The major histocompatibility complex-restricted antigen receptor on T cells: Distribution on thymus and peripheral T cells

Cell, Vol. 38, 577-584,

September

1984. Copyright

0 1984 by MIT

0092.8674/84/090577-08

$02.00/O

The Major Histocompatibility Complex-Restricted Antigen Receptor on T Cells: Distribution on Thymus and Peripheral T Cellsn Neal Roehm,* Lynne Herron,’ John Cambier,**§ David DiGuisto,* Kathryn Haskins,* John Kappler,*** and Philippa Marrack*” * Department of Medicine National Jewish Hospital and Research Center + Veterans Administration Hospital * Department of Medicine 5 Department of Microbiology and Immunology ‘I Department of Biochemistry, Biophysics and Genetics University of Colorado Health Sciences Center Denver, Colorado

Summary A monoclonal antibody, KJ16-133, which binds to antigen-specific, major histocompatibility complexrestricted (Ag/MHC) receptors on about 20% of BALB/c peripheral T cells has been used to examine the expression of these receptors on thymocytes and different subpopulations of peripheral T cells. Although KJlG-133~reactive receptors were found on mature thymocytes at similar frequencies and levels as on peripheral T cells, these molecules were absent from the first cells to enter the thymus, and in less mature thymocyte populations KJ16-133~reactive cells were less frequent than in the periphery and bore lower quantities of receptor. These results showed that Ag/MHC receptors are present on the surfaces of immature thymocytes, albeit at variable levels, during the time that the repertoire of these cells for Ag/MHC is thought to be selected. Additional experiments showed that KJ16-133 could not be used to distinguish T-cell receptors with different restriction specificities, i.e., for Class I or Class II products of the MHC. Introduction The experiments of Zinkernagel et al. (1978) Bevan (1977) Bevan and Fink (1978) and many others suggested that the thymus plays an important role in determining the specificities of peripheral T cells. In chimera experiments, for example, it has been shown that (H-2” x H-2b)FI precursor thymocytes passing through a thymus bearing only H-2” have a definite preference later on for recognizing antigen (Ag) in association with products of H-2” rather than of H-2b. Although there has been some controversy about how absolute the rules of thymic restriction are (Doherty and Bennink, 1979) it is clear that the major histocompatibility complex (MHC) type of the thymus does affect the repertoire of peripheral T cells to some extent. The process by which repertoire is selected in the thymus is of tremendous interest; presumably it involves

ll This IS paper number seven in the series entitled “The Mqor patlbility Complex-Restricted Antlgen Receptor on T Cells.”

Histocom-

the expression of T-cell receptors for Ag/MHC on the surface of thymocytes. Recently, Acute et al. (1983a) have described experiments involving coprecipitation of receptors with the T-cell-specific glycoprotein T3, which suggested that these molecules are not expressed on immature thymocytes. On the other hand, Yanagi et al. (1984) have shown that mRNA encoding what is probably one chain of Ag/MHC receptors is expressed at high levels in unseparated preparations of thymocytes. Expression of mRNA does not necessarily imply expression of functional protein-especially in the case of the T-cell receptor, which requires that at least two and probably more different mRNAs be expressed (Meuer et al., 1983b; Haskins et al., 1983; Allison et al., 1982) so these two sets of results are not necessarily mutually exclusive. On the other hand, it has been shown that immature cortical thymocytes comprise 75%-85% of the lymphocytes in this organ; medullary cells, which have the phenotypes of mature peripheral T cells, comprise only 15% of the thymocytes (reviewed by Droege and Zucker, 1975; Scollay and Shortman, 1983). It would perhaps be surprising to immunologists if no Ag/MHC receptors were found on the large population of immature thymocytes, especially since there is some controversy over the idea that medullary cells are immediate precursors for peripheral T cells (Elliott, 1973; Cantor and Weissman, 1976; Stutman, 1977). Peripheral T cells may be restricted by either class I or class II products of the MHC. Receptors capable of interacting with these two classes appear not to overlap. Although class l-specific or restricted T cells have often been shown to cross-react with other class I products (Lemmonnier et al., 1977; von Boehmer et al., 1979) and class II-specific or restricted T cells may cross-react with other class II products (Sredni and Schwartz, 1980; Kappler et al., 1981; Hedrick et al., 1982) no example of crossreaction by a cloned T cell between the two classes has yet been reported. In addition, class l-reactive cells are usually cytotoxic, whereas class II-reactive cells usually have helper activity (Bach et al., 1976). These data have led to the idea that T-cell receptors themselves differ markedly, depending on their MHC restriction specificity and, indeed, may use different variable and/or constant regions. We have recently described a monoclonal antibody, KJ16-133, that binds to Ag/MHC receptors on about 20% of peripheral T cells in BALB/c and most other strains of laboratory mice (Haskins et al., 1984). The determinant recognized by KJI 6-l 33 is absent in a few strains, including, most importantly for the results in this paper, SJL/J. Since this is the first well-characterized anti-receptor antibody to react with a reasonable percentage of T cells, we have used it to examine the expression of Ag/MHC receptors on populations of T cells and thymocytes. Here we describe the ability of the antibody to react with receptors on mature and immature thymocytes and on thymocytes from fetuses of different ages. We have also examined the expression of receptors on both L3T4+ and Lyt 2+ peripheral T cells.

