Expression of HLA antigens by human thymic epithelial cells

Expression of HLA antigens by human thymic epithelial cells

Expression of HLA Antigens by Human Thymic Epithelial Cells Robert V. Rouse, Peter Parham, F. Carl Grumet, and Irving L. Weissman ABSTRACT: Human thy...

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Expression of HLA Antigens by Human Thymic Epithelial Cells Robert V. Rouse, Peter Parham, F. Carl Grumet, and Irving L. Weissman

ABSTRACT: Human thymuses were examined by tissue section staining with antibodies s p ~ + fez monomorphic and polymorphic HLA-A. B. C. and DR determinants. The principal call (~+p* expressing high levels of HLA antigens has the distribution of epithelial cells, lmmuna~l, ctr+~ microscopy confirnted their epithelial nature. As in the mouse, both medullary and cortical epithelial cells express high levels of class II (DRJ antigens, a finding that is remarkable in t/m~ these antigens were originally thought to be restricted to lyntphoid and accessory cells. Class I ¢A. B, and C) antigens are also present on thymic epithelial cells. They are easily detectab[¢ +n medallary epithelial cells, but two distinct patterns of cortical staining were observed. One gr+u,~ of antibodies produced intense dendritic staining throughout the cortex: the other group pmducd only faint or no cortical dendritic staining at all. These different staining patterns do not corrddf, with known properties of the antibodies and thus appear to be due to intrinsic properties of t~" different A, B. and C antigens.

ABBREVIATIONS MHC major histocompatability gene complex HLA human MHC DPBS Dulbecco's phosphate-buffered saline, pH 7.2

lg PBS IEM H-2

immunoglobulin phosphate-buffered saline immunoelectron microscopy mouse MHC

I NTRODUCTIO N The thymus plays an important role in the differentiation and developmem o f T lymphocytes. Rare circulating thymic lymphocyte precursors enter the thymus and undergo an extensive proliferation and selection process produdng numbers of thymic emigrants already differentiated into functional subtypes [ 1,2]. The thymus is divided into a cortex and a medulla. The framework o f the cortex is an interconnecting network of long, thin dendritic processes of thymic e p i t h e ~ cells. T h e spaces between these processes are filled with large numbers of |ymphocytes. The framework of the medulla is also formed by epithelia] cells ~,h~:h range from ce~is with short dendritic processes to cohesive clusters o f flattened cells (Hassal's corpuscles). Since the spaces between medullary epithelial cell processes are smaller than in the cortex, lymphocytes are present there only in

From the Lal oratory of Experimental Ontology. Departmentof Pathologo,eRVR. FCG. ILW~ a ~ +tk~ Department of Structural Biology (PPL Stanford University Schoolof Medicine. Stanf6rd. Califer~. Address requestsfor reprints to Dr. Robert V Rouse. Department of Patho/ogr. Stanford Uni~.,~+it~ Schoolof Medicine. Stanford. CA 94305. ReceiredNorember2. 1981; acceptedFebruary 19. 1982. I+lum~-i Immunology 5.21-34 ( ! 982) ~ El~'v~r Sch:nce Publishing Co., Inc., 1982

52 VanderbiltAve.,New York,NY 10017

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R.V. Rouse et al. small clusters or individually. Macrophages and interdigitating reticular cells are present in the medulla and deep cortex [3,4]. In general, peripheral T cells functionally recognize foreign antigens in the context of MHC * self-antigens on target or accessory cell surfaces, resulting in genetic restriction of the immune response [5,6]. In the mouse, it has been proposed that this requirement (and ability) o f T cells as a population to recognize MHC self-antigens is genetically determined not by the thymocytes themselves but by radioresistant elements of the thymus [7]. In previous studies on the mouse thymus we found an unusual distribution of MHC antigens on thymic epithelial cells and proposed that these MHC-bearing epithelial cells may be instrumental in the acquisition of MHC restriction by T lymphocytes [8]. With the development of heterologous and monoclonal antibodies to human MHC antigens of both class I (HLA*-A, B, and C composed of a 45,000-dalton chain complexed to ~O2-microglobulin) and class II (DR or la composed of 28,000- and 33,000-dalton chains), we have studhed normal human thymuses by tissue section immunologic staining and IEM* to determine if a distribution of MHC antigens similar to that seen in mice exists in the human thymus. In this communication we demonstrate that the major cells in the thymus that express high levels of MHC antigens have the pattern and distribution of epithelial cells. IEM using monoclonal anti-c~ass I and II MHC antibodies confirms that these antigen-bearing cells are epithelial in nature. Occasional macrophagcs (nondendritic) also are positive by IEM with the same antibodies. As in the mouse, it is remarkable that cells that are clearly epithelial in derivation and structure expres~ high levels of class Il MHC antigens, antigens originally thought to be restricted to lymphoid and accessory cells. In addition, there is a marked difference in cortical epithelial staining patterns produced by different groups of antibodies directed against both monomorphic and polymorphic determinants of the class i MHC molecule.

