The differentiation of T-lymphocytes

CELLULAR

IMMUNOLOGY

27, 256-273 (1976)

The Differentiation III. The Behaviour in

of Subpopulations Short-Term H~PI?ER

KELVIN

of T-Lymphocytes of Mouse Thymus

Cell Culture1

AND

KEN

SHORTMAN

The Walter and Eliza Ho11 Ilzstitzlte of Medical Hospital P.O., Victoria Royal iWclbozrr~zc Rcr&led

Cells

Jwe

Rcscarciz, dlclhurne, 31150,Amtralia

9, 1976

Adult mouse thymus cells were incubated at 37°C in culture medium in order to The level of dividing cells fell examine various aspects of T-cell differentiation. rapidly after culture, and there was a reduction in cell size. A striking effect was the loss of thymocyte viability bvhich occurred in two phases. A total lysis of some cells occurred over the first 2 hr, but this Jvas less extensive with higher cell concentrations, or in the presence of serum. This phase was followed over the next 70 hr by a steady loss of cell viability, even under optimised conditions. Thymocyte death was more extensive than the death of spleen or lymph node cells under the same conditions. Thymocyte death in culture \vas limited to a particular subset of cells. Rapid loss of viability was a constant feature only of the major thymocyte population (characterised as high Thy 1 alloantigen (e), low histocompatibility antigen (H2), thymus leukemia alloantigen (TL) + ve, and cortisone sensitive). Within this group survival time was a function of intrathymic age, with maximum lability in older cells. Cells initially large survived better than small cells. In contrast, the minor thymocyte subpopulation (characterised as low 8, high HZ, TL - ve, and cortisone resistant) survived well, with an occasional increase after 5-24 hr of culture. Isolated populations of low-8 cells gave the same good survival in culture, showing they were not simply being replenished from the high-8 elements. The results document another basic difference between the major (high 0) and minor (low 8) thymocyte subpopulations, namely, differential survival in cell culture. The results are in line with the view that the major and minor subpopulations represent largely independent lines of development, rather than having direct precursorproduct relationship. The extreme lability of the major, high-0 subset could reflect a programming for intrathymic death.

INTRODUCTION

The differentiation of T-cells occurs within the thymus gland, but the actual developmental pathway and the role of the thymic environment and thymic hormones are not yet clear. Some basic but unresolved questions are the extent of intrathymic cell death compared to emigration from the thymus, and the inter-relationships between thymocyte subpopulations. It is known that stem cells seeding in the thymus lead to a small population of rapidly cycling cells (mean cycle time 8-9 hr) which generate nondividin g thymocytes (1-S). Following an intrathymic life1 This is Publication

Copyright All rights

0 1976 by of repwduction

No. 2137 from

Academic Press, Inc. in any

form

reserved,

the Walter

and Eliza

Hall

Institute.

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span of about 3 days, cell emigration occurs, but estimates of the rate of cell exit vary widely (1, 5-14). In general, there appears to be a disparity between the high rate of cell production and rate of seeding in peripheral tissues, leading to the suggestion by Metcalf (2) that most cells born in the thymus die there. Recent evidence from thymidine reutilization studies is in line with this view (15). Further information is obtained from a study of antgenically defined thymocyte subpopulations. Two general classes are readily distinguished, the major population of immunologically inactive cells (high 192 (Thy 1)) TL positive, low H2, generally cortisone sensitive, and located in the cortex) and the minor, active subpopulation (low 0, TL negative, high HZ, generally cortisone resistant, and located in the medulla) (16). These were originally believed to have a precursor-product relationship. However, Shortman and colleagues (17-21) and Schlesinger (16, 22) have suggested the two subpopulations may represent separate streams of development. In the first paper of this series (20)) kinetic studies on intact animals, using a thymidine labelling and autoradiographic approach, indicated that the two subpopulations developed independently. This has been supported by the recent work of Fathman et al. (23), although in their model the two separate lines develop from a common high-8 dividing precursor. 1Ye have proposed that the low-8 de\-elopment pathway leads to active peripheral T-cells, whereas the high-0 pathway could lead to a terminal cell programmed for intrathymic death, this being the reaction mechanism for deleting self-reactive cells (17-21). Studies on thymoc:\te maturation using intact animals have been limited by the difficulty of quantitatively measuring cell exit and entry from the thymus, by the difficulty in assessing cell death rates, and by lack of suitable cell transfer systems for following directly the precursor-product relationships of isolated thymocyte subsets. This study was designed to follow the behaviour of thymocytes over short periods of cell culture, in a closed and manipulable system where accurate balance sheets can be maintained. Clearly in such cultures the normal steady state of the thymocyte population is disrupted, and important components of the microenvironment may be lacking. However, some of the basic characteristics of the developed subpopulations can readily be examined. l\‘e report further evidence for the independent behaviour of the low-8 subpopulation, and document the rapid death cells under a range of culture conditions. of hi&0 MATERIALS