Cell

578

Results In this paper we examine the distribution of Ag/MHC receptors on thymocytes and peripheral T-cell subsets, using the monoclonal antibody KJ16-133. We have shown that this antibody binds to the receptors on about 20% of the Ag-specific, MHC-restricted T cells in BALB/c mice (Haskins et al., 1984). The reason that KJ16-133 binds only one-fifth of all Ag/MHC receptors is not known, but several possibilities are listed in the Discussion. Clearly, many of our experiments would be more reasonably done using a monoclonal antibody that binds all Ag/MHC receptors. Such a reagent is not available at the moment. KJl6133 is the only monoclonal antibody described so far with an ability to bind many different receptors. As previously documented (Haskins et al., 1984) and shown here, there is no evidence that the KJIG-133.binding determinant is expressed preferentially at a particular time in T-cell ontogeny. Thus we feel that the observations made in this paper on KJlG-133-binding receptors probably reflect also the properties of the other 80%, KJ16-133 nonreactive Ag/ MHC receptors. This is certainly the assumption underlying our interpretation of the data. lmmunoprecipitation of Ag/MHC Receptors from Thymocytes We have previously shown that the rat monoclonal antibody KJI 6-l 33 precipitates Ag/MHC receptors from about 20% of Ag-specific, MHC-restricted T-cell hybridomas and from normal BALB/c lymph node T cells (Haskins et al., 1984). We wished to find out whether similar molecules could be isolated from thymocytes with this antibody and, if so, whether the receptors were expressed only on mature cells or whether they could also be found on immature thymocytes. Thymocyte suspensions were therefore prepared and fractionated using PNA or LAgI. Thymocytes aggregated by PNA (PNA+) or not aggregated by LAgl (LAgl-) have been shown to be immature (Kruisbeek et al., 1980; Herron et al., 1983; Scollay and Shortman, 1983). Peanut agglutinin-negative (PNA-) cells are mature thymocytes. Thymocytes were also isolated from BALB/c mice pretreated with cortisone acetate. These cortisoneresistant (CR) cells also comprise the mature population. All these cell populations, and whole thymocytes, were surface-labeled with “Y, lysed, and their lysates incubated with KJ16-133 or the antibody specific for the receptor on the T-cell hybridoma DO-1 1 .I 0, KJl-26. After thorough washing, material bound by the antibodies was solubilized with sodium dodecyl sulfate (SDS) and analyzed on nonreducing SDS-polyacrylamide gel electrophoresis (PAGE). Autoradiograms of these electrophoreses are shown in Figure 1. KJ16-133 precipitated the diffuse 85 kd band characteristic of Ag/MHC receptors from whole thymocytes, PNA+ and PNA- thymocytes, LAgI- thymocytes (LAgl+ thymocytes could not be prepared in sufficient quantities to be useful in these experiments), and CR thymocytes. The band precipitated from whole thymocytes and PNA+ cells was light compared with that isolated from

KJI-26

Figure 1. lmmunoprecipitation mocytes

of Ag/MHC

Receptors

from BALB/c

Thy-

BALB/c thymocytes were fractionated from normal thymuses using PNA. Thymocytes were also obtained from cortisone-treated mice. These cells, unseparated thymocytes, and the T-cell hybridoma DO-1 1 .I0 were surfacelabeled with ‘? and lysed. Lysates were incubated with the cross-reacting anti-receptor antibody KJ16-133. bound to rabbit anti-rat immunoglobulin coupled to sepharose beads, or with the clone-specific antibody KJI-26 directly coupled to sepharose beads. Bound material was removed by boiling in SDS and run on nonreducing SDS 10% PAGE. Molecular weight markers are indicated by the arrows.

PNA- and CR thymocytes, suggesting that the receptor might not be expressed as well on immature as on mature thymocytes. A similar band was precipitated from DO11 .iO, the T-cell hybridoma donating the Ag/MHC receptor used to raise KJ16-133 (data not shown). Although the clone-specific antibody, KJl-26, precipitated the receptor from DO-1 1 .lO, no material was brought down by this antibody from thymocytes, illustrating the specificity of KJI-26 and the cross-reactivity of KJ16-133. From these results we concluded that KJI 6-l 33reactive Ag/MHC receptors were expressed on both mature and immature thymocytes although the percentages of cells bearing receptors, and the amount they bore, could not be estimated from this type of experiment. Distribution of KJlG-133-Reactive Material on Thymocytes To find out how much KJ16-133~reactive receptor was expressed on how many cells of the different thymocyte subpopulations we used flow cytometry. Peanut agglutinin+ and PNA- thymocytes, CR thymocytes, whole thymocyte populations, and peripheral lymph node T cells from BALB/c mice were incubated with saturating concentrations of KJl6-133 followed by staining with fluorsceinconjugated mouse anti-rat kappa monoclonal antibody (FLMAR-18 or FL-RG7/9.1). As controls, cells were incubated

Distribution 579

of Antigen-Specific,

H-2-Restricted

c

Receptors

PERIPHERAL 1 CELLS (BALB/c)

aa

I

/ 2, PERIPHERAL 1 CELLS (SJL) I

a.*

i ,

‘.-

RELATIVE FLUORESCENCE 7 I 1 J 10 100 IWO RELATIVE FLUORESCENCE Figure 2. The Reaction of BALB/c Lymph Node T Ceils with KJ16-133

BALB/c or SJL lymph node T cells were incubated at 37°C rn PBS-FCSazide in anti-Thy1 or KJ16-133. After washrng they were incubated with FLmonoclonal mouse anti-rat kappa. Control cells were incubated without antibody (not shown) or with FL-ant&rat kappa. Fluorescence was then analyzed with a Cytofluorograf. Fluorescence profile of cells incubated wrth FL-anti-rat kappa; --- fluorescence profile of cells Incubated wrth KJ16-133 and then FL-anti-rat kappa.