METHODS Tissue. Small portions of tissue were obtained from thymuses incidentally removed during cardiac surgery and from tonsils removed surgically. The ages of the patients ranged from 3 months to 5 years, with one 19 years of ~ge. The tissue was immediately frozen for light microscopy. Small (1 mm square) fragments of fresh thymus were prepared for IEM by successive immersion in fresh 0.1 or 0.2'~/~ glutaraldehyde in DPB$* (overnightL 7% sncrme-DPBS (3 hr), 15% sucrose-DPBS (3 hr), and 25% sucrose-10~ glycerol-DPBS (overnight) and then frozen. HLA A, B, and C typing. Ckrated whole blood was centrifuged over HypaqueFicoll layers to isolate peripheral blood iymphocytes for HLA t/ping by microlymphocytotoxicity as previously described [9]. Alloantisera for typing all full and workshop specificities except Aw43 and CwS--Cw8 were obtained from the authors' own laboratories, from the N I H Serum Bank, and by exchange with other investigators. Antibodies. The following antibodies were used for staining of tissue: Anti-H: a rabbit antibody that reacts with free class I HLA heavy chains of all haplotypes but not with surface HLA compiexed with ~2-microglobulin [ 10] (kindly supplied by J. Strominger, Harvard University).

HLA Antigens on Human Thymic Epithelial Cells


Anti-p23,30: a rabbit antibody that reacts with the invariant portion of tb~ ~ II MHC molecule [ 11] (kindly supplied by J. Strominger, Harvard University ~.

Rabbit anti [32-microglobulin: a rabbit antiserum that reacts with/3,-m~crCgk~ulir~ [12] (kindly supplied by J. Strominger, Harvard University).

Human alloantisera: locally screened sera from previously pregnant bkx~d dormers, which had been selected for reactivity with specific HLA-A, B, or C p~fiymorphic determinants.

L203: a monoclonal mouse antibody that reacts with a monomorphic determinml~ of the human class II MHC molecule [13] (kindly supplied by L I . a m ~ o University of Pennsylvania, and R. Levy, Stanford University).

Monoclonalanti-class I HLA antibodies: monoclonal mouse antibodies reactive with monomorphic and polymorphic determinants of the class I HLA molecule are listed in Tables 1 and 2. Monomorphic antibodies are those that appear, within the sensitivity of quantitative cellular radiobinding assays, to react with all A, B, and C gene products with the same affinity [22]. Any antibodies that devi~e from this definition are classified as polymorphic. The validity of this distincti~m has been confirmed by analysis of cross-reactivity patterns of all these an¢i~ bodies in other species. Monomorphic antibodies retain this property in ~1 species, whereas polymorphic antibodies are invariably polymorphic in cros~ reactive species [23].

Second-stage reagents: Fluorescent rabbit anti-mouse lg* (prepared in our Laboratories), fluorescent sheep anti-rabbit Ig (Institute Pasteur), peroxidase rabbi~ anti-mouse Ig (Dako), peroxidase swine anti-rabbit Ig (Dako), and goat an~ihuman K light chain with peroxidase rabbit anti-goat Ig (both gifts from R. Warnke, Stanford University).