AXD

METHODS

Mice. In most experiments male, inbred CBA/Ca H Wehi mice, G-7 weeks of age were used to provide thymus cells. In the experiment using TL as a marker, 7-week-old male A/J mice were used. Prefiaration of
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(viable plus dead)/ml in COz-bicarbonate buffered, mouse osmolarity, Eagle’s minimal essential medium (MEM) containing 10% FCS and supplemented with pyruvate (1 g/liter), penicillin (25 mg/liter), streptomycin (20 mg/liter), and Z-mercaptoethanol ( 1O-4M) . Cortisone-resistant cells. Mice were given one intraperitoneal injection of 2.5 mg of cortisone acetate (Merck, Sharp & Dohme (Australia) Pty. Ltd.) and the thymus was removed 48 hr later. The cells were then isolated as above, and any damaged cells were selectively removed by the low ionic strength aggregation procedure (26). Bulk isolation of the low-8 cells. The high-8 subpopulation was selectively killed by controlled anit- antiserum and absorbed rabbit complement treatment, as described previously (20), and the damaged cells were then selectively removed by low ionic strength aggregation (26). Cell cuItzrre conditions. Unless otherwise described, cells were cultured in Marbrook vessels (27)) ( 1.2 cm inner chamber diameter, 2.2 cm diameter of outer vessel) fitted with Nuclepore membranes (Nuclepore Corp., Pleasanton, Calif.) having l-pm pore size (N 100 CPR 04700). The cells in 1.0 ml of MEM-FCS were placed in the inner chamber and in 4 ml of MEM-FCS in the outer chamber. The cultures were maintained in a humidified incubator at 37°C with continuous passage of 10% CO, in air. At time intervals the cells were removed, the inner chambers were washed with 0.5 ml BSS-10% FCS, and the suspensions were made up to 2 ml with BSS-10% FCS. Short-term cultures of up to 3 hr containing 1, 5, 10, and 20 X lo6 cells/ml in a total of 5 ml MEM-BSS (1 : 1) plus 10% FCS and 1O-4M 2-mercaptoethanol were set up in Falcon plastic tubes and kept in suspension by gentle intermittent agitation. Aliquots of 0.1 ml were removed at 3-min intervals up to 20 min and at lo- to 15-min intervals up to 3 hr. Total viable and dead cells. Numbers of viable and dead nucleated cells were counted in a haemacytometer under phase contrast optics after addition of 0.1 ml of eosin (1% w/v in normal saline) to 0.1 ml of the cell suspension, Counts of 200300 cells were made for each sample. Low-0 and high-0 cells. Low-8 cells (and high-0 cells by difference from controls), were determined by a differential cytotoxic assay using diluted AKR mouse antiCBA thymocyte serum and absorbed rabbit complement, according to the method of Shortman and Jackson (20). Controls of AKR serum plus complement were run in parallel. Nonspecific lysis due to complement was negligible under the conditions used. Some cell loss due to incubation at 37°C alone did occur, corresponding to the labile cell population described earlier (20). Normal thymocyte populations contained 15 i 4% low-0 cells in 20 routine assays. High-HZ and low-H2 cells. The division of thymocytes into two categories depending on the surface levels of H2 alloantigen was performed by the immunofluorescent technique of Cerottini and Brunner (28), as described elsewhere (17)) using a C57BL/6 anti-CBA antiserum. To eliminate the background low level nonspecific staining by damaged cells, all dead and damaged cells were eliminated prior to assay by a neutral pH density cut procedure (25). TL + ve and TL - ve cells. The division of A/J mouse thymocytes into a major population expressing surface TL alloantigen and the minor population lacking TL, was also performed by immunofluorescence. The anti-TL antiserum was obtained from Dr. U. Hammerling via Dr. T. Mandel, and was produced by the injection of

