CELLULARIMMUNOLOGY 119,85-100(1989)

Nature of the Thymocytes Associated with Dendritic Cells and Macrophages in Thymic Rosettes

KEN SHORTMAN,* DAVID VREMEC,* ANGELA D’AMIco,* FRANK BATTYE,* AND RICHARD BOYD~

*The Walter and Eliza Hail Institute of Medical Research, Melbourne, Australia, and tDepartment of Pathology and Immunology, Monash University Medical School, Melbourne, Australia

Received August 12, 1988; accepted October 25,198s

Thymic rosettes, structures consisting of 3-30 thymic lymphoid cells attached to a central macrophage or dendritic cell, were released from mouse thymus tissue by collagenase digestion. They were shown to be preexistent structures within the thymus, but to be subject to extensive exchange with free thymocytes under certain conditions. An isolation procedure was developed, using a new technique of zonal unit-gravity elutriation, which minimized exchange and pro- duced a completely pure sample of the larger rosettes. The rosette-associated thymocytes were analyzed by two- and three-color immunofluorescent staining and flow cytometry. The domi- nant cell type was a small, CD4+CD8+, cortical-type thymocyte. However, all of the established thymus subpopulations defined by CD4 and CD8, including CD4-CD8+ and CD4+CD8- ma- ture thymocytes and CD4-CD8- early thymocytes, were also present in rosettes. Very few of the cells present were of an intermediate or transitional phenotype. Rosette-associated thymo- cytes were somewhat enriched in large dividing thymocytes, in CD4-CD8- thymocytes, and in mature thymocytes expressing the T-cell antigen receptor-CD3 complex. Their most striking characteristic was a marked depletion in small thymocytes lacking surface H-2K expression, a major population among free thymocytes. The physiological role of the rosette structure is dis- cussed, and it is suggested that the heterogeneity of the associated thymocytes in part reflects the existence of different types of rosettes in different areas of the thymus. 0 1989 Academic PISS, IK.

INTRODUCTION

The stromal cells of the thymus are believed to provide a series of microenviron- ments which dictate various stages of T-cell development. Despite the likely impor- tance of stromal elements, most attempts to discern the intrathymic pathway of T-cell development from thymocyte subpopulation analysis have used data from mechanically disrupted thymus suspensions with the stromal cells and lymphoid- stromal cell complexes being eliminated as debris. Key intermediate stages in T-cell development may have been missed by this selective approach. However, it is possible by using enzymic digestion to isolate from the thymus lymphocyte-stromal cell com- plexes, and these isolated complexes may serve as a model of the events occurring in a particular thymic microenvironment. Notable among these are thymic nurse cells (l-3), complexes of epithelial cells and lymphoid cells, and thymic rosettes (4, 5), complexes of macrophages or dendritic cells and lymphoid cells. Thymic rosettes are the subject of this study.

The strongest evidence that thymic rosettes are involved in early stages of T-cell development comes from the kinetic experiments of Kyewski (5), who demonstrated

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0008-8749189 $3.00 Copyright 8 1989 by Academic press, Inc. All rights of reproduction in any form reserved.

86 SHORTMAN ET AL.

rapid entry of bone marrow precursor-derived cells into thymic rosettes, after entry into the pool of CD4CD8- thymic precursor cells but before entry into the pool of mature thymocytes. If such rosettes are crucial at early developmental stages, the surface phenotype of the lymphoid component might reasonably be expected to differ from that of the bulk of thymocytes. However, the dominant component of thymic rosettes has been found to be a CD4+CD8+ or “double positive” cell, expressing low levels of H-2K and CD5, and binding high levels of peanut agglutinin (6-8); unfortu- nately this is also the phenotype of the typical cortical thymocyte, the major compo- nent of thymus suspensions. The majority of such cortical cells are destined for intra- thymic death, although a very small proportion might develop into mature T cells (9, 10). It has been suggested (7,8) that the lymphoid cells ofthymic rosettes, although of cortical phenotype, are in fact an “intermediate” form en route to mature T cells, differing from most cortical cells in functional capability and expression of the T-cell antigen receptor (TcR).’

An alternative approach to studying lymphocyte-stromal interactions has been to reassemble such complexes in vitro. Papiemik and colleagues ( 12- 14) have demon- strated the formation of rosettes between mainly cortical-type thymocytes and cul- tured thymic macrophage/dendritic-type cells and have suggested a way the interac- tion might rescue cortical thymocytes from programmed death. However, in this type of study it is not clear if the interacting cells would ever have associated within the thymus itself. Such rosette formation is not restricted to cells of thymic origin (15, 16), and clearly the rosettes formed by interacting splenic macrophages and thymic lymphocytes are not reconstructions of a complex existing in vivo.

In the present study we follow the Kyewski approach of isolating the rosettes found within the thymus itself, since a good understanding of such preexisting structures should provide a basis for assessing the significance of interactions reassembled in vitro. We particularly wished to check for the existence on thymic rosettes of cells intermediate in phenotype between cortical-type CD4+CD8+ thymocytes and ma- ture, medullary-type CD4CDV or CD4+CD8- thymocytes, and to determine if any early CD4CD8- thymocytes or their subsets were preferentially located in these structures. Since we aimed to go beyond the earlier studies and characterize not only the dominant lymphoid component of thymic rosettes but also any minor subpopula- tions as well, we employed the same two- and three-color immunofluorescent stain- ing and flow cytometry techniques recently applied to the total free thymocyte popu- lation (17-23). Because of our interest in minor subpopulations we required a high level of rosette purification and minimal exchange of lymphoid cells after tissue diges- tion, and we have developed an isolation procedure that meets these requirements.