Light microscopic staining. Six-micrometer frozen sections were fixed I0 rain in acetone at 4°C and stored in a dessicator at - 20°C. Sections were rinsed in PBS ~ for 5 rain, incubated with the first-stage antibody 10 min, washed with PBS for 5 rain, incubated with the second-stage antibody for 10 min, and washed with PBS for 5 min. If stained with immunoperoxidase-labeled antibodies, the sections were then incubated with 0.1% diaminobenzidine in 0.3% H20_, in PBS° washed in PBS for 15 rain, incubated with 0.5 M copper sulfate in 0.9f¢~NaCl for 5 rain, washed and dehydrated in graded ethanols and xylene, and mounted. All staining was performed at room temperature. Slides stained with fluorescent reagents mounted in 9:1 glycerol-Tris buffer. All antibodies were titered from maximal staining. lmmunoelectron microscopy. IEM was performed as previously described [24]. BHe~y, lt)-/zm frozen sections of glutaraldehyde-fixed tissues (see above) were stained as for light immunoperoxidase study. After dehydration, sections were embedded in Epon and removed from the glass slides; then thin (0.05/zm) sections ~x:re cut for transmission electron microscopy.

Controls. Sections of each thymus were also incubated with normal rabbit serum and with MOPC21, a m'~,elomaprotein of the same isotype as L203. No stainip.g was observed by either light microscopy or electron microscopy. Specific controls for the staining with h*aman alloantibodies and polymorphic monoctonal an~ibodies are described below.



BT,B40 BT,B40 B 7 , B 4 0 plus t~tl~,t B,|o~us ant~ens All A , B a n t i g e n s except Aw32,BI3 15

14 15 16 i" 15 t8 19 19 17


of monoclonal


A2 A2 A2,B i 7 A2 B7





+ + +


+ + +

+ + +


+ + +


Ctx + + + +

+ + + +


NT NT NT + + +

"¢ + + +


+ + +

NT + + + + + + + + + + + ÷ + + NT



NT + + + + + + + + + + + + + NT


M (Aw24,B7,B8,Bw6)

(NT). Staining pattern: medullary confluent (Med)


+]+/+ !NT NT NT + !-



¢A 1 , A 2 , B 5 5 , B 6 2 , C w I , C w 3 , B w 6 )

T h y m u s stained



t A2,Aw30,B i 3,B40,Bwa)


+Staining h~t~'n~iW from f~iut ~ + ~ tu vcr~. steong ~ + + + + L m:gative ~- ~. variable faint anti negative I ~-/- ), and not tested or t'ortical dendritic ICtx).


PA2.1 BBT,2 MA2,1 MA2.2 BBT.I MB40,I MB40,2 MB40,~ MB40A

Monochmal antibody



++++ NT NT +++ NT


++++ NT NT +++ NT


T Ctx


++++ ++++ ++++ +++ ++ NT ++++ + ++++ +++ ++ +++ + + + + + + + + "/ NT


Ctx ++++ NT +++ +++ NT


++++ +++ +++ +++ NT


Ctx ++++ +++ + +++ NT


++++ +++ ++++ +++ NT


Ctx ++++ +++ + ++ NT


++++ +++ ++++ +++ NT


Ctx ++++ +++ +/+++ NT


++++ ++++ ++++ ++++ NT



++++ ++++ ++++ ++++ NT


'~AIso produced moderate to strong cortical lymphocyte staining.

'HLA + B2, HLA-~2-microglobulin complex~ B2,/8:-microglobulin.

bReferences for anti~'odies: BB'~. -7 and PA2.6 [ [ 5 ~, .MB40.5 [ 1 ~], W6/32 [201, BBM. 1 f21 ].

~Staining intensity from faint ( + ) to very strong ( + + + + ), negative I - ), variable faint and negative ~+ / - ). and not tested tN'f~; Staining pattern: medullary confluent (Med~ or cortical dendritic (Ctx).

BB7.7 MB40.5 PA2.6 W6/32 BBM. 1


T h y m u s Stained"

Staining patterns of monoclonal antibodies detecting monomorphic HLA determinams

Monocional a n t l b o d y h Specificity



R.V. Rouse etal.

