also common to find these cells very close or adjacent to other intracellular-positive cells (Figure 2b). The second type of KJ16-133 reactive thymocyte in the cortex exhibited both surface and perinuclear staining and was only rarely observed (Figure 2~). The staining was patchy, with large areas of the cell surface and perinuclear space devoid of label. Finally, the third type of KJ16-133 reactive cortical thymocyte exhibited surface staining only (Figures 2d and 2e) and was observed with frequencies intermediate to the other two types. There were two striking features of the staining pattern exhibited by cortical thymocytes bearing

cell-surface antigen receptors reacting with KJ16-133. First, these cells were almost always associated with processes of thymic epithelial cells. Second, the expression of antigen receptors by these cells appeared restricted to areas of thymocyte membrane in contact with epithelial cells or their processes. The average length of labeled thymocyte perimeter in contact with thymic epithelium was 80% of the total labeled thymocyte perimeter (n = 29). This value was significantly greater than the percentage of labeled thymocyte perimeter expected to be in contact with epithelium if the receptor labeling was random on the thymocyte perimeter (8%; P < 0.0005). In other samples of thymus tissue, it was shown that these epithelial cells in contact with cortical thymocytes express la antigens (Figure 2f). Reaction product was also observed on the surface of epithelial cells adjacent to KJ16-133’ cells, but this has been considered to be a diffusion artifact (Novikoff et al., 1972). Occasionally epithelial cells displayed intracellular vacuoles which contained peroxidase reaction product (Figure 2d). Because of the endogenous peroxidase activity sometimes associated with these structures, it was not possible to determine the basis for this intracellular epithelial staining. Medullary Distribution of Antigen Receptors Reacting with KJ16-133 In contrast to the cortical thymic environment, which contained lymphocytes with several different patterns of antigen receptor expression, the medullary region of the thymus contained one type of KJ16-133+ cell that exhibited cell surface labeling without intracellular labeling (Figure 2g), a pattern similar to that seen on peripheral T cells (Farr and Anderson, unpublished observations). The reaction product associated with medullary cells was often heavier and more intense than that seen on cortical cells (compare Figures 2d and 29). In contrast to the distribution of receptors on cortical thymocytes, medullary cells in contact with epithelial cells or cells resembling dendritic cells or macrophages did not exhibit patchy distribution of receptors on their cell surface (Figures 2g and 2h). As in the cortex, KJ16-133’ cells in the medulla were observed both as solitary profiles and in clusters of positive cells. Controls for the ultrastructural studies were the same as those used for the light microscopy studies, i.e., treating SWR thymus tissue with biotinylated KJ16-133 antibodies followed by avidin-peroxidase conjugates, omitting the biotinylated antibody during processing of C3H thymus tissue, and the demonstration of endogenous peroxidase activity in the thymus. The results of ultrastructural controls were identical to those obtained at the light microscopic level and are not shown here. Discussion One of the most intriguing aspects of T cell differentiation is the phenomenon of MHC restriction. The current view of T cell differentiation in the thymus is that immunoincompetent cells committed to the T cell lineage enter the thymus cortex where they expand clonally and express

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Figure

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(a) Cortical thymocyte with an intracellular labeling pattern. Reaction product is present in the perinuclear envelope (arrows); the cell surface (arrowheads) is not labeled. (b) A cluster of three cortical thymocytes with cytoplasmic but no surface staining (asterisks). (c)A cortical thymocyte expressing both perinuclear (arrows) and surface (arrowheads) staining. Surface staining is punctate. (d) A cortical thymocyte with surface labeling (arrows) but devoid of intracellular reaction product. Note that the reaction product is predominantly associated with the lymphocyte membrane in contact with an epithelial cell process (E). Adjacent lymphocytes are not labeled. The epithelial nature of the cell process is indicated by the presence of