MATERIALS AND METHODS

Mice. Specific pathogen-free 5-week-old female CBA CaH Wehi mice, bred at the Hall Institute animal facility, were used throughout.

Thymocyte suspensions. Suspensions of thymocytes from mechanically disrupted tissue were prepared as described elsewhere ( 17).

’ Abbreviations used: MHC, major histocompatibility complex; TcR, T-cell antigen receptor; FCS, fetal calf serum; EDTA, ethylenediaminetetraacetate; HSA, the heat stable antigen recognized by the mono- clonal antibodies B2A2, M l/69, and J 11 d; IL-ZR, the interleukin-2 receptor determinant recognized by the monoclonal antibody PC6 1.

THYMIC ROSETTES 87

Fluorescein labeling of dissociated thymocytes. Thymocytes in suspension were directly labeled with fluorescein isothiocyanate (20 &ml, 20 min, room tempera- ture), using procedures described elsewhere (2,22).

Immunofuorescent staining of surface antigens. Full details of the fluorescent re- agents, monoclonal antibodies, and staining protocols are given elsewhere (17, 18, 20-22). The fluorochromes used were green, fluorescein; red, phycoerythrin; and blue, coumarin. One-color analyses generally used green fluorescent staining, two- color analyses, green and red. Three-color analyses using red, green, and blue fluo- rescent staining were used for subpopulations of CD4-CD8- thymocytes, with both CD4 and CD8 being stained for blue fluorescence. Propidium iodide was included in the final cell wash to selectively stain dead cells. The monoclonal antibodies used were anti-CD4, GUS; anti-CD8, 53-6.7; anti-H-2K, 11.41; anti-Thy 1, 3OiH12; anti-CDS, 53-7.3; anti-HSA, M1/69; anti IG2R, PC61; anti-CD3 (anti-T3-E chain), 145-2Cll; and anti-V/38, F23.1.

Flow-cytometric analysis. Full details of the use of the modified, two-laser, FACS II instrument (Becton-Dickinson, Sunnyvale, CA) are given elsewhere (18, 20, 22, 24). Dead cells, debris, and any cells much larger than large lymphocytes (including most macrophages) were gated out on the basis of low-angle light scatter and of “off- scale” bright red fluorescence from propidium iodide uptake. For the two-laser, three- color analysis of CD4-CD8- subsets, all blue positive (CD4+ and/or CD8+) cells were gated out and red and green fluorescence data were collected only on the blue negative cells, Most analyses were based on files of 50,000 cells. Two-parameter analyses were based on a 64 X 64 grid, with the contour levels representing threefold differences in cells per grid square.

Collagenase digestion of thymic tissue. Thymuses, usually 12, were cut into small pieces with sharp scissors, and the fragments were suspended in 10 ml medium in a 30-ml glass bottle at room temperature (22°C). The medium was RPM1 1640, isoos- motic with mouse serum, with additional Hepes buffering to maintain a pH around 7.2, and supplemented with 2% fetal calf serum (FCS). The fragments of tissue were kept in suspension and in continuous movement using a stainless-steel wire agitator connected to a small electric motor. Every 5 min the medium, containing released thymocytes in suspension but no detectable rosettes, was removed using a stainless- steel gauze filter. The thymus fragments were resuspended in fresh medium, and again agitated. This was continued until the release of thymocytes was visibly very low. This usually took four such washings, and removed 80% of the releasable free thymocytes. The thymic fragments were then suspended in 5 ml of the same medium containing 1 mg/ml collagenase (Boehringer-Mannheim, West Germany) and 0.02 mg/ml DNAse (Grade II bovine pancreatic DNAse I, Boehringer-Mannheim). Di- gestion was carried out at 22°C for 20-25 min, with continuous agitation as before. The extract, containing free thymocytes and rosettes, was removed from any remain- ing tissue debris and filtered through a fine stainless-steel gauze.

Purification of thymic rosettes by zonal unit-gravity elutriation. Full details of the new technique of unit-gravity elutriation have been published elsewhere (25). The principle is illustrated in Fig. 1. The mixture of rosettes and free thymocytes was placed in a conical chamber, and medium was then pumped in continuously, at con- stant input velocity, at the base. The medium was stabilized by a serum gradient, and by running the separation in a cold room. A baffle at the chamber base prevented turbulence at the inflow disturbing the upper zones. As the medium rose in the cham-

88 SHORTMAN ET AL.

FIG. 1. Schematic diagram of the sepqration of different sized celk or multicellular complexes by zonal unit-gravity elutriation. Full details are in the text and Ref. (25). The cone-shaped chamber contains a bafIle to prevent the rapid inflow of medium causing a fountain effect. The inflowing medium is stabilized by a serum gradient. Cells or multicellular complexes sediment against the upflowing medium, which forms a continuously decreasing velocity gradient. Slowly sedimenting small ceils are elutriated from the chamber before faster sedimenting multicellular complexes.