with anti-Thy1 followed by FL-anti-rat kappa, with FL-antirat kappa only, or with no antibody at all (to measure autofluorescence). Additional controls were provided by SJL/J thymocyte subpopulations or peripheral T cells since we have previously shown that the determinant recognized by KJI 6-l 33 is not on SJL/J T cells (Haskins et al., 1984). The results of typical experiments are shown in Figures 24. As far as the controls for these experiments are concerned, in all cases greater than 95% of the cells were Thyl+ (data not shown). Furthermore, the fluorescence profiles of unstained cells were identical with those of cells incubated with FL-anti-rat kappa (data not shown). As shown in Figure 2, and previously reported (Haskins et al., 1984) staining BALB/c peripheral T cells with KJ16133 revealed a discrete population of cells that bound a uniform quantity of antibody. No stained cells were seen in the SJL preparation. There was no evidence for a population of BALB/c peripheral T cells that expressed intermediate amounts of receptor. This result contrasted with the KJ16-133 staining profile of BALB/c thymocytes (Figure 3). Although a significant portion of thymocytes was stained with the antibody, the staining intensity was very heterogenous; many cells stained significantly but weakly, and a continuum of cells stained at increasing levels reached staining intensities comparable to those of positive peripheral T cells. Overall, a smaller percentage of thymus cells than of peripheral T cells was stained by KJ16-133 and FL-anti-rat kappa (10.3% versus 17.7%). When different thymocyte subpopulations were analyzed, the fluorescence profile of more mature cells was found to be quite like that of peripheral T cells. Thus discrete, strongly staining populations of KJl6-133-binding cells were seen in both the CR and PNA- cells (Figure 3). The percentage of cells staining with KJI6-133 in these

Figure 3. The Reaction 133

of BALB/c

Thymocyte

Subpopulations

with KJ16-

BALB/c thymocytes were fractionated using PNA or were isolated from cortisone-treated mice. They were stained with KJ16-133 or Thy1 as described in the legend to Figure 2 and Experimental Procedures. All populations were 295% ThyI+. Fluorescence profile of cells incubated with FL-anti-rat kappa only; --- fluorescence profile of cells incubated with KJ16-133 followed by FL-anti-rat kappa.

preparations was similar to that of peripheral T cells. Both mature thymocyte preparations contained some cells that exhibited intermediate levels of staining with KJI 6-l 33. This might have been due to contamination by immature cells or might have reflected a real heterogeneity in receptor density on mature thymocytes that was not apparent on peripheral T cells. Not unexpectedly, since immature thymocytes represent about 90% of the cells in the thymus, the KJI 6-I 33staining profiles of immature, PNA+ thymocytes were similar to those of unseparated thymocyte preparations. As shown in Figure 3 among PNA+ cells, the KJI 6-133-positive cells were very heterogenous in their ability to bind the antibody ranging from barely detectable levels to the levels on peripheral T cells. Similar percentages of cells stained significantly, 10.3% of unseparated cells, and 9% of PNA+ cells, The KJI 6-133-binding cells in the PNA+ population were not contaminating mature cells because their staining profile was quite distinct from that of PNA- or CR thymocytes. Also, since we considered this an important point, we checked the purity of the populations by measuring their ability to respond to concanavalin A (Con A). Immature thymocytes have been shown not to respond to Con A by proliferating, whereas mature thymocytes, like peripheral T cells, divide vigorously in response to the mitogen (Kruisbeek et al., 1983; Herron et al., 1983). Measurement of the proliferation of titrated cells in response to Con A showed that the PNA- cells contained less than 2% mature cells (results not shown). No KJ16-133 staining of any description was seen in SJL/J thymocytes (Figure 4). From these results we concluded that Ag/MHC receptors were expressed on some immature thymocytes. The fact that the percentage of staining cells was about half that of mature thymocytes or peripheral T cells suggested that about half the immature thymocytes expressed no Ag/MHC receptors at all. The levels of staining suggested

Cell 580

KI

loi RELATIVE

Imoo

10

u)o

Iwo

FLUORESCENCE

Frgure 4. The Reaction of SJL/Thymocytes 133

and Subpopulations

with KJ16-

Thymocytes were isolated from SJL/J mice, fractionated, and stained as described above: fluorescence profile of cells incubated with FL-antirat kappa only; --- fluorescence profile of cells incubated with KLJ16-133 followed by FL-anti-rat kappa.

LOG RELATIVE RuoREscuKT

that as thymocytes mature they express increasing amounts of surface receptor until levels reach those found on peripheral T cells. Other explanations, discussed more fully below, are also possible. Expression of KJlG-133~Reactive Receptors during Thymus Ontogeny In another series of experiments we stained thymocytes from BALB/c fetuses and adult mice with KJI 6-I 33 or antiThyl, followed by FL-anti-rat kappa or FL-anti-rat kappa alone. As before, cells incubated with no FL-anti-rat kappa had the same staining pattern as those incubated with FLanti-rat kappa alone, so only the latter profiles are shown. Greater than 99% of the cells stained with anti-Thy1 except when day 16 fetal thymocytes were analyzed. These were 86% Thy1 -bearing (data not shown). Our results are shown in Figure 5. No thymocytes from fetuses 16 days after conception reacted with KJI 6-I 33. Binding cells were seen at low frequency (1.3%) in 17 day embryos. The percentages of staining cells gradually increased to almost adult levels in newborn mice (10.4%). In the experiment shown here 14.6% of adult BALB/c thymocytes stained with KJI 6-133, a somewhat higher percentge than that reported above. As before, SJL thymocytes did not react with KJ16133. Two points from these results are noteworthy. First, the earliest Thyl+ cell found in fetal thymus does not bear surface receptors for antigen plus MHC. As the cell matures in the thymus it acquires receptors. Second, fetal thymocytes that bear receptors bear considerably fewer per cell than do peripheral T cells. The profile of receptors per cell on fetal and newborn thymocytes is similar to but perhaps reflects even fewer molecules per cell than the profile shown above for immature, PNA+ cells from adult thymocytes. The profile for newborn thymocytes has a striking shoulder, representing fewer receptors per cell than peripheral T cells or mature, PNA- cells.