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receptors for antigen/MHC. During the maturation of these cells, a dramatic selection takes place, such that only those T cell clones with receptors capable of recognizing antigens in combination with the allelic forms of the MHC proteins expressed by the thymic epithelial cells can mature fully (Bevan, 1977; Zinkernagel et al., 1978). These cells progress to the thymic medulla and/or exit the thymus to the periphery as functional T cells, whose antigen specificity is now said to be “restricted” by self-MHC proteins. In addition, there is evidence that in the course of differentiation in the thymus, T cells are eliminated that would be capable of responding in the periphery directly to self/MHC proteins in the absence of antigen (Jordan et al., 1985). The molecular basis of either restriction or tolerance to self-MHC is not known, but it is reasonable to hypothesize that both require a direct interaction between the newly expressed T cell receptor and the MHC proteins expressed within the thymic cortex. In order to understand the role of the antigen/MHC receptor in these selection events it is important to know the details of receptor expression by thymocytes. This study was the first to examine the in situ expression of these receptors by T cells within the thymus, and was accomplished with immunohistochemistry utilizing the monoclonal antibody KJ18-133. This antibody detects an allelic determinant expressed on the beta chain of the receptor of about 20% of peripheral T cells in positive strains (Haskins et al., 1984; Epstein et al., 1985; Roehm et al., 1985). Our major findings on the expression of this determinant were: surface expression was most frequent, uniform, and at highest levels on medullary thymocytes; surface expression was infrequently detected on cortical thymocytes and was nearly always patched on that part of the thymocyte surface in contact with epithelial cell processes; in cortical thymocytes, cytoplasmic expression was detected more frequently than surface staining, and was only rarely seen with simultaneous surface expression. Although the developmental relationship between different populations of thymocytes expressing the KJ16-133 determinant cannot be established on the basis of these morphologic studies, the pattern of antigen receptor expression would be consistent with initial cytoplasmic accumulation in cortical thymocytes, followed by a brief simultaneous cytoplasmic and low surface expression, then low level of surface expression alone, and finally, high level surface expression concommitant with maturation and peripheralization. Such a scheme would have a number of obvious parallels to B cell immunoglobulin expression during ontogeny. lmmunocytochemical localiza-

tion of immunoglobulin in developing B cells has demonstrated the same pattern of expression, i.e., some cells with cytoplasmic only, some with surface only, and some with both (Raff et al., 1976; Cooper et al., 1977). Furthermore, the earliest identifiable B cell progenitor, the pre-B cell, expresses cytoplasmic mu chain only. Only at a later stage, when light chain genes are expressed, does cytoplasmic mu chain decline with simultaneous expression of a complete surface immunoglobulin (Levitt and Cooper, 1980; Siden et al., 1981). The biochemical nature of the cytoplasmic KJ16-133 binding material associated with cortical thymocytes is not known. Its perinuclear localization was distinctive and similar to that described for immunoglobulins in plasmablasts (Leduc et al., 1968) Class I MHC products in mitogen activated spleen cells (Farr et al., 1980), and Class II MHC products in thymic epithelial cells (Farr and Nakane, 1983). Since this perinuclear envelope is contiguous with the rough endoplasmic reticulum and contains ribosomes on the cytoplasmic face, these staining profiles have been considered to reflect newly synthesized protein. The analogy to B cells might suggest that the material represents an accumulation of beta chain in those thymocytes that have not as yet rearranged and expressed their alpha chain complex. The available data on expression of the alpha and beta complexes in developing thymocytes is consistent with the expression of the alpha complex after the beta complex (Raulet et al., 1985; Born et al., 1985; Snodgrass et al., 1985). Arguing against the possibility of free cytoplasmic beta chain is the finding that, although KJ16-133 binding survives reduction and alkylation of the receptor, reactivity is lost if the alpha and beta chains are separated (Haskins et al., 1984). However, we can not rule out the possibility that the denaturing conditions used to separate alpha and beta chains irreversibly destroy the KJ16-133 determinant or that free beta chain synthesized in situ would remain reactive with KJ16-133. Rather than free beta chain, an intriguing alternative would be that the cytoplasmic material is for the most part made up of complete alpha/beta receptors whose surface expression awaits the expression of one of the proteins that make up the T3 complex, since a tight and perhaps obligatory association between T3 and the receptor on the cell surface has been demonstrated (Meuer et al., 1983; Weiss and Stobo, 1984). The patchy or discontinuous distribution of KJ16-133 reactive receptors on cortical thymocytes was in contrast to the continuous pattern of receptor expression displayed by medullary thymocytes. The basis for these differences is not known, but the observation that the cortical recep-

tonofilaments (arrowhead). The epithelial cell also contains a vacuole that exhibits peroxidase activity (asterisk). (e) Cortical thymocytes expressing surface and no cytoplasmic KJS133 reactive antigen receptors. As in (d), note that antigen receptor localization (arrows) is primarily at the area of contact between the lymphocyte and adjacent epithelial cell process(E). (f) Localization of la antigens associated with an epithelial cell process in the thymus cortex. la antigens are uniformly expressed on the epithelial cell surface. (g) Expression of KJ16-133 reactive antigen receptors by a medullary thymocyte. Reaction product appears evenly distributed over the thymocyte cell surface, even though in contact with a thymic epithelial cell (E). (h) Expression of KJ16-133 reactive antigen receptors by a medullary thymocyte. Reaction product appears evenly distributed over the thymocyte cell surface, even though in contact with both an epithelial cell (E) and a dendritic cell (DC). The epithelial cell is identified as such by the presence of a desmosome (arrow), while the dendritic cell is identified on the basis of electron lucent cytoplasm, euchromatic nucleus, and distinctive cellular processes (arrowheads). Bar = 1 pm.