ber to regions of increasing cross-sectional area, the upward velocity of the medium fell, creating a continuous velocity gradient. The cells and the rosettes sedimented at unit gravity against this ascending counterstream, and so could be separated into regions where their sedimentation velocity was equal to the upward velocity of the medium. In practice the medium input velocity was too fast to allow equilibrium banding, so first the free thymocytes, then later the rosettes, were flushed from the chamber. The uncentrifuged collagenase digest of the thymuses, containing rosettes and free thymocytes, still in the collagenase medium, was placed directly in the base of a 70-mm-wide (maximum diameter), 175ml volume chamber (25). The gradient used was from 20 to 60% FCS, in a Hepes-buffered balanced salt solution isoosmotic with mouse serum. The gradient generated was steep initially then shallower later and was pumped in at the rate of 165 ml/hr. On exit of medium from the chamber, 12 X 1 O-ml fractions were collected and screened directly for rosettes and free thymo- cytes by phase contrast microscopy. An example of the separation achieved is given in Fig. 2. The rosette-enriched fractions were then pooled, generally those corre- sponding to elutriation volumes 50- 120 ml in Fig. 2. The separation was sufficiently reproducible for rosettes to be collected as a single large fraction in the later ex- periments.

Dissociation of thymocytes from thymic rosettes. The rosettes were centrifuged to a pellet then suspended in a saline solution, isoosmotic with mouse serum, Hepes buffered at pH 7.2, lacking Ca2+ and Me but containing ethylenediaminetetraace- tate (EDTA), 5 mM. The suspension was incubated for 4 min at 37”C, with mixing with a Pasteur pipet. Cold FCS was then layered underneath the suspension, and the cells were recovered by centrifugation (4OOg, 7 min).

Correction offluorescence distribution projles for eflects of contaminants in thymic rosette fractions. To obtain a sample of the thymocytes normally contaminating the rosette fraction, thymic tissue was digested with collagenase and the cells released were separated by unit-gravity elutriation, exactly as for thymic rosette preparation except that the cells and rosettes were centrifuged to a pellet and the rosettes were completely dissociated with EDTA medium, as above, just before elutriation. The fractions normally pooled as rosette-enriched fractions then contained no rosettes, but contained free mononuclear cells taken to represent the free cell contaminants.

THYMIC ROSETTES 89

I’ rt 1 1 -Free Thymocytes

\

FIG. 2. Separation of thymic rosettes by zonal unit-gravity elutriation. Full details of the conditions are given in the text. The results represent one experiment typical of over 10 performed using this protocol.

The yield in these fractions amounted to a mean of 20% of the cells usually recovered after dissociation of rosettes postelutriation. To apply a correction based on these cells, each thymic rosette isolation was paired with an exactly parallel “contaminant” isolation, with exactly the same fractions pooled. The separated “contaminant” thy- mocytes were then stained under exactly the same conditions as thymocytes from rosette fractions. Normal thymocyte controls were run to ensure that near identical staining profiles were obtained. Using a computer program for this purpose, the num- bers of cells accumulated in the 256 channels (single parameter) or the 64 X 64 grid squares (two parameters) from 50,000 cell analyses of two to four such stainings were averaged. Twenty percent of the average value for each channel or grid square of the “contaminant” thymocyte staining was subtracted from the average of the “rosette” fraction staining. The corrected data, analogous to a difference spectrum, were then plotted. Such corrected profiles were used to assess the phenotype of the lymphoid cells actually in the complexes. An example of this correction process is given in Fig. 3.

Tests for phagocytic activity within thymic rosettes. Isolated rosettes were resus- pended in a RPM1 1640 medium containing 10% FCS and placed in slide chambers (LAB-TEK, Miles Scientific, Mulgrave Nth, Australia). The concentration was ad- justed so that individual rosettes were well spaced after sedimentation onto the slide. The slide chambers were then incubated for 2-6 hr, at 37°C in a 10% COz-in-air incubator. The medium was then gently removed, the rosettes attached to the slide surface were fixed with methanol:acetic acid:water 90: 1: 10, and then the slide was lightly stained with Giemsa. The proportion of rosettes in which the macrophage-like central cell had engulfed lymphocyte nuclei into the cytoplasm was counted. Over 200 rosettes were assessed per slide chamber, and at least two slide chambers were used per point.

Radioautographic assessment of L3Hj TdR-labeled dividing cells in thymic rosettes. Groups of 10 mice were injected once intravenously with 100 &i of r3H]TdR and 1

90 SHORTMAN ET AL.

1

All thymocytes Rosette-fraction thymocytes

‘;i;;gg;gii ._? :::.::::::::::::::::iiiiiiiiiiiiiiili_

E

i, ,~~~~

COntLminMtS in Rosette-associated Rosette-fraction thymocytes [Corrected I

CO4

FIG. 3. Two-color fluorescence analysis of the expression of CD4 and CD8 on rosette-associated thymo- cytes. The results give an example of the process of correcting the thymocytes in the rosette-enriched fraction for the contribution of the estimated 20% free mononuclear cell contaminants present. Full details are given in the text. Results are the distributions obtained from the pooled data ofeight analyses ofseparate preparations for all thymocytes (teased out thymocyte suspensions), four experiments on the elutriated rosette-enriched fractions, and four experiments on the contaminants isolated by destroying rosettes before elutriation. The contour levels represent threefold differences in cells per grid square.

hr later the animals were killed and rosettes isolated from the thymuses. Smears of cells from the rosettes were subjected to radioautography as described elsewhere (26).