Figure 5. Appearance Ontogeny

of KJIG-133~Reactive

Thymocytes

during Thymus

Thymocytes were Isolated from BALB/c fetuses at different days of embryogenesis, from newborn BALE/c or from adult BALB/c or SJL mice. These were stained as described above. Fluorescence profile of cells Incubated with FL-anti-rat kappa only; fluorescence profile of cells Incubated with KJ16-133 followed by FL-anti-rat kappa.

Distribution of KJlG-133-Reactive Receptors on Peripheral T Cells Peripheral murine T cells have been divided into two categories based on expression on their surfaces of L3T4 (called Leu3 or T4 in man) or Lyt2 (called Leu2 or T8 in man). It has been shown that L3T4+ cells are generally restricted by or specific for class II products of the MHC, and Lyt2+ cells are restricted by or specific for Class I products (Engleman et al., 1981; Swain, 1981; Spitz et al., 1982) although these rules are not absolute (Greenstein et al., 1984). KJ16-133 was raised against the receptor for cOVA/l-ad on an L3T4+, Lyt2- T-cell hybridoma. If the MHC restriction of the receptor were in any way reflected in its structure or antigenicity, it might be expected that KJ16133 would react preferentially with Class II-restricted L3T4+ T cells. BALB/c peripheral T cells were therefore exposed to biotin-conjugated KJI 6-I 33 or biotin-conjugated antiThyl, followed by phycoerythrin-conjugated avidin (PEavidin). Staining was done in the presence or absence of FL-conjugated anti-L3T4 fluorescence or anti-Lyt2. These cells were then analyzed for forward light scatter (to facilitate fluorescence analysis of only live cells) and red and green fluorescence using the Cytofluorograf. Sample isometric displays of data are shown in Figure 6 and quantitative data in Table I. No cells were stained by PE-avidin alone (Figure 6). Nearly 87% of the cells stained with biotin-conjugated anti-Thy1 and PE-avidin (Table 1, not shown in Figure 6). Nearly 70% of peripheral T cells stained with FL-anti-L3T4 (Figure 6). Similarly, nearly 18% of the cells stained with FL-anti-Lyt2 (Figure 6). If cells

DiIiributron

of Antigen-Specific,

H-2.Restricted

Receptors

Table 1. Distribution Peripheral T Cells

CONTROL

KJl6-133 ALONE

of KJIG-133.Reactive

T-Cell Population

% of Total Lymph Node T Cells

Total

100

Receptors

on BALB/c

% of Population Binding KJ16-133

Amount of KJ16-133 Bound

19.3 + 0.3

25.1

Lyt2+

17.8 f 0.4

33.8 f 0.3

22.0

L3T4+

69.6 k 1 .I

19.6 f 0.3

28.1

Lyt2+ + L3T4+

91.1 + 1.1

(22.5)

Thy1 +

86.9 + 0.6

(22.2)

Data were calculated from the isometrics shown in Figure 5. Shown are the percent of BALB/c lymph node cells staining with Lyt2, L3T4, Lyt2 + L3T4 or Thyl, and then the percentage of each of these populations that also stained with KJI6-133. Also shown is the amount of KJ16-133 bound by each of these subpopulations, expressed as the mean channel number of red (KJ16-133) fluorescence. Bracketed numbers are predicted rather than observed data.

somewhat more of this antibody per cell than cells in the Lyt2 population that bound KJ16-133 (Table 1). These data suggested that, contrary to our expectations, KJ16-133 receptors were more often found on Lyt2+, mostly Class l-reactive T cells, than on L3T4+, mostly Class II-reactive cells. Also, L3T4’ cells appeared to have somewhat more receptors per cell than Lyt2+ cells.

^ Figure 6. Reaction

of BALB/c

Lymph Node T Cells wrth KJ16-133

T cells were isolated from the lymph nodes of normal BALB/c mice and Incubated with biotrn-conjugated KJ16-133 or biotin-conjugated anti-Thy1 followed by PE-avidin, and/or with FL-anti-L3T4 or FL-anti-L@? Red and green fluorescence of these cells was then analyzed using a Cytofluorograf. The Isometric displays show the red fluorescence (left hand axis) and green fluorescence (right hand axis) of cells stained with the indicated antibodies.

were stained with both FL-anti-L3T4 and FL-anti-Lyt2, 91% fluoresced, suggesting that an insignificant percentage of cells bound both antibodies (Table 1, data not shown). Biotin-conjugated KJI 6-l 33 followed by PE-avidin stained about 19% of the peripheral T-cell preparation (Figure 6). The percentage of cells staining with KJI 6-I 33 (red) was unaffected by simultaneous staining with FL-antiL3T4 or FL-anti-Lyt2 (green) (Figure 6). When stained with both anti-L3T4 and KJ16-133, 14% of cells exhibited both red and green fluorescence (Figure 6). This represented 19.6% of the L3T4+ cells. Approximately 7% of cells stained with both anti-Lyt2 and KJ16-133 (Figure 6). This represented almost 34% of all the cells binding Lyt2. The fact that 19.6% and 34% are both higher percentages than 19.3%, the percent of the total T-cell population that bound biotin-coupled KJ16-133 and PE-avidin probably reflects the fact that only 91% of the cells in this isolate were Lyt2 and/or L3T4 positive. The contaminating 9% of cells appear to be Thy1 -, L3T4-, Lyt2-, and KJI 6-133-. Cells in the L3T4+ population that bound KJ16-133 bound

Discussion There is considerable interest in the expression of Ag/ MHC receptors both on T cells in the periphery and in the thymus. These receptors are assumed, of course, to exist on peripheral T cells, but even in this case there is debate over whether T cells with different functions or different types of specificities might bear different types of receptors. In the thymus, where the repertoire of T cells for Ag/ MHC is supposed to be established, it is clearly important for immunologists to know how much receptor is expressed, and by which cells, and how rapidly it diversifies. This paper describes the first experiments to have examined some of these problems at the single-cell level. In the past some contradictory results have been reported about the expression of Ag/MHC receptors on thymus cells. Acute et al. (1983a) showed that glycoproteins with the physical properties of Ag/MHC receptors, i.e., disulphide-linked heterodimers with subunit molecular weights of about 50 and 43 kd, could be identified on human peripheral T cells and on the mature, T3+ population of human thymocytes, which comprise about 25% of the lymphocytes in the thymus (Reinherz et al., 1980). Proteins with these properties could not be identified on immature thymocytes or on thymomas with an immature phenotype. McIntyre and Allison (1983) showed that an anti-receptor antiserum could precipitate the appropriate molecules from thymocytes. There was no indication in their work that the receptor was distributed heterogenously on different subpopulations of thymocytes, nor that thymocytes bore significantly fewer receptors than peripheral T cells.