RESULTS Expression of Class I MHC Antigens Frozen sections of 24 human thymuses were stained with W6/32, a monoclonal antibody that recognizes a monomorphic determinant of the HLA-/32-micrnglobulin complex. The antib~ly intensely stained dendritic cells throughout the cortex with only faint staining of cortical lymphocytes (wherever they could be identified separately from the positively staining processes) (Fig. 1). The medulla demonstrated extensive confluent staining involving positive cells with features characteristic of medullary epithelial cells (abundant cytoplasm and thick cytoplasmic processes); because of the confluent nature of these cells, the staining characteristics of other medullary cells, including lymphocytes, and interdigitadng reticular cells could not be determined. Parallel sections of fot,r of the same thymuses were stained with anti-H antiserum (which detects free class I HLA chains), resulting in a pattern similar to that seen with W6/32 except for a slightly less pronounced dendritic pattern in the cortex. When thymuses were stained with monoctonal PA2.1, which recognizes a polymorphic determinant of HLA-A2, a distinctly different pattern was seen. Of 16 thymuses of unknown HLA type studied with this antibody, 13 were completely negative and the remaining 3 were positive. In order to verify that the positively staining thymuses were derived from A2-positive individuals, peripheral blood lymphocytes from the next 8 patients were A,B,C-typed, and 3 were found to be positive for A2. When these 8 thymuses were stained with PA2.1, only the thymuses from the 3 patients typed A2 were positive. In each of these 3 cases, the medulla was s,rongly positive, similar in appearance to tissue stained with W6/32. The cortices of these thymuses, however, did not exhibit the extensive, intense dendritic staining observed with W6/32 (Fig. 2A). Instead, the cortices demonstrated a variable, faint pattern ranging from completely unstained areas to scattered, very faintly positive dendritic cells. This did not appear to Ix. an effect of antibody coru:entradon, for extensive dilution of W6/32 failed to produce a similar pattern. Sections of thymus from an HLA-AI,A1 I,BS,Bw35,Bw6,Cw4 patient were tested with a set of human alloantisera. Six sera were used that recognize, respectively, HLA antigens AI and Bw49; Bw35 and Bw51; A1 I; B8 and Bw59; Aw23 and Aw24; and Aw52 and A25. Two of these and~ra (anti-Al and Bw49 and anti-Bw35 and Bw51) produced a pattern similar to that described above for antibody PA2.1, with ~onfluent medullary staining and variable, faint cortical staining. When these t'~vo antisera were tested on a thymus fi'om an Aw23,Aw32,B8,B27,Cw2,Bw4,Bw6 patient, no detectable staining was observed. The anti-A 11 and anti-B8 and Bw59 sera did not produce interpretable staining because of high nonspecific background staining. The anti-Aw23 and Aw24 and anti-Aw32 and A25 sera did not produce detectable staining. In order to examine further these different staining patterns, several human thymuses were stained with a battery of monodonal and-human HLA antibodies of different specificities and affinities. As seen in Table l, all the HLA-A2-specific antibodies tested produced the same asymmetric staining pattern as described above for antibody PA2.1, with moderate to strong confluent medullary staining and variable, faint cortical staining. A single thymus in our ser/es was from a patient typed as B7-positive. This thymus v-as stained wkh the four antibodies that recognize BT, producing a staining pattern quite similar to that described above for W6/32. In no instance did we observe the m~ked asymmetry in cortical and medullary staining intensities seen with anti-A2 antibodies. Two antibodies in this group, MB40.2 and MB40.3, with a specificity for B40 and B7 antigens,

HLA Antigens on Huraan Thymic Epithelial Cells


stained the medulla and cortex of the B7 thymus but failed to stain either reghm in the single thymus typed as B40. (B40 includes a number of related but distinguishable allelic products, and these two monoclonal antibodies may not re~:t with all of them [19]). With one exception, the antibodies that recognize monomorphic determinants consistently produced staining patterns similar to those described above fi~r W~'~* 32. The PA2.6 antibody alone produced variable staining patterns not related to differences in age of the subject or to any known differences in tissue handlingTwo o f the thymuses stained with PA2.6 demonstrated a pattern similar to that for W6/32 with strong confluent medullary and dendritic cortical staining. T h e same antibody, on four other thymuses, however, produced a pattern not unlike that seen with HLA-A2-reactive antibodies. In these four thymuses, although the medulla was strongly stained in a confluent pattern, there was only faint cortical dendritic staining (Table 2). Cortical lymphocyte staining was faint to nondetectable in all cases tested with antibodies specific for monomorphic or polymorphic HLA-A, B, or C specificities, except where these lymphocytes were adjacent to the positive epithelial cells. Sections of thymus stained with a monoclonal mouse anti-~2-microglobu|in (BBM. 1) or rabbit antisera specific for ~2-microglobulin produced a pattern similar to that seen with W6/32 except for more staining of the cortical lymph~ytes, which ranged from moderate to strong in intensity. Even with extensive dilution of the antisera, the lymphocyte staining was easily observed with these reagents. Tonsil B-zone lymphocytes stained intensely with L203 and W6/32, and Tzone lymphocytes stained intensely with W6/32. In tonsils that stained with PA2. I or BB7.1, the pattern was identical to that seen with W6/32.