RESULTS

Thymic Rosettes as Preexistent Structures Which Exchange with Free Thymocytes

Kyewski et al. (4) have demonstrated that rosettes are largely preformed within the thymus rather than assembled after tissue digestion; however, a small amount of exchange was noted (personal communication). It was important to check this aspect under our conditions, since a significant degree of exchange could distort the analysis of minor subpopulations. To assess the exchange of rosettes with free thymocytes when they were forced into contact by centrifugation to a pellet, rosettes were purified as described under Materials and Methods, and then mixed in suspension with free thymocytes which had been directly fluoresceinated, adding in twice as many free as rosette-associated thymocytes. Even without centrifugation, 1 hi-incubation at O&C led to 27% of all rosettes incorporating a fluorescent cell. In most cases only a single fluorescent cell was present, and the overall level of exchange was about 5% of the rosette-associated thymocytes. A single cycle of centrifugation and resuspension led to 68%,of rosettes including at least one fluorescent cell, and the overall proportion of fluorescent cells among rosette-associated thymocytes was 3 1%. Two centrifuga- tions led to 40% fluorescent cells in rosettes, and five centrifugations led to an equilib- rium exchange figure of 69% fluorescent cells.

It was clear that to avoid exchange conventional isolation procedures would need to be modified to eliminate any step where released rosettes were mixed with high

THYMIC ROSE’ITES 91

concentrations of free thymocytes, and to eliminate any step involving centrifugation together of rosettes and free thymocytes. To determine the effectiveness of the final procedure in avoiding exchange, three intact thymuses were first mixed with fluores- ceinated free thymocytes derived from three mechanically disrupted thymuses. When isolated according to the procedure in Materials and Methods, only 3% of rosettes included one possible fluorescent cell, and even here this may have been background autofluorescence. In contrast when a more conventional approach of teasing, diges- tion, concentration by centrifugation then purification by elutriation was used, 64% of rosettes included a fluorescent cell and 18% of rosette-associated thymocytes were fluorescent.

Purification of Thymic Rosettes

Each thymus treated with a mixture of mechanical and enzymic disruption re- leased about 2 X lo8 free thymocytes, about 6 X lo5 total rosettes (any structure with three or more thymocytes attached to a central cell), and about lo5 large rosettes (15- 20 thymocytes attached to a central cell). The challenge in isolation of these thymic rosettes was to obtain a maximum purification of a representative sample, under gentle conditions which maintained the structures intact and which minimized the opportunities for exchange of cells. Mechanical release and washing of free thymo- cytes from the tissue fragments prior to collagenase digestion gave the equivalent of a fivefold enrichment of rosettes. The zonal unit-gravity elutriation procedure was then used directly on the collagenase digest, without concentration by centrifugation, to separate released rosettes from free mononuclear cells. This procedure (illustrated diagramatically in Fig. 1) was designed to accomplish in a single step the same level of purification obtained in the standard procedures (3, 27) by repetitive sedimenta- tion over layers of serum. The effectiveness of the separation is shown in Fig. 2.

Rosettes sedimented much faster than most free thymocytes and mononuclear cells (Fig. 2). The rate of sedimentation increased with rosette size, as shown by the increasing medium volumes required to elute the larger structures containing 15-20 attached thymocytes. The faster sedimenting fractions also contained some rosettes with a very large central cell.

At the peak of rosette concentration, where rosette size averaged 6-8 thymocytes attached to the central cell, there were about 5 free mononuclear cells per rosette.(Fig. 2). This represented a 12-fold enrichment in rosettes over the input material and an estimated 70-fold enrichment over a whole.thymus suspension. Although some fur- ther enrichment of these peak fractions could be obtained by a concentration fol- lowed by a second elutriation, many rosettes then dissociated and the chance for exchange was increased, accordingly only the single elutriation approach was used. In the faster sedimenting regions, where rosette size averaged 15-20 thymocytes at- tached to the central cell, there was only one free m,ononuclear cell present per 3 to 10 rosette structures when the sample was examined before further handling or concentration; even these few free mononuclear cell “contaminants” may have de- tached from rosettes in the last stages of handling. This represented an estimated-330- fold enrichment of rosettes over a whole thymus suspension, and a purity of 99% for rosette-associated thymocytes. However, this highly pure region contained only 20% of all rosettes, and about 40% of all rosette-associated thymocytes, In m’ost experi- ments fractions were pooled so approximately 50% of all rosettes, generally in the 6-

92 SHORTMAN ET AL.

TABLE 1

The Distribution of CD4- and CD&Defined Subpopulations among Rosette-Associated Thymocytes

Fraction

Distribution of subpopulation (percentage) Ratio CD4+CD8-

CD4+CD8+ CD4-CD8- CD4+CD8- CD4-CD8+ CD4-CD8+

All thymocytes Rosette-enriched fraction Contaminants Rosette-associated thymocytes

(corrected) Large rosette fraction

(pure; uncorrected)

83.1 3.7 9.4 3.8 2.5 75.2 7.4 10.4 7.0 1.5 73.1 7.1 k4.0 5.8 2.4

75.7 7.5 9.5 7.3 1.3

75.2 7.9 12.1 4.8 2.5

Note. Results are the means of 4 experiments for the rosette-enriched fractions and contaminant fmc- tions, 10 experiments for total thymocytes, and 2 experiments for the large rosette fraction.