Cell 548

tors were almost always patched in the region of the surface adjacent to an epithelial cell process makes it tempting to suggest that we have visualized in situ either the process of selection of MHC restriction, or perhaps tolerance induction caused by the interaction of the receptor and the MHC proteins expressed on the epithelial cell surface. With regard to Class II MHC proteins, there is some additional support of these notions. As shown here and previously (Hoffman-Fezer et al., 1978; Rouse et al., 1979; Farr and Nakane, 1983) these cortical epithelial cells are rich in la antigens and the experiments of Sharrow et al. (1981) utilizing radiation chimeras have shown that donor thymocytes can express recipient la antigens bound on their cell surface. It is worth noting that despite an extensive search, we did not observe any patching of receptor in areas of contact between thymocytes and the two other types of cells expressing Class II MHC antigens in the thymus: macrophages and dendritic cells. This is perhaps relevant to the controversy over the role of these other cell types in selecting MHC restriction (Long0 and Schwartz, 1980). A patching of the T cell receptor caused by interaction with epithelial MHC antigens could be an activating signal similar to that delivered to mature T cells when receptor is cross-linked by immobilized antireceptor antibody (Kappler et al., 1983). The failure to see thymocyte/epithelial interactions in the medulla may reflect the maturational state of the thymocytes or differences between cortical and medullary epithelial cells (Haynes et al., 1983; Van Vliet et al., 1984; Kingston et al., 1984; Farr and Anderson, 1985). KJ16-133 antibody was used in a previous study to analyze thymocyte subpopulations in suspension by flow cytometry (Roehm et al., 1984). This study also showed high level surface expression by mature medullary and peripheral T cells. Also in agreement with this study, immature cortical thymocytes displayed much lower levels of surface receptor. However, about 10% of the cortical thymocytes had detectable surface expression, a proportion higher than that observed in the present study. The most likely explanation for this difference is the sensitivity of the two staining procedures and the surface distribution of the receptor. We can estimate from the flow cytometry experiments that the cortical cells express on their surface only about 2000-5000 molecules of receptor/cell (lo%-20% of the values obtained with peripheral T cells). Thus, in a thin section, we could expect less than 25 molecules of receptor to be present on the cell surface contained within the volume of an ultrathin tissue section. It is unlikely that the peroxidase method used here could detect this number of molecules randomly distributed on the cell surface. Therefore, it is likely that we detected receptors on cortical cells only when they had patched to one area of the cell surface and the plane of the section happened to pass through this patch. Thus, our observations here probably represent an underestimate of the frequency of cortical surface expression and are skewed toward cells that have patched their receptors. Although previous flow cytometric experiments have documented the dramatic rise in the level of Surface receptor expression upon maturation of thymocytes, there

is no obvious explanation for this increase. The patching of antigen receptors on cortical cells to areas of contact with epithelial cells raises the possibility that this interaction may lead to shedding of the receptor by the thymocytes, resulting in low levels of surface expression by cortical thymocytes. Once this interaction ceases upon maturation, the receptor may rapidly accumulate to a much higher level on the cell surface. In both the cortex and the medulla, KJ16-133’cells often appeared in clusters of two to eight cells. We are currently adapting morphometric analysis to determine the statistical significance of these clusters. This observation raises several interesting points. First, the presence of such clusters implies local clonal expansion. The size of the cluster presumably reflects the rate of expansion and rate of movement of the progeny out of the local area. This expansion could result from specific cell interaction, as in the case of the observed cortical T cell/epithelial cell contact or might be hormonally driven, as in the case of cortical thymocytes without surface receptor expression. The observation of cell clusters in the medulla was particularly surprising, since there has been no previous suggestion of clonal expansion within the medulla. Although it is clear that a beta chain polymorphism contols KJ16-133 reactivity with the receptor, the region of the chain bearing the determinant has not been conclusively identified. Recent data, however, suggest that the determinant is encoded by a family of V beta genes present in BALBlc and absent in SJL mice (Sim and Augustin, 1985; D. Loh, personal communication; unpublished observations). There is no reason to suppose that the expression of these V beta genes in the thymus is not representative of all others. While these particular V beta gene segments are used by T cells of very many different specificities (Haskins et al., 1984) our conclusions about the generality of our results for all T cell receptors, not just those binding KJ16-133, are certainly constrained by this assumption. Experimental Procedures Mice