Expression of Class II M H C Antigens All 24 thymuses in this series were stained with monoclonal L203 antibody, and 11 of the thymuses were also stained with rabbit anti-p23,30 antiserum: both o f these antibodies are reactive with monomorphic determinants of the class II M H C molecule and both demonstrated a pattern of expression similar to that seen with W6/32 (Fig. 2B). The staining of the dendritic array throughout the cortex was intense, as was the confluent staining in the medulla. In general, the cortical lymphocytes expressed even less class II antigen than class I antigen, being detectable only where they were adjacent to positively staining dendritic cell processes; staining of medullary lymphocytes could not be determined because o f the confluent nature of the positive medullary epithelial cells.

lmmunoelectron Microscopy T h e pattern of cortical and medullary staining observed wish both class | and II M H C antigens is consistent with the distribution of thymic epithelial cells. In order to identify the natm'e of the cells detected with these reagents, we performed IEM on two o f the thymuses. IEM using both monoclonal anti-class I and II MHC antibodies (L203 and W6/32) produced similar findings, demonstrating that the predominant cells expressing M H C antigens in the thymus are epithelial cells (Fig. 3). In the cortex, positive staining was demonstrated virtually only on cells with long processes that exhibited well-formed desmosomes with tonofilaments and vesicles, characteristics o f epithelial cells [3]. Virtually all such cells and processes identified were positive. Cortical lymphocytes were noted to be stained only on surfaces adjacent to positively staining epithelial cells.


R.V. Rouse et al.

F I G U R E 1 Frozen section of 4-ye~lr-oJd human ~hymus s~ained wigh W6/32 ;¢~umodonal anti-class I H L A monomorphic determinantS. ~A)The mc-dul]a !/[~) s in~c ~.~cly s~ained with a confluent pattern; the corte~; demonstra,oes inten~- dendritic stainin,~. ,,B) Higher magnification of the cor:ex shows stained dendritic processes of epithelial (clds; mos~ ~f the intervening l$,mphocytes express detectable antigen only where the~" arc adiacen~ ~o the epithelial cell processes.

In the medulla, the predominant positive cells were also fi~und to be- cpithcli~d in natu-e, with broad proce.,ses, ~el|-fi~rmed desmosomes, vesicles, z~d t~mofilaments. Occasional epitheli~d cells did not bear visible l~bel. More lymphocytic staining was noted in the r,ledulla than in the cortex, ~Ithough again only on surfaces adjacent to the positive epithelial cel~ processes. Scattered macroph~ges w~re identified in the medulla and deep c~rtex; some were clearly positive, other~, were not. In the absence of definite uItras~ructur~ll






y 2~

F I G U R E 2 Parallel section o f same human thymus as Figure 1. (A) Stained with PA2. (mt~noclonal anti-HLA-A2 polymorphic determinant). The medulla is stained with a c~nfluent pattern, but the cortex demonstrates very little staining. (B) Stained with L2g~g (monoclonal anti-class II M H C monomorphic determinant). Contiuent medullary ,~d dendritic cortical staining is similar to that seen with W6/32.

R. V. Rouse et al.

"::21: ...-,

......... ,




~i~<, i,i,~i~i!'