20 thymocytes per central cell size range, contributed to the rosette-enriched sample (corresponding to elutriation volumes 50- 120 ml in Fig. 2). There was then an aver- age of 2-3 free mononuclear cells per rosette in the pooled fractions. After concentra- tion by centrifugation the rosettes in these pooled samples contained a mean of 15 f 8 thymocytes attached to the central cell, and an average of 7 f 3 free mononu- clear cells (mainly lymphoid) for every rosette, two-thirds of these free thymocytes having apparently dissociated from the rosettes during concentration and resus- pension.

Corrections for Mononuclear Cell Contaminants in the Rosette-Enriched Fractions

As an additional method of ascertaining how many of the free cells in the final preparations were contaminants, rather than cells shed from the rosette structures themselves, rosettes were disrupted by EDTA treatment prior to elutriation (see Ma- terials and Methods). The cells appearing in what would normally be the rosette- enrichment fractions were then considered to be the contaminants, present due to the limitations of the separation procedure itself. Eleven paired experiments were run, where the only difference between the pairs was whether rosettes were disrupted just prior to elutriation, or just after pooling the postelutriation rosette-enriched frac- tions. Disruption of rosettes prior to elutriation produced fractions containing 20 + 9% of the total mononuclear cells in the regions normally pooled for rosette enrichment. Thus one in five of the thymocytes harvested from the normal pooled, rosette-enriched fractions was likely to be a nonrosette contaminant.

The phenotype of the mononuclear cells normally contaminating the rosette prep- arations was determined by examining the cells present in these pooled fractions when rosettes were disrupted prior to elutriation. Compared to a control thymocyte suspension the “rosette contaminants” included a higher proportion of large and medium sized cells, as would be expected from the selection for high sedimentation rate; however, small thymocytes were still the dominant population. The “rosette contaminants” included all four of the thymic populations defined by CD4 and CD8 expression (Fig. 3 and Table l), and by no other surface marker did they show any

THYMIC ROSETTES 93

CD8

FIG. 4. Two-color fluorescence analysis of the expression of CD4 and CD8 by the thymocytes associated with highly pure samples of large sized rosettes. The controls are mechanically teased out preparations of thymocytes, or the same preparations subject to the same collagenase digestion used to tease rosettes. The high purity, faster sedimenting, large rosette fraction was used. The figure displays one of two experiments giving near identical results.

extreme difference in phenotype from the total thymocyte population, or from the thymocytes of the “rosette-enriched fraction.”

The information on both the number and the fluorescence staining intensity of the “contaminants” allowed a correction to be made to the fluorescence distribution profiles of the “rosette-enriched fraction” thymocytes (using the procedure described under Materials and Methods) to produce an estimate of the “rosette-associated thy- mocyte” distribution. An example of the correction process is given in Fig. 3 and Table 1. It should be emphasized that in no case did the corrected “rosette thymo- cyte” profiles differ radically from the original data on the “rosette-enriched fraction thymocytes.” In all cases the effects of various degrees of correction (assuming con- taminant levels of between 10 and 40%) was tested, to determine what differences in calculated phenotype would have occurred if the actual level of contaminants differed from the measured average of 20%. No extreme differences were apparent in any of these analyses. In subsequent figures only the corrected “rosette-associated thymo- cyte” results are presented, and these are compared to results on a normal, mechani- cally dissociated thymocyte preparation. Additional control experiments (such as those given in Fig. 4) established that exposure of thymocytes to collagenase-DNAse did not detectably change the fluorescent distribution profiles of any of the surface markers used.

The Expression of CD4 and CD8 on Rosette Thymocytes

The free thymocytes of mechanically dissociated thymus suspensions could be di- vided into four distinct populations on the basis of CD4 and CD8 expression (Fig. 3). These were the major population of “double positives” (cortical-type cells), the two populations of “single positives” (mainly mature, medullary-type thymocytes), and the population of “double negatives” (mainly early thymocytes). All four popula- tions were also found in the thymocytes of the isolated rosette-enriched fraction and were present after correction of the profiles for contaminants (Fig. 3). These results agree with the published work showing that cortical-type CD4+CD8+ cells are the dominant thymocytes associated with rosettes, but extend this by clearly demonstrat- ing the presence of the three other groups. The rosettes were not restricted to a single mature T-cell lineage, since both types of single positives were present. It was impor-

94 SHORTMAN ET AL.

All thymocytes

Forword Light Scatter

FIG. 5. Low-angle light scatter distribution of rosette-associated thymocytes. The results are derived from two sets of paired experiments, on rosettes and contaminants, and are corrected for the contribution of contaminants as in Fig. 3. The broken line is the control data from mechanically disrupted thymus suspen- sions.

tam to note that the four CD4- and CDS-defined populations of rosette-associated thymocytes showed fluorescence intensity identical to that from free thymocytes, and there was no marked concentration of any subpopulation of “intermediate” pheno- type, especially after correction for contaminants.

The proportions of the four subpopulations did however differ somewhat between rosette-associated and free thymocytes, as shown in Table 1. There were proportion- ally more of the mature single positives associated with rosettes, and the ratio of the CD4XD8+ to the CD4+CD8- cells was generally closer to 1: 1 than to the 2 or 3: 1 ratio characteristic of both thymic and peripheral mature T-cell populations. There were also proportionally more of the early double negatives among the rosette-associ- ated thymocytes.