Cell 582

Molecular biological studies have also suggested that the genes encoding Ag/MHC receptors are expressed at least to some extent by thymocytes. Both Hedrick et al. (1984) and Yanagi et al. (1984) have shown that mRNA coded for by a rearranging, immunoglobulin-like gene(s) is present in thymocytes-in fact at higher levels than in peripheral T cells. Our own results show that Ag/MHC receptors are indeed expressed on thymocytes. The frequency of mature thymocytes’ reacting with the cross-reacting anti-receptor antibody was the same as that of peripheral T cells (20%) although there was some evidence that not all these cells expressed quite as much receptor as peripheral T cells. Immature thymocytes, on the other hand, although they were to some extent receptor-bearing, had very heterogenous amounts of KJ16-133~reactive receptor per cell, ranging in a continuum from barely detectable levels to those expressed on peripheral T cells. KJ16-133.bearing cells occurred at about half the frequency (10%) among immature thymocytes as in mature thymocytes or peripheral T cells. In spite of the relatively low levels of receptor on immature thymocytes, however, it was clear that some of these molecules were present on these cells. This contradicts the report of Acute et al. (1983a), but probably is not surprising to most cellular immunologists, Our results showed that during development of the thymus KJ16-133reactive receptors were not present on the first Thyl+ cells to appear in the fetal thymus. These cells are first found in the organ at about day 14 of embryogenesis, yet even 2 days later (on day 16 of embryogenesis) we were still unable to detect KJ16-133binding cells. After this day, the percentage of binding cells gradually increased, reaching levels equivalent to those of adult, immature, PNAf cells by about the day of birth. The interpretation of these results depends to some extent on the nature of the determinant recognized by KJ16-133. The antibody may bind an allelic determinant on a constant region of T-cell receptors present on 20% of peripheral T cells, it may bind an allelic determinant on one of several J regions, or it may bind an allelic determinant on a family of V regions. Of these possibilities we favor the first, because of the temperature sensitivity of the antibody binding to cells and because KJI 6-133 clearly binds to the T-cell receptor at a site far from the “idiotypic” determinant recognized by clone-specific antibodies (Haskins et al., 1984). Nevertheless, whatever the nature of the determinant recognized, several ideas can be suggested. The fact that KJI 6-I 33 reacted with a lower percentage of immature thymocytes than mature thymocytes or peripheral T cells, and none of the first Thyl+ cells to appear in the thymus, can lead to several interpretations. Antigenspecific, MHC-restricted receptors may be absent from prothymocytes and about 50% of immature thymocytes, and may be expressed only after sufficient proliferation and differentiation has been incurred by the maturing cell to allow the rearrangement and functional expression of genes encoding (at least) both chains of the Ag/MHC

receptor. On the other hand, KJI6-133nonreactive Ag/ MHC receptors may be present on very early thymocytes and the KJ16-133 determinant may appear only after maturation and selection in the thymus. It is also possible that mutation of receptor genes in immature thymocytes might lead to loss of expression of receptors because, for example, of the generation of termination codons Either interpretation of these results leads to the conclusion that the number of Ag/MHC receptors on developing or immature thymocytes is lower than on peripheral T cells (Figures 3 and 5). This result should be considered by any theory that seeks to account for the selection for Ag/selfMHC reactivity in the thymus, apparently in these populations of immature cells. A number of previous papers have addressed the problems of whether T cells with different specificities or different surface markers bear different types of receptors. As far as Ag/MHC receptors are concerned, the answer to this problem so far seems to be that they do not. The gross biochemical properties of Class I- or Class Il-restricted receptors on OKT8+ or OKT4+ cells seems to be similar in man and mouse (Meuer et al., 1983a; Kappler et al., 1983a). Also, peptide maps or ‘251-surface-labeled receptors do not distinguish between the two types (Acuto et al., 1983b; Kappler et al., 198313). The data presented in this paper support these results. KJI6-133, raised against a Class Ii-restricted receptor reacts with both L3T4+ (mostly Class Ii-restricted) and with Lyt2+ (mostly Class Irestricted) peripheral T cells, Unexpectedly, the antibody reacts with a rather higher percentage of Lyt2+ than L3T4+ cells. Also unexpectedly, the L3T4+ population seemed to bear rather more receptors per cell than the Lyt2+ population. Whether this means that only the specificities of the variable portions of the receptor will distinguish these populations remains to be seen. Experimental

Procedures

Animals SJL/J mace were purchased from the Jackson Laboratories, Bar Harbor, ME. BALB/cBy animals were bred in the vivarium here from parents acquired from the same sources. All adult mice used in these studies were between 8 and 12 weeks old. BALB/c fetuses were obtained from timedpregnancy animals mated in our own vivarium, or at the Jackson Laboratories, Preparation of Peripheral T Cells T cells were isolated from lymph node cell suspensions nylon wool columns (Julius et al., 1973).

by passage

over

Preparation of Thymocytes Subpopulations Cortisone-resistant thymocytes were prepared from the thymuses of animals treated 2 days previously wrth 2.5 mg of cortisone acetate intraperitoneally. This procedure reduced the number of thymocytes per organ to less than 10% of untreated controls. Peanut agglutinin-positrve (PNA+) and -negative (PNA-) thymocytes were prepared from suspensions of normal thymocytes as previously described (Kruisbeek et al., 1960). Bnefly, thymocytes at 2.5 x 108 cells/ml were incubated in balanced salts solution (BSS) containing 0.5 mg/ml PNA at room temperature for 15 min. Cell suspensions were then adjusted to 5% fetal bovine serum (FBS) and layered over a drscontinuous gradient consisting of layers of IO%, 25%, 50%, and 100% FBS in BSS. Afler 60 min incubation at room temperature cells from the 5% and 10% FBS layers were collected and termed PNA-.