FIGURE 3 IEM view of human thymus sta/ned with L203 ~monoc|ona~ anti-class 11 MHC monomorphic determinant). Label (#rrou'.~) is identifiable on the s u r ~ e of cells with long processes and desmosomes ¢imaL markers (e.g., desmosomes, tonofiiaments~ for interd/gitating reticular cells, they could not be recognized with certainty in the IEM study o f this tissue.

DISCUSSION These studies demonstrate that the predominant cells expressing high levels of both class I and II M t t C antigens in the human thymus are epithelial in nature. These findings are similar to our previous findings in the mouse thymus and in

HLA Antigens on Human Thymic Epithelial Cells


agreement with the reports ofJanossy et al. [25] and Bhan et al. [26] but ut~Ek,~ that of Brown et al. [27] who found no thymic cortical staining with W(d~2° In addition, we have demonstrated that two distinct patterns of class i M H C a n ~ : n distribution are detectable on thymic epithelial cells; while some determi~angs are easily detectable on both medullary and cortical epithelial cells, others ~ easgly detectable only on medullary epithelial cells, with only very faint sta/ning of cortical epithelial cells. All but one of the antibodies that recognize mem,~morphic HLA determinants produced the former pattern; both patterns o f staining were observed with antibodies recognizing polymorphic HLA determinants. These ultrastructural findings are similar to those obtained in our p r e v i ~ IEM studies on the mouse thymus [24]. The presence of desmosomes and t~nofilaments confirms the epithelial nature of the predominant cells bearing M H C antigens. The occasional stained macrophages identified were too infrequent to account for the extensive patterns of MHC antigen expression seen. The | ~ k o¢ M H C antigen expression by some macrophages and medullary epithelial cel|s may be due to failure of penetration of the antibody (although a 3-hr incub~/~n ga'-le the same results) or may represent distinct subpopulations of macroph~g~:s and epithelial cells in the thymus. We have not been able to address directly whether the M H C antigens observ~M in the human thymus are synthesized by the cells that bear them. We behev¢that thymic epithelial cells synthesize the MHC antigens they bear t'or two re~sons. First, in murine bone marrow chimeras the thymic epithelial cells express thymus-type, not bone marrow-type, MHC antigens (Rouse, unpublished data~. Second, anti-H antiserum appears, at least in lymphoid cell lines, to recogn~¢ the unassociated heavy chain that is a precursor of the surface HLA-A,B,C molecule [ 10]. This suggests that the staining of thymic epithelial cells observed with anti-H may represent an intracytoplasmic synthesized precursor, or that epitheli'A cells may express free HLA heavy chains on their surfaces. The different staining patterns observed with the various anti-class I ant/bodies could be either the result of differences in the properties of the antibodies or o¢ the properties of the HLA-A, B, and C antigens expressed by the cells in the medulla and cortex. We will consider these two possibilities in turn. Asymmetric staining patterns might be observed with two populations of cells if their antigen densities are different and antibodies of low affinity are used. Stable binding o f low-affinity antibody would require bivalent attachment, a state more readily attained on a cell with high antigen density. The effectiveness with which ~m antibody can bind to the c~.~ilsurface thus depends upon (a) the affinity o f the binding interaction, (b) the valency of the antibody, and (c) the class o f antibody, in that flexibility of the antibody may affect its ability to form multivalent a~tachment sites. The results of experiments that assess the capacity of soluble HLA antigens to compete with cells for monoclonal anti-HLA antibodies ~Parham, unpublished data) show no correlation with the staining patterns observed. It appears as though the different reaction patterns are due to intrinsic properties o f the HLA-A, B, and C antigens rather than to differences in the antibodies. Our results suggest that all antigenic determinants of A, B, and C mo|ecules can be detected on medullary epithelial cells, whereas only a subset c~q be detected on cortical epithelial cells. "/'his could be due to differences in the molecular environment in which A, B, and C antigens exist in the membrane o f the two cell types. Masking of H-2* antigens by complex glycoprotein moleculLes has been reported in the mouse [28]. More subtle effects of this type might only certain antigenic determinants on the same molecule. Alternatively, the expression of certain allelic products could be reduced in cortical cells compared to cells in the medulla. It is also possible that the A, B, and C molecules exprt-ssed