These findings were then checked using the highly purified large rosette fractions, where no correction for contaminants was required, and where any selective loss of cells from the rosette structures might have been less extensive. The results (Fig. 4) confirmed the basic findings that all four subpopulations were present, and that the rosette-associated thymocytes were somewhat enriched for “double negatives” and “single positives” (Table 1). However, in this faster sedi’menting fraction the ratio of CD4+CD8- to CD4-CD8+ thymocytes was higher than in the more representative rosette fraction.

The Size Distribution and Incidence ofDividing Cells among Rosette-Associated Thy- mocytes

The relative size of rosette-associated thymocytes was assessed from the low-angle light scatter measurements collected during fluorescence analysis. Scatter distribu- tions from’the rosette-enriched fractions were corrected as before for the contribution of contaminants, although the corrections involved were slight; an example of the resultant low-angle light scatter distribution profiles is given in Fig. 5. The predomi- nant cells in rosettes were clearly the same size as the small cortical thymocytes. This indicated that the CD4+CD8+ cells in rosettes were mainly small, nondividing cells rather than being all large, dividing cells. However, rosettes contained a higher pro- portion of medium and large sized cells, compared to normal free thymocytes. This

THYMIC ROSETTES 95

CD 3

FIG. 6. The expression of CD3 on rosette-associated thymocytes. The results are derived from two sets of paired experiments on rosettes and contaminants and are corrected for the contribution of contaminants as in Fig. 3. The broken line is the control data from mechanically disrupted thymus suspensions.

is in accordance with the higher proportion of single positives (medium sized) and double negatives (large sized) as shown in Table 1. These light scatter distributions were checked on the pure sample of large sized rosettes, and very similar results were obtained. The scatter from the very large sized macrophage-like cells in rosettes was much greater than for the lymphoid component, and “off scale” in these diagrams.

To determine the incidence of dividing cells among rosette-associated thymocytes, animals were given a I-hr pulse of [3H]TdR, thymuses were removed, and the rosette- enriched fractions (and paired contaminants only fractions) were isolated, and the associated thymocytes were subjected to radioautography. The rosette-enriched frac- tions were corrected for the contribution of contaminants, as before, although this correction was relatively small. In three experiments rosette-associated thymocytes were found to contain a mean of 12.2 -t 0.4% labeled cells (cells in S phase) whereas the free thymocytes included 10.0 + 0.4% labeled cells. Thus the proportion of cells in division was slightly higher than among free thymocytes.

The Expression of the T-Cell Antigen Receptor by Rosette-Associated Thymocytes

The interaction of thymocytes with macrophages or dendritic cells has been pro- posed as a key event in repertoire selection, but this would demand that the thymo- cytes being selected display their TcR. We checked if rosette-associated thymocytes included a high proportion of cells expressing some TcR, by using the monoclonal antibody 145-2C 11 which detects the E chain of the CD3 complex (28). As shown in Fig. 6, the majority of rosette-associated thymocytes displayed negative-to-low levels of CD3 similar to free thymocytes, a pattern consistent with the major cell type in rosettes being a typical small CD4CDV cortical thymocyte. Rosette-associated thy- mocytes also gave a distinct but smaller peak of cells expressing high levels of CD3, and there were proportionally more of these than present in a typical thymocyte sus- pension. This is in accordance with the higher proportion of “single positives” within the rosette-associated population (Table l), the majority of which are CD3+. These findings were checked with the highly pure sample of faster-sedimenting rosettes, which gave near-identical results.

Expression of H-2K by Rosette-Associated Thymocytes

H-2K is expressed at a high level on mature single positive thymocytes ( 17,23) and on some early or double negative thymocytes (18), but is either at a low level, or is

96 SHORTMAN ET AL.

FIG. 7. The expression of H-2K on rosette-associated thymocytes. The results are derived from two sets ofpaired experiments on rosettes and contaminants and are corrected for the contribution ofcontaminants as in Fig. 3. The broken line is the control data from mechanically disrupted thymus suspensions.

completely absent, on cortical double positive thymocytes (17). The H-2K distribu- tion of a normal, mechanically disrupted thymus suspension (Fig. 7) therefore gave a main peak of negative-to-low cells, and a minor peak of cells showing bright H-2K fluorescence. The rosette-enriched fraction thymocytes and the contaminant-cor- rected rosette-associated thymocytes (Fig. 7) gave a different distribution. The H-2K bright cells were clearly present in rosettes, and at a higher incidence, as would be expected from the higher incidence of single positive and double negative cells (Table 1). However, the main peak of rosette-associated thymocytes, corresponding to small CD4+CD8+ thymocytes, was now of cells showing low-to-intermediate levels of H- 2K rather than low-to-negative levels. Thus the small CD4+CD8+ thymocytes in ro- settes differed from the general small cortica1 thymocyte population in expressing higher levels of H-2K. These findings were checked in two experiments using the highly purified fast-sedimenting rosette fraction. The results were nearly identical, with the predominant rosette-associated thymocyte expressing intermediate levels of H-2K.

To check if the pattern of H-2K expression on rosette-associated thymocytes was likely to be generated by selective death of the cells which were negative for H-2K during rosette isolation, a suspension of free thymocytes was incubated at 37°C for periods from 0 to 2 hr in a culture medium, or for 30 min with collagenase-DNAse, then stained and analyzed for H-2K expression, excluding dead cells from analysis by propidium iodide staining. No shift of the main peak to higher levels of H-2K expression was obtained (data not shown).