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A’l’T,-ncg;ltivc nlicc (congenic with A/J mice) with ASL 1 tumor cells. A/J mmse tllytnocytcs lvere incubated with this antiserum (diluted 1 : 8) then binding of t11e antiserum was revealed using a commercial polyvalent goat anti-mouse fluorcsceinated antiserum (Kallestad, diluted 1 : 16). Fluorescent cells were comited using Leitz Pleon epi-illumination. Control studies with TL-negative strains showed no fluorescent cells in the thymus. To eliminate the background, low level nonspecific staining by damaged cells, all dead and damaged cells were eliminated prior to assay by a neutral pH density cut procedure (25). Nzwzbcrs of dividing cells. The uptake of [“H]TdR by the cells in culture was used as an estimate of the frequency of dividing cells. Aliquots of cell suspension containing 3-5 X lo6 cells were pulsed for 30 min with 5 &i of [3H] TdR ( [6-‘HIthymidine 22Ci/mol, the Radiochemical Centre, Amersham, U.K.) in small plastic tubes in a water bath at 37°C. The reaction was terminated by placing the tubes in ice and the cells were washed by centrifugation three times through a stepwise gradient of FCS. Smears of the cells in FCS were made on gelatin-coated slides, then prepared for autoradiography, and counted as previously described (20). In viva labcllilzg of cells uf various ages. In experiments designed to follow the fate and development in culture of cells of various ages, G-week-old mice were injected with [SH]TdR under conditions giving close to continuous labelling of the thymus cells, as described in an earlier paper (20). Dividing cells were labelled by injection of 50 &i iv 30 min before killing. Cells up to 1 day old were labelled by 5O+Ci iv injections 24 hr and 30 min before killing. Cells up to 2 days old were labelled by 50-&i iv injections 48 hr, 24 hr, and 30 min before killing. The thymus cells were then isolated and cultured in triplicate. At time points the cells xvere recovered and damaged cells were removed by a neutral pH density cut procedure (25). The cells were counted and smears and autoradiographs were prepared as previously described (20). Scdinfentation velocity separation. Cells were separated by sedimentation velocity at unit gravity for 2 hr using the basic procedure of Miller and Phillips (29), as described elsewhere (21) . RESULTS Swvival

of Tlzymzu Cells in Culture

Even when conditions were optimized, the most marked characteristic of adult mouse thymocytes in culture was the extensive loss of viable cells. The balance sheet for viable (eosin excluding) and nonviable (eosin stained) cell recovery over 70 hr of culture under the optimized conditions given in Materials and Methods is shown in Fig. 1. The loss of viable cells was in two phases. The initial phase comprised a rapid loss of (under these conditions) up to 2070 of the original viable cells over the first 2 hr. During this phase there was a net loss in all cells, not just viable cells, indicating complete cell disintegration. Adherence of cells to the vessel walls was minimal, as verified by microscopy. This first phase was followed by a second more gradual drop in viable cells over about 50 hr. During this second phase the total cell number remained constant, the loss of viable cells being accountable by an increase in dead cells that remained intact throughout the culture. These two phases of cell death will be considered separately in more detail.

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VIABLE

0

CELLS

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TIME IN CULTURE

[HOURSI

FIG. 1. Kinetics of death of adult CBA mouse thymus lymphoid cells in culture. Points and error bars represent means and standard deviations (not standard errors of the mean) from each containing duplicate cultures for each time pooled data of 16 separate experiments, point and assayed in duplicate. Cells were obtained from the pooled thymus glands of three mice for each experiment.

Rapid Loss of Labile Thysocytes Short-term cultures were set up as described in Materials and Methods to study factors affecting the initial phase of cell loss. The effect of the two major factors is shown in Fig. 2. Rapid cell loss was greatly enhanced by the absence of serum from the medium. Under these conditions, cell loss was very cell-concentration dependent, the higher cell concentrations being protective. Note that the perNO SERUM

WITH SERUM

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FIG. 2. The effect of serum and cell concentration on survival of thymus cells during shortterm incubation. Different concentrations of thymus cells were cultured in suspension in Falcon tubes in the absence or in the presence of 10% FCS. Numbers refer to millions of cells in 1 ml of medium. Each curve is based on cell counts from single aliquots of 0.1 ml taken at 0-, S-, 15, 30-, 60-, loo-, and 160-min time points from single cultures of 5 ml. The upper curves give percentage viability of those cells recovered. The lower curves show the actual total viable cells recovered as a percentage of the total placed in culture.