R.V. Rouse et al. by thymic cortical epithelial.cells are structurally different from those.expressed by medullary cortical epithelial cells. Changes in posttranslational modifications such as glycosylation, phosphorylation, and glycolipidlzation might be involved, although this is unlikely [29,30]. Finally, this may be determined at the genetic level if class I M H C antigens are encoded by more than one nonallelic but closely related set o f genes as recently suggested [31]. T h e distribution o f class II M H C antigens is similar in human and mouse thymuses. We have observed polymorphic class I M H C determinants in mouse thymuses in a distribution similar to that observed in the human thymus with antibodies specific for HLA-A2 but not with the distribution observed with antibodies specific for B7 or the monomorphic class I determinants [8]. We have not yet had the opportunity to stain mouse thymus with antibodies specific for monomorphic H-2 determinants. Whether these differences in class I M H C antigen distributions in mouse and human thymuses are due to species differences or are merely a result of the limited number of determinants so far examined awaits further investigation. T h e observation that rabbit anti-~2-microglobulin and monoclonal anti-132microglobulin, BBM. 1, stain cortical thymocytes more intensely than antibodies against other class I determinants is consistent with our previous estimates of the relative amounts of/32-microglobulin and HLA-A,B,C on thymocytes [21]. These results suggest that/32-microglobulin or a molecule cross-reactive with it may be present on these cells in a non-A,B,C-associated form. We have previously discussed mechanisms by which cells expressing M H C antigens in the thymus might determine tile M H C restriction o f peripheral T cells and proposed a model o f T-cell differentiation [8]. If the expression of M H C antigens by thymic epithelial cells is instrumental in T-cell maturation, the study o f thymuses from cases of immunodeficiency disorders and other immunologic diseases may be worthwhile.


This work was supported by NIH grants CA-05838, HL-21662, NSF grant PCM-8017834 and USPHS contracts NOI A1-82558 and A1-09072. We thank Ms. DeDe Bremer for untiring and excellent technical assistance. We wish to thank Drs. Jack Strominger, Abe Fuks, Ron Levy, and Lois Lampson for generous gifts of antibodies withi hich this study couldnot have taken place. We especially thank Dr. Jack Strominger for many valuable discussions.

REFERENCES 1. Cantor H, Weissman I: Development and function of subpopulations of thymocytes and T lymphocytes. Prog Aller~,y 20:1, 1976. 2. Scollay R, Kochen M, Butcher E, Weissman I: Lyt markers on thymus cell migrants. Nature 276:79, 1978. 3. Bearman RM, Levine GD, Bensch KG: The ultrastructure of the normal human thymus: A study of 36 cases. Anat Rec 190:75% 1978. 4. Kaiserling E, Stein H, Muller-Hermelink HK: Interdigitating reticulum cells in the human thymus. Cell Tiss Res 155:47, 1974. 5. van Rood JJ, deVries RRP, Bradley BA: Genetics and biology of the HLA system. In: ME Doff, Ed. The Role of the Major Histocompatibility Complex in Immunobiology. New York, Garland STPM Press, 1981, pp. 59-113.