Distributron 583

of Antrgen-Specific,

Table 2. Monoclonal

H-2-Restncted

Antibodies

Receptors

Used in These Studies

Antrbody

Specificity

Source

Reference

T24140.7

Thyl.2

Dr. I, Trowbridge

Dennert et al., 1979

GKt .5

L3T4

Drs. D. Dialynis and F. Fitch

Dialynis et al., 1983

Dr. P. Gottleib

Gotilerb et al., 1980

US

Haskins et al., 1984

ADH4 (15)

w

KJ16133

15%-20%

MAR-18.5

Rat kappa

Drs. L. Arnold and L. Lanier

RG7/9.1

Rat kappa

Dr. T. Sprrnger

BALE/c

Ag/MHC

receptors

Cells from the 100% FBS layer were collected and layered again on similar gradients. After another 60 min at room temperature cells that had again settled to the 100% FBS layer were collected and used as PNA+. Lobster agglutrnin l-negative (LAgl-) thymocytes were prepared in similar fashion to the PNA- cells after incubation for 1 hr on ice in 0.5-I 5 mg/ml soluble LAgl (Herron et al., 1983). When necessary, red blood cells were lysed wrth Gey’s solution (Gey and Gey, 1936). Radiolabeling and lmmunoprecipitation of Cells T cells, thymocytes, and thymocyte subpopulations were ‘251-surface-labeled by standard methods using lodogen (Fraker and Speck, 1978; Markwell and Fox, 1978). They were lysed using NP-40 as previously described (Haskins et al., 1983), and receptors were isolated by incubating the lysates overnight with Sepharose 48 beads to which rabbit anti-rat rmmunoglobulin had been coupled directly, and the monoclonal rat antibody KJ16-133 bound by incubation. These beads were thoroughly washed after incubation with lysates; the bound material was removed by bolting in sodium dodecyl sulphate (SDS) and analyzed by nonreducrng SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970). Monoclonal Antibodies The monoclonal antibodies used in these studies are listed, together wrth their sources and some of their properties, in Table 2. Anti-Thyl, anti-Lyt2, and anti-L3T4 have already been well characterized In the literature (Dennert et al., 1979; Gottlieb et al., 1980; Dialynas et al.. 1983). The properties and Initial characerizatron of KJ16-133 have already been described by Haskins et al. (1984). Briefly, the hybridoma secreting thus antibody was produced from a fusron of lymph node cells from a rat hyperimmunized with the Isolated receptor for chicken ovalbumin (cOVA) and I-Ad of the cloned Tcell hybridoma. DO-l 1 .lO. Upon examination KJ16-133 proved to bind the Ag/MHC receptors on 15%-20% of peripheral T cells rn BALB/c mice but no receptors rn SJA/20 or SJL/J animals. lmmunofluorescence Staining and Analysis Cells were suspended at 107/ml in phosphate-buffered saline containing 1% FBS and 0.2% sodium azrde (PBS-FBS-azide). For staining, appropriate amounts (determined to give optimal staining in preliminary experiments) of primary antibody were added to IO6 cells in 100 pl and incubated at 37°C for 20 mm. This temperature was used because cell bindrng by KJ16133 proceeds poorly at 4°C (Haskins et al., 1984). Cells were then washed twrce in PBS-FBS-azide and incubated for 15 min at 4°C with the appropriate concentration of secondary fluorescetn (FL)-conjugated antibody or phycoerythrin (PE)-conjugated avidin (Becton-Dickinson, Mtn. View, CA). Finally, cells were washed three times with PBS-FBS-azide and analyzed using an Ortho Cytofluorograf System 50H equipped with a 2150 datahandling system. The Cytofluorograf is equipped with a 5 watt Coherent argon laser. For all measurements the 488 nm line was used for fluorochrome excrtation. Green (FL) fluorescence was measured usrng the Instrument in standard configuration. Red, PE. fluorescence was also measured using the standard instrument configuration except that a 590 nm bandpass filter (10 nm band wrdth, Ditric Optics, Boston, MA) was placed before the red detector. In all experiments, dead cells were excluded from the analysis by gattng based upon forward light scatter. Data were analyzed and corrected for red-green fluorescence cross-over using standard 2150 computer software. Two reagent combrnations were used for KJ16-133 staining. For studies of antigen expressron on thymocytes, KJ16-133 in the form of tissue culture