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FK. 3. Comparison of percentage viability and numbers of viable cells in thymus, spleen, and lymph node lymphocyte cultures. Means and standard deviations from duplicate assays of triplicate cultures in a single experiment are shown. Cells from the pooled lymphoid organs of three mice were used.

viability (ratio of viable cells recovered to all cells recovered) remained relatively constant, despite the net loss of viable cells. More debris was noted in those cultures showing a net loss of viable cells, indicating that the lost cells disintegrated completely, rather than appearing as eosin-stained dead cells. In all further experiments culture conditions were optimized (10 x lo6 cells/ml ; 10% FCS present) to minimize this early death phenomenon. In addition, most subsequent results have been normalized to a l- or 2-hr time point, to direct attention only to the second more gradual phase of cell loss where complete cell balance sheets could be maintained. centage

Conzparison of Cell Loss and Cell Division Node Cells

Rates of Thymus,

Spleen,

and Lymph

To determine if rapid death in culture was a phenomenon common to all lymphocytes, cultures of spleen, lymph node, or thymus cells were compared. As shown in Fig. 3, thymus cells died more rapidly in culture, both as seen by the net loss in viable cells, and the change in viable count amongst those cells recovered. Maintenance of cell numbers in culture by spleen or lymph node cells could have been due to increased cell generation by cell division. Accordingly, [3H]thymidine pulses and autoradiographic assessment were used to compare the incidence of dividing cells in the cultures at various time points. However, as shown in Fig. 4,

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TIME IN CULTURE [HOURSI FIG. 4. Comparison of the cell division rate of thymus, spleen, and lymph node cells in culture. At each time point a proportion of cells from duplicate cultures was pulsed with [aH]TdR and the percentage of viable cells containing label was determined by autoradiography. In each case 400 total viable cells were counted on duplicate smears from duplicate cultures. The thymus cell curve is the mean (2 standard deviation) of two separate experiments, while the spleen and lymph node curves represent single experiments. Pooled lymphoid organs from three mice were used for each experiment.

thymus cells had by far the highest cell division rate over the first 10 hr, although this dropped off rapidly to a negligible level by 20 hr. Clearly, since more new cells were being generated early in the thymocyte cultures, the actual death rate must have been even higher to maintain the net cell loss shown in Figs. 1 and 3. The Relationship

between Cell Size and Cell Survival

TO determine if all thymocyte subpopulations behaved the same in culture, the survival of cells differing primarily in cell size was compared by fractionating thymocytes by sedimentation velocity at unit gravity, then culturing cells from the individual fractions for 10 hr. The sedimentation velocity profile of the starting population, and the percentage survival of cultured fractions is given in Fig. 5. Clearly, the faster-sedimenting larger cells survived better than smaller cells, and in certain fractions an increase in total cells was obtained, a consequence of cell division. Thus, cell death was a characteristic of the major, small thymocyte population, whereas survival and proliferation was a charcteristic of large cells. To determine if cells changed during the period of culture, sedimentation velocity separation was carried out after a IO-hr culture of unfractionated thymocytes. In culturing these cells mouse osmolarity medium was used, and no evaporation of the medium was recorded. As shown in Fig. 6, the sedimentation profile of the viable cells after culture showed a marked shift to more slowly sedimenting, presumably smaller, cells. The results of Figs. 5 and 6 combined show that smaller cells tend to die, whilst larger cells develop into smaller cells, helping to maintain the smaller cell population.

Antigen&

Markers for Thymocyte Subpopulations

To determine if the antigenically defined thymus subpopulations had the same survival characteristics, samples of thymocytes after various times of culture were

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’ 0

SEDIMENTATION RATE (mm/h)

FIG. 5. Sedimentation velocity separation of viable thymus lymphocytes showing the percentage survival of separated cells after culturing individual fractions for 10 hr. Thymus cells from three mice were used and single cultures for each fraction were prepared and counted. Under the conditions used (mouse osmolarity, 6°C) the sedimentation rates were about 10% higher than would be observed under the standard conditions used by others (human osmolarity, 4°C). The size of most thymocytes of the modal sedimentation rate (about 3 mm/hr) was loo-130 pm’.

assayed for the relative proportion of cells bearing different markers. The main assay used was differential sensitivity to anti-0 antiserum and complement, an assay designed to reflect the different levels of 0 (Thy 1) on the surface of the major (high 19) and minor (low 6) subsets. This assay was rapid, quantitative, and objective. It offered the advantage that the same technique, scaled up, could be used to isolate the minor subpopulation, by first killing all highly sensitive cells and then eliminating dead cells from the suspension. To verify the results from these experiments, immunofluorescent assays directly reflecting the surface levels of HZ and TL antigens were also used.

SEDIMENTATION

FIG.

culture.