Expression of Thy 1 and Heat Stable Antigen on Rosette-Associated Thymocytes

Two surface antigens present on most thymocytes, but at different levels on cortical and mature thymocytes, are Thy 1 ( 17) and HSA, the heat stable antigen recognized by the monoclonal antibodies B2A2, M1/69, and J 1 Id (10, 19, 20, 22). Both Thy 1 and HSA were expressed at high levels on the main group of free thymocytes (pre- dominantly the CD4+CD8+ cortical cells), with a trail of cells of lower expression (predominantly the CD4+CD8- and CD4CD8+ mature cells with some CD4CD8- cells). In contrast to the results with H-2K, the majority of rosette-associated thymo- cytes showed the same high levels of Thy 1 and HSA as free thymocytes (data not

THYMIC ROSETTES 97

shown), indicating the major group of small CD4+CD8+ in rosettes expressed normal levels of these markers. However, the rosette-associated thymocytes included more cells expressing low levels of both antigens, in accordance with the higher incidence of CD4-CD8+, CD4+CD8-, and CD4CD8- cells (Table 1).

Surface Phenotype ofRosette-Associated CD4-CDS- Thymocyte Subpopulations

The CD4CD8- “double negative” thymocytes can be divided into a series of sub- populations, some of which have been shown to represent early stages of T-cell devel- opment (10,20). In defining these subsets the surface antigens HSA, Thy 1, CD5 (Ly l), and IG2R (the determinant on the chain of the interleukin-2 receptor recognized by the monoclonal antibody PC6 1) are particularly useful (20). In addition, a propor- tion of the CD4-CD8- thymocytes which are HSA- express the TcR-CD3 complex, with a very high utilization of V/38 variable region genes (10, 2 1). To check if the CD4-CD8- subpopulations associated with rosettes differed from those free in the thymus, a series of two- and three-color flow-cytometric analyses was performed. Rosette-associated thymocytes were stained with CD4 and CD8 together in one color, to allow gating for CD4CD8- cells. The other colors were used for single-parameter analyses for the antigens above, or for two-parameter analysis of HSA versus CD3, or HSA versus VP8 (using F23.1). The results (data not shown) in all cases showed no significant difference in subpopulation distribution or TcR-CD3 complex expression between rosette-associated and free CD4-CD8- thymocytes. There was no evidence that TcR+ CD4-CD8- thymocytes selectively associated with rosettes.

Extent of Phagocytosis ofRosette-Associated Thymocytes

If the formation of a rosette structure was a prelude to the elimination of thymo- cytes by the central macrophage-like cell, it should be possible to find evidence of phagocytosis of the associated thymocytes. If smeared and stained immediately after isolation, the central cell of a minor proportion of rosettes had nuclear debris within the cytoplasm, but the origin of this nuclear material was unclear. As a more direct test, thymic rosettes were isolated, suspended in culture medium, and incubated in slide chambers for various periods, under conditions where phagocytic cells would be fully active. After various periods the medium was gently removed, and the ro- settes and cells which had settled onto the slides were fixed and stained with Giemsa. The proportion of rosettes where the central cell had engulfed one or more whole thymocytes was then counted. After 2 hr a mean of 23% (range 16-30%) of rosettes showed thymocyte engulfment by the central cell. After 6 hr the proportion was un- changed (mean 22%). It appeared that phagocytosis of thymocytes was a process oc- curring only in a minority of rosettes.

DISCUSSION

This study has supported the view of Kyewski et al. (4) that the rosettes isolated following collagenase digestion of the thymus reflect an association between lym- phoid and nonlymphoid cells that preexists within the thymus. We cannot exclude the possibility that the central cell picks up some or all of the associated thymocytes immediately alter tissue disruption begins, but at least this pickup must be from cells in the immediate vicinity, rather than occurring randomly after release from the tis-

98 SHORTMAN ET AL.

sue structure. However, our study has also emphasized the ease with which random pickup and exchange can occur during rosette isolation after the initial release from the tissue, and special precautions are needed to keep this exchange well below a level of I %. An essential aspect of our work has therefore been to devise a separation and purification procedure where random exchange is minimal, so that the rosette-associ- ated thymocytes can be analyzed with some confidence that any subpopulations found represent genuine components (or at least close neighbors) of the structure in vivo.

Our unit-gravity elutriation technique (25) has provided a very gentle procedure for rosette isolation and allowed us to avoid the centrifugation steps that cause rosette dissociation and exchange. Using this technique the larger sized rosettes were ob- tained virtually pure, in a single step. The smaller sized rosettes were somewhat con- taminated with free mononuclear cells, and our efforts to include them in the study involved us in the tedious process of purification of a sample of the contaminants and subsequent correction of the data for their presence. Although there was evidence for a difference in the ratio of the mature thymocyte subsets present in large versus small rosettes, the overall results were very similar. For many purposes it should in future be possible to adopt a simpler approach, and study only the purer, larger sized rosette fractions. The structures in the faster sedimenting fractions probably corre- spond to the type of rosettes normally obtained using the conventional approach of repetitive sedimentation over serum, since the smaller rosettes would be selectively eliminated with the free thymocytes.