Male and female mice of CBA/J, C3H/HeJ, and SWR/J strains were purchased from Jackson Laboratories (Bar Harbor, Maine). BALBlc mice were purchased from Life Sciences (St. Petersburg, Florida). All mice were maintained in the vivarium of the Department of Biological Structure. Reagents The production and specificity of the monoclonal antibody KJ16-133 has been described previously (Haskins et al., 1964; Roehm et al., 1965). It labels approximately 20% of peripheral T cells and can immunoprecipitate a disulfide-linked heterodimer structure from T cells. This monoclonal antibody was purified from ascites fluid by affinity chromatography using goat anti-rat immunoglobulin antibodies coupled to Sepharose 48 (Pharmacia, Piscataway, New Jersey), eluted from the column with 2 M sodium thiocyanate, and then biotinylated according to the method of Bayer et al. (1979). Enzyme-labeled anti-la antibodies (A. TH anti-A. TL) used in this study have been described previously (Farr and Nakane, 1963). Peroxidase-conjugated avidin was prepared by the periodate/borohydride method of Wilson and Nakane (1978). Hydrogen peroxide (30%) was purchased from Mallinckrodt. Inc. (Paris, Kentucky). Except where noted, other reagents were obtained from Sigma Chemical Co. (St. Louis, Missouri).

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Staining Procedures The techniques used for immunohistochemistry have been described in detail previously (Farr and Nakane, 1981; 1983). Briefly, for light microscopic analysis, frozen sections of thymuses were mounted on gelatin coated slides and allowed to air dry overnight before fixation (10 min in acetone at -20%). After rinsing in phosphate buffered saline (0.02 M PO,, 0.9% NaCI, pH 7.4) (PBS), the sections were incubated for 1 hr at room temperature with biotinylated antibodies diluted in PBS containing 1% bovine serum albumin (PBSIBSA). In preliminary experiments, antibody concentrations of 5-10 fig/ml were found to provide maximal specific staining with low background. To visualize antibody binding, sections were washed three times with PBS and then incubated with the avidin-peroxidase conjugate. This conjugate was used at a concentration of 20 pg/ml. Following additional washes in PBS to remove unbound avidin-peroxidase, the sections were reacted to demonstrate peroxidase activity using 3.3’ diaminobenzidine as the electron donor (Graham and Karnovsky, 1966) dehydrated and mounted with coverslips. Thymus tissue for ultrastructural studies was obtained from mice fixed by vascular perfusion. While mice were under ether anesthesia, the thoracic cavity was opened and the heart exposed. A cannula was inserted into the left ventricle and, after cutting the right atrium, 10 ml of Hanks balanced salt solution (Gibco Laboratories, Grand Island, New York) was infused, followed by 50 ml of a mixed aldehyde fixative (Farr and Nakane, 1983). At the end of the perfusion, the thymus was removed, washed in PBS containing 5%~ sucrose (PBS/S), and then cut into 50 p thick sections with a Vibratome (Ted Pella, Tustin, California). Thymus sections were incubated overnight with biotinylated antibodies diluted in PBS/S, then washed three times with PBS/S before incubation with avidin-peroxidase (20 Fglml) for 6 hr at room temperature. At the end of this incubation, the sections were again washed three times with PBS/S, reacted to demonstrate peroxidase activity, and then processed for transmission electron microscopy. Ultrathin sections were examined and photographed without additional counterstaining. Quantitation of lmmunohistochemical Labeling The extent of surface labeling of cortical thymocytes and the relationship of that staining to areas of epithelial cell contact were quantitated on micrographs with the aid of a ZIDAS digitizing board (Carl Zeiss, Thornwood, New Jersey). Photographs included in this analysis fulfilled three requirements. First, sections through labeled thymocytes included the nucleus. Second, the epithelial nature of the adjacent cell could be clearly identified on the basis of morphologic features. Third, all micrographs were of the same final magnification, 13,800x. Using the digitizing board, the following measurements were made on micrographs: thymocyte perimeter, length of thymocyte perimeter that bore peroxidase reaction product, length of thymocyte perimeter in contact with epithelial cells or their processes, and length of thymocyte perimeter that was in contact with thymic epithelium and expressed peroxidase reaction product. Observed percentages of labeled thymocyte perimeter in contact with thymic epithelium was compared to the expected length if the distribution of reaction product was random. Statistical significance was determined with a Student’s t test. Acknowledgments We thank Dr. Neal Roehm for providing initial biotinylated KJ16-133, Mr. Mark Hartley for excellent photographic assistance, Drs. Edwin Boatman and John Prothero for assistance in digitizing and analyzing the resulting data, and Dr. Claire Robles for reviewing the manuscript. This work was supported in part by USPHS Grants AG-04360 and IA-18785 from the National Institutes of Health, University of Washington Graduate Research Funds (RR05432). Contract 225-AT-06-79EV 10270 from the Department of Energy, and by Grants IN-26Y and IM-49 from the American Cancer Society. This work was done while J. W. K. was supported by Faculty Research Award 216 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

July 8, 1985; revised

August

26, 1985

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