HLA Antigens on Human Thymic Epithelial Cells


6. McMichael AJ: Genetic restrictions in the immune response.J d i n Paths)| ~2 <+u+++|);~), 1979. 7. Zinkernagel RM: Thymus and lymphohemopoietic cells: Their role in T ceil rrmcuration in selection of T cells' H-2-rt.~striction-speciticity and in H-2 linked Ir ger~e control. Immunol Rev 42:224, 1978;. 8. Rouse RV, van Ewijk W, Jones PP, Weissman IL: Expression ~f MHC antigens by" mouse thymic dendritic cells. J Immunol 122:2508, 1979. '~911Lizak GE, Grumet FC: A new micromethod for in z'itro detection of antiplatelet antibodies: C-FDA thrombocytotoxicity. Hum Immunol 1:87, 1980. 10. Krangel MS, Orr HT, Strominger JL: Assembly and maturation of HLA-A and HLAB antigens in vivo, Cell 18:979, 197'9. 11. Humphreys RE, M¢CuneJM, Chess L, Herrman HC, Malenka DJ, Mann DL, Parham P, Schlossman SF, Strominger JI.: Isolation and immunologic characterization of a human B-lymphocyte-specific, cell surface antigen. J Exp Med 144:98, 1976. 12. Robb RJ, StromingerJL Mann DL: Rapid purification of detergent-solubilized HLA antigen by affinity chromatography employing anti-B2-microglobulin serum. J Biol Chem 251:5427, 1976. 13. Lampson LA, Levy R: Two populations of la-like molecules on a human B cell line. J lmmunol 125:293, 1980. 14. Parham P, Bodmer WF: Monoclonal antibody to a human histocompatibility alloantigen, HLA-A2. Nature 276:397, 197:L 15. Brodsky FM, Parhatfi P, Barnstable CJ, Crumpton MJ, Bodmer WF: Monoclonal antibodies for analysis of the HLA s';stem. Immunol Rev 47:3, 1979. 16. McMichae AJ, Parham P, Rust N, Brodsky F: A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and BIT. Hum Immunol 1:121, 1980. 17. Parham P, Brodsky FM: Mouse monoclonal antibodies against HLA-,k,B,C antigens. ln: GJ Hammerling, U Hammerling, JF Kearney, Eds. Monoclonai Antibodies and T Cell Hybridomas. Amsterdam, Elsevier, 1981, pp. 67-76. 18. Parham P, McLean J: Characterization, evolution and molecular basis of a polymorphic antigenic determinant shared by HLA-A and B products. Hum lmmunol 1:131, 1980. 19. Parham P: Monochmal antibodies against two separate alloantigeaic sites of HLAB40. Immunogenetics 13:509, 198L. 20. Barnstable CJ, Bodmer WF, Brown G, Gaifre G, Milstein C, Williams AF, Zieg]er A: Production of monoclonal antibodies to group A e,ythrocytes, HLA and other human cell surface antigens~New tools fi)r genetic analysis. Cell 14:9, 1978. 21. Brodsky FM, Bodmer WF, Parham P: Characterization of a monoclonal anti-~8,microglobulin antibody and its use in~ the genetic and biochemical analysis of major histocompatibility antigens. EurJ immunol 9:536, 1979. 22. Brodsky FM, Parham P: Monomorphic anti-HLA-A,B,C monoclonal antibodies detecting molecular subunits and combinatorial determinants; J lmmunol 128:129, 1982. 23. Brodsky FM, Parham P: Evolution of HLA antigenic determinants: Species crossreactions of monoclonal antibodies, lmmunogenetics 15:151, 1982. 24. van Ewijk W, Rouse RV, Weissman IL: Distribution of H-2 microenvironments in the mouse thymus: lmmunoelectron microscopic identification of I-A and H-2K bearing cells. J Histochem Cytochem 28:1089, 1980. 25. Janossy G, Thomas JA, Bollum FJ, Granger S, Pizzolo G, Bradstock KF, Wong L, McMichael A, Ganeshaguru K, Hoffbtand AV: The human thymic microenvironment: An immunohistologic study. J Immut,ol 125:202, 1980.


R.V. Rouse et al. 26. Bhan AK, Reinherz EL, Poppema S, McCluskey RT, Schlossman SF: Location of T cell and major histocompatibility cemplex antigens in the human thymus. J Exp Med 152:771, 1980. 27. Brown G, Biberfeld P, Christensson B, Mason DY: The distribution of HLA on human lymphoid, bone marrow and peripheral blood cells. Eur J Immunol 9:272, 1979. 28. Codington JF, Klein G, Cooper AG, Lee N, Brown MC, Jeanloz RW: Further studies on the relationship between large glycoprotein molecules and ailotrac,splantability in the TA3 tumor of the mouse: Studies on segregating TA3-Ha hybrids. J Natl Cancer lnst 60:811, 1978. 29. Parham P, Alpert BN, Orr HT, Strominger JL: Carbohydrate moiety of HLA antigens--Antigenic properties and amino acid sequences around the site ofglTcosylation. J Biol Chem 252:7555, 1977. 30. Ploegh HL, Orr HT, Strominger JL: Biosynthesis and cell surface localization of nonglycosylated human histocompatibility antigens. J Immunol 126:270, 1981. 31. Steinmetz M, Winoto A, Minard K, Hood L: Clusters of genes encoding mouse transplantation antigens. Cell 28:489, 1982.