There is a wide range of views of the physiological significance of thymic rosettes. They may be considered a garbage disposal system, collecting then phagocytosing reject thymocytes, or as sites of induction of early steps in T-cell development, or as sites of selection of cells with appropriate TcR specificity, or simply as structural elements with no developmental role. Our results are pertinent to all these views without pointing exclusively to one or the other.

Some of our data are compatible with a garbage disposal unit model. We find that most of the cells associated with rosettes are, as others have found (6-8), typical small cortical thymocytes, the majority of which are fated to die within the thymus (29), and presumably to be disposed of by phagocytic cells. However, not all the rosette- associated thymocytes are of this “end cell” type. Our in vitro experiments on isolated rosettes did indicate that the central macrophage-like cell was in some cases able to engulf attached thymocytes, as expected of a cell disposal system. However, only about one in five rosettes showed this behavior, a result similar to that reported by Epstein et al. (6). So garbage disposal may be part of the role of only some of these structures.

The view that rosettes represent a special microenvironment for inducing early steps in T-cell development receives some support from our observation that there are early, CD4-CD8- thymocytes associated with rosettes, and that rosette thymo- cytes are somewhat enriched for larger dividing cells. There is little evidence that rosettes direct development exclusively down one T-cell lineage, since both CD4-- CD8+ and CD4+CD8- mature phenotype cells are included in rosettes. However, the presence in rosettes of these mature single positive thymocytes, and of small double positive thymocytes, argues against rosettes being exclusively concerned with early steps of development.

If rosettes were involved in the selection of the TcR repertoire, it might be expected that the contact between the central cell and the associated thymocytes would be

THYMIC ROSETTES 99

mediated via the TcR. Our data indicate that there is some enrichment of CD3+ (and thus TcR+) thymocytes within rosettes, and to this extent we support the view of Kyewski et al. (8) that the data are compatible with a repertoire selection model. However, this is an optimistic interpretation at this stage, since the majority of rosette-associated thymocytes are CD33, their interaction is not receptor mediated, and they cannot be engaged in a process of repertoire selection.

Our subpopulation analysis data overall do not support the view (7,8) that rosette- associated thymocytes are a special transitional thymocyte subpopulation at some intermediate, partially mature stage of development. While the analyses do show a very small proportion of cells of intermediate phenotype in terms of CD4 and CD8 expression, the majority of rosette-associated thymocytes fall into precisely the same categories as the total free thymocyte population. Thus the finding that rosette-associ- ated thymocytes are predominantly CD4+CD8+ like cortical thymocytes, but overall have some immunological function, can now best be explained by the presence of a small group of functional, CD4CD8+ and CD4+CD8- cells within rosettes. Likewise the thymocyte subpopulation within rosettes expressing high surface levels of TcR are likely to be these “single positives,” rather than the predominant group of “double positives.”

The one really striking difference we have found between rosette-associated and free thymocytes is the relative absence on rosettes of those thymocytes which lack surface H-2K expression. This result serves as an internal control showing that associ- ation of a thymocyte with a rosette is not just due to some random pickup process. The result indicates selectivity between different types of small cortical CD4+CD8+ thymocytes, those that are H-2K’“” being present in rosettes and those that are fgqpegative being absent. This selective absence of the 50% H-2Knegati” thymocytes in rosettes is enough to explain the relative depletion of double positive thymocytes and enrichment of double negative and single positive thymocytes shown in Table 1. However, the significance of this selectivity is not clear. The result is compatible with the existence of an “intermediate” CD4+CD8+ cell en route to becoming a mature “single positive” (7, 8) but we consider this the least likely interpretation in view of the other results discussed above. The most direct interpretation would be that the H-2K’“” cells eventually develop into H-2K”“p”ti’” cells which then leave the rosette structure, or perhaps are phagocytosed. We cannot entirely exclude the more trivial explanation that the H-2Knegative thymocytes are the more fragile, and have died or been selectively lost during rosette isolation, although our control experiments argued against this. Nor can we exclude the possibility that the thymocytes have picked up H-2K from the central dendritic cell or macrophage, much as has been described for surface Class II expression by thymocytes (30). However, we have found no clear correlation between TcR expression and H-2K expression by these cells, making this explanation less likely.

Overall these results, apart from those on H-2K expression, fail to provide a unique phenotypic pattern that could point to the physiological function of rosettes, since by most parameters the rosette-associated thymocytesare disappointingly like the total free thymocyte population. However, as Kyewski (3, 5-8) has pointed out, thymic rosettes are of at least two distinct types, those with a macrophage-like central cell which are located in the cortex, and those with a dendritic-like central cell located in the medulla or at the cortico-medullary junction. These different types of rosette could well have entirely different functions, and could be associated with quite different thymocyte populations. Indeed, our own preliminary data have indicated

100 SHORTMAN ET AL.

that the thymocytes associated with the macrophage rosettes are phenotypically dis- tinct, and the differences in the CD4+CD8- to CD4CD8+ ratio in the faster sedi- menting fractions, which seem to include more of the macrophage-associated ro- settes, are a reflection of this distinction. This study therefore serves as a starting point for further investigation involving separation of the different classes of rosettes prior to analysis of the associated thymocytes.

ACKNOWLEDGMENTS

We are grateful to Roland Scollay and Anne Wilson for their advice and assistance, and to Mark Cozens for help with the flow cytometry. The experiments were supported by the National Institutes of Health, USA, Grant No. AI 173 10; by the C. H. Warman Research Fund; and by the National Health and Medical Research Council, Australia.

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