supernatant was used as the primary antibody followed by fluoresceinconjugated MAR18.5 or RG7/9.1 (FL-anti-rat kappa) (a monoclonal mouse anti-rat kappa chain antibody, a gift from Dr. Larry Arnold, University of North Carolina). For a positive control anti-Thy1 (T24/40.7) was used in conjunction with FL-anti-rat kappa. For two-color analysis, KJ16-133 isolated by ammonrum sulfate precrprtation was biotin-conjugated using hydroxybiotinsuccinimide (Sigma, St. Louis, MO; Pohlit et al., 1979) and used in conjunction with PE-avidin. Monoclonal anti-Lyt2.2 (ADH/14) was purified by adsorption to monoclonal rat anti-mouse kappa chain antibody derivatrzed Sepharose 48 and elution with 3.5 M magnesium chloride. Monoclonal anti-L3T4 (GK.15) was isolated by ammonium sulfate precipitation. Both anti-Lyt2 and anti-L3T4 were directly fluoresceinated as previously described using fluorescein on celite (Sigma, St. Louis, MO). The percentage of antigen-expressing cells was determined using the Model 2150 Computer data-processing system. Fluorescence histograms containing equal numbers of cells, which were either unstained, stained with the fluorescein-conjugated secondary antibody alone, or stained with both the primary and secondary antibody reagents, were constructed and superimposed. Using the data-processing system, the control values were subtracted from the experimental values on a channel-by-channel basis. The percentage of cells positively stained was determined by integration of the area to the right of the point of intersection of the two curves. Acknowledgments We would like to thank Ralph Kubo and Charles Hannum for their help with this project; Janrce White, James Leibson, and Eleanora Kushnrr for therr excellent technical assistance; and Edna Squillante for her secretarial help. This work was supported rn part by a Damon Runyon-Walter Winchell Cancer Fund postgraduate fellowship to N. R., by an American Cancer Society, Inc. fellowship to K. H., and by U. S. Public Health Service grants Al-18785 and Al-20519, and American Cancer Society Grant IM-49. Thus work was done while J. K. was supported by an American Cancer Society Faculty research award. 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 USC. Section 1734 solely to indicate this fact. Received

May 4, 1984

References Acute, O., Hussey, R. W., Fitzgerald, K. A., Protentis, J. P., Meuer, S. C., Schlossman, S. F., and Reinherz, E. L. (1983a). The human T cell receptor: appearance in ontogeny and brochemical relationship of a and p subunits on IL-2 dependent clones and T cell tumor. Cell 34, 717-726. Acute, O., Meuer, S. C., Hodgdon, J. C., Schlossman, S. F., and Reinherz, E. L. (1983b). Peptide varrability exists within ~1and p subunits of the T cell receptor for antigen. J. Exp. Med. 758, 1368-1373. Allison, J. P., McIntyre, of murrne T-lymphoma 2293-2300.

B. W., and Bloch, D. (1982). Tumor-specific antigen defined with monoclonal antibody. J. Immunol. 129,

Bach, F H., Bach, M. L., and Sondel, P. M. (1976). Differential function of major histocompatibility complex antigens in T lymphocyte activation. Nature 259, 273-281.

Cell 584

Bevan, M. J. (1977). In a radiation chimera host H-2 antigens determine the immune responsiveness of donor cytotoxic cells. Nature 269, 417-419. Bevan, M. J., and Fink, P. J. (1978). The influence of thymus H-2 antigens on the specificity of maturing killer and helper cells. Immunol. Rev. 42, 319.

Kappler, J., Kubo, R., Haskrns, K., White, J., and Marrack, P. (1983a). The mouse T cell receptor. Comparison of major histocompatibility restricted receptors on two T cell hybridomas. Cell 34, 727-737.

Cantor, H., and Weissman, I. (1976). Development and function of subpopulations of thymocytes and T lymphocytes. Prog. Allergy 20, l-64.

Kappler, J. W., Kubo, R., Haskins, K., Hannum, C., Marrack, P., Pigeon, M., McIntyre, B., Allison, J., and Trowbridge, I. (1983b). The major histocompatibility complex-restricted antigen receptor on T cells in mouse and man. V. Identification of constant and variable peptides. Cell 35, 295-302.

Dennert G., Hyman, R., Leslie, J., and Trowbndge, I. S. (1979). Effects of cytotoxic monoclonal antibodies specific for T200 glycoproteins on functional lymphoid cell populations. Cell. Immunol. 53, 350.

Kruisbeek, A., Zijlstra, J. J., and Krose, T. (1980). Distinct effects of T cell growth factors on thymic epithelial factors on the generation of cytotoxic T lymphocytes by thymocyte subpopulations. J. Immunol. 125, 9951002.

Dialynas, D. P., Wilde, D. B., Marrack, P., Pierres, A., Wall, K. A., Havran, W., Otten, G., Loken, M. R., Pierres, M., Kappler, J., and Fitch, F. W. (1983). Characterization of the murine antigenic determinant designated L3T4a, recognized by monoclonal antibody GKI .5: expression of L3T4a by functional T cell clones appears to correlate primarily with Class II MHC antigenreactivity. Immunol. Rev. 74, 29-56.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophase T4. Nature 227, 680-685.

Doherty, P. C., and Bennink, J. R. (1979). Vaccinia-specific cytotoxic T-cell responses in the context of H-2 antigens not encountered in thymus may reflect aberrant recognition of a virus-H-2 complex. J. Exp. Med. 749, 150-157.

Markwell, M. A. K.. and Fox, C. F. (1978). Surface-specific iodination of membrane proteins of virus and eucaryotic cells using 1346tetra chloro3a,6aY-diphenylglycoluril. Brochemistry 7 7, 4807-4817.

Droege, W., and Zucker, R. (1975). thymus. Transplant. Rev. 2.5, 3-25. Elliott, E. V. (1973). A persistent Nature New Biol. 242, 150.

Lymphocyte

lymphoid

subpopulations

cell population

in the

in the thymus.

Engleman, E. G., Benike, C. G., Glickman, E., and Evans, R. L. (1981). Antibodies to membrane structures that distinguish suppressor/cytotoxic and helper T lymphocytes subpopulations block the mixed leucocyte reaction in man. J. Exp. Med. 154, 193-198. Fraker, P. J., and Speck, J. C. (1978). Protein and cell membrane iodinations with a sparingly soluble chloramide 1,3,4,6-tetrachloro-3a,6n-diphenyl-glycoluril. Biochem. Biophys. Res. Commun. 80, 849-857. Gey, G. O., and Gey, M. K. (1936). The maintenance of human normal cells and tumor cells in continuous culture. I. Preliminary report: cultivation of mesoblastic tumors and normal tissue and notes on methods of cultivation. Amer. J. Cancer 27, 45. Gottleib, P. D., Marshak-Rothstein, A., Auditore-Hargreaves, K., Berkoben, D. B., August, D. A., Rosche, R. M., and Benedetto, J. D. (1980). Construction and properties of new Lyt-congenic strains and anti-Lyt2.2 and antiLyt3.1 monoclonal antibodies. Immunogenetics IO, 545-556. Greenstein, J., Kappler, J., Marrack, P., and Burakoff, S. J. (1984). The role of L3T4 in recognition of la by a cytotoxic H-2D%pecific T cell hybridoma. J. Exp. Med. 159, 1213-1224. Haskins, K., Kubo, R., White, J., Pigeon, M., Kappler, J., and Marrack, P. (1983). The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J. Exp. Med. 157, 11491169. Haskins, K., Hannum, C., White, J., Roehm, N., Kubo, R., Kappler, J., and Marrack, P. (1984). The major hrstocompatibility complex-restricted antigen receptor on T cells. VI. An antibody to a receptor allotype. J. Exp. Med. 160, 452. Hedrick. S. M., Matis, L. A., Hecht, T. T., Samelson, L. E., Longo. D. L., Heber-Katz, E., and Schwartz, R. H. (1982). The fine specificity of antigen and la determinant recognition by T cell hybridoma clones specific for pigeon cytochrome c. Cell 30, 141-152. Hedrrck, S. M., Nrelsen, E. A., Kavaler, J., Cohen, D. I., and Davis, M. M. (1984). Sequence relationships between putative T-cell receptor polypeptrdes and immunoglobulins. Nature 308, 153-158. Herron, L. R., Abel, C. A., Vanderwall, J., and Campbell, P. A. (1983). Immature thymocytes isolated using a sialic acid-specific lectin are unresponsive to concanavalrn A. Europ. J. Immunol. 13, 73-78. Julius, M. H., Sampson, E., and Herrenberg, for the isolation of functional thymus-derived J. Immunol. 3, 645.

L. A. (1973). A rapid method murine lymphocytes. Europ.

Kappler, J. W., Skidmore, B., White, J., and Marrack, P. (1981). Antigeninducible H-2-restricted, interleukin-2 producing T cell hybridomas. Lack of Independent antigen and H-2 recognitton. J. Exp. Med. 753, 1198-1214.

Lemmonnier, F., Burakoff, S. J., Germain, R. N., and Benacerraf, B. (1977). Cytolytic thymus-derived lymphocytes specrfic for allogeneic stimulator cells crossreact with chemically modified syngeneic cells, Proc. Nat. Acad. Sci. USA 74, 1229-l 233.

McIntyre, B. W., and Allison, J. P. (1983). The mouse T cell receptor: structural heterogeneity of molecules of normal T cells defined by xenoantisera. Cell 34, 739-746. Meuer, S. C., Acute, D., Hussey, R. E., Hodgdon, J. C., Fitzgerald, K. A., Schlossman, S. F., and Reinherz, E. L. (1983a). Evidence for the T3associated 90K heterodimer as the T cell nntigen receptor. Nature 303, 808-810. Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Schlossman, S. F., and Reinherz, E. L. (1983b). Clonotypic structures involved in antigenspecific human T cell functron. Relationship to the T3 molecular complex. J. Exp. Med. 157, 705-719. Pohlit, H. M., Haas, W., and von Boehmer, H. (1979). Haptenation of viable biological carriers. In Immunological Methods, I. Lefkowitz and B. Pernis, eds. (New York: Academic Press), p. 181. Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H., and Schlossman, S. F. (1980). Discrete stages of human rntrathymic differentiation: analysis of normal thymocytes and leukemic lymphoblasts of T cell lineage. Proc. Nat. Acad. Sci. USA 77, 1588-1592. Scollay, R., and Shortman, K. (1983). Thymocyte subpopulations: an experrmental review, including flow cytometrrc cross-correlations between the major murine thymocyte markers. Thymus 5, 245-295. Spitz, H., Borst, J., Terhorst, C., and deVries, J. E. (1982). The role of T cell differentiation markers in antigen-specific and lectin-dependent cellular cytotoxrcity mediated by T8+ and T4+ human cytotoxic T cell clones directed at class I and class II MHC antigens. J. Immunol. 129, 1563-1569. Sredni, B., and Schwartz, R. H. (1980). Alloreactivity T cell clone. Nature 287, 855-857.

of an antigen specific

Stutman, 0. (1977). Two main features of T cell development: thymus traffic and post-thymrc maturation. Contemp. Top. Immunobiol. 7, 1. Swain, S. L. (1981). Significance of Lyt phenotypes: Lyt2 antibodies block activity of T cells that recognize class I major histocompatibility complex antigens regardless of their function. Proc. Nat. Acad. Sci. USA 78, 71017106. von Boehmer, H., Hengartner, H., Nabholz, M., Lernhardt, W., Schreier, M. H., and Haas, W. (1979). Fine specificity of a continuously growing cell clone specific for H-Y antigen. Eur. J. Immunol. 9, 592-596. Yanagi, Y., Yoshikai, Y., Leggett, K., Clark, S. P., Aleksander, I,, and Mak, T. W. (1984). A human T cell-specific cDNA clone encodes a protein having extensive homology to immunoglobulin chains. Nature 30, 145-149. Zinkernagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Klein, P. A., and Klein, J. (1978). On the thymus in the differentiatron of H-2 selfrecognition by T cells: evidence for clonal recognition? J. Exp. Med. 747, 882-896.