RATE Immlh

1

6. Sedimentation velocity separation of thymus lymphocytes before and after 10 hr in The same total number of cells (120 X 10’) was separated in each case. Only the viable

cells were countedin the distribution curves.

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CBA mice: N0r1Tlal Cortisone resistant Low-8 cells, isolated by killing high-8 A/J mice: Normal Cortisone resistant Low-8 cells, isolated by killing high-8

~IOI’I’ER

AND

Cytotoxic differential

assay B level

Low B

Hi&

SIIORTMAN

Immunofluorescent assay differential HZ level 6

Immunofluorescent assay presence of TL

High HZ

Low H2

TL - ve

TL + ve

16 61

84 39

24 67

76 33

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The inter-relationships between these different assays were tested, to check if they were reflecting basically the same two thymus subpopulations. It could be argued that the cytotoxic assay reflects ease of lysis by complement rather than the surface 8 level, and indeed the ‘high-@ cell population does contain a subset of very labile cells (20). A number of experiments, some summarized in Table 1, argued against this interpretation. (i) The selective killing of high-8 cells with anti-8 and complement has been shown to reach a clearly defined plateau, showing that at least by this criterion two distinct subpopulations were revealed (20). (ii) The levels of anti-8 and complement-resistant and -sensitive cells in thymus using the cytotoxic assay were compared with the levels of high-H2 and low-H2 cells using an immunofluorescent assay, and with the levels of TL - ve and TL + ve cells using an immunofluorescent assay (Table 1). In general, the results were in agreement, but the minor subset always appeared numerically smaller with the cytotoxic assay. This was possibly due to our setting conditions to ensure that all of the major subset was killed, since we felt that contamination of the ‘low4 category with ‘high-e’ cells was more likely to be misleading than the converse. (iii) Density separation studies have shown the ‘high-e’ and ‘low-# cells, defined by the cytotoxic assay, are physically distinct and largely separable populations (21). Earlier separation studies had shown close correspondence between the high-8 and low-H2 cells, and between the low-8 and high-H2 cells, both sets being defined by immunofluorescence (17). More recent experiments have shown close correspondence between the detailed density distribution profiles of ‘low4 cells defined by cytotoxic assay and high H2 cells defined by immunofluorescence, and between the ‘high-o’ cells defined by the cytotoxic assay and low-H2 cells defined by immunofluorescence (21; Shortman, unpublished data). This applies to both the CBA and C57BL/6 mouse thymocytes, which show small but characteristic differences in the density separation profiles for the two subsets (17). (iv) Selective killin, u and removal of all cells highly sensitive to anti-8 antiserum and complement, under conditions where the anti-8 resistant cells were recovered in high yield, removed all low-H2 cells defined by fluorescence, and all TL- positive cells defined by fluorescence (Table 1) .

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FIG. 7. Comparison of the survival kinetics of ‘high-O’ and ‘low-O’ cells in cultures of total thymus cells from CBA mice. The results were obtained from viable cell counts and assay of the differential sensitivity of cells surviving in cultures to anti-8 antiserum and complement, as discussed in text. The normalized curves shorn the means and standard deviations of data from nine separate experiments, each containing duplicate assays of duplicate cultures for each time point. Approximately 15% of the total viable cells were low-0 at 2 hr of culture, the starting point for the kinetic study. Three mice \vere used for each experiment.

The conclusion from these studies was that, although exact cell for cell correspondence cannot be proven for the cells defined by each separate assay, they nlust at the least show extensive overlap. At the level of a preliminary breakdown into two (major and minor) subsets, the assays are almost equivalent. Nevertheless, all three assays were employed to characterize the thymocytes in culture. Cortisone

Semitivity

and Antigcnically

Defined Subsets

Additional information on the relationships of the subsets defined by different assays was obtained by characterizing the cortisone-resistant and cortisone-sensitive thymocytes. Cortisone treatment reduced the overall thymus weight to arounrl 20%, but viable lymphoid cell recovery was only 3-S%, indicating that at least some of the minor (antigenically defined) subset was lost by hormone injection. Elsewhere, we have argued that there is not complete correspondence between the popslations defined by surface antigens and those defined by in vivn steroid treatment, some low-0 cells being steroid sensitive, and some high-6 cells being steroid resistant (17, 20, 21). However, the results of cortisone treatment did demonstrate the close correspondence between the different assays designed to measure surface alloantigens. (Table 1). Cortisone resistance was also used to further characterize thymocyte survival in culture. Comparison

of the Szmhal

of High-0

avzd Low-0

Subpopzllntions

The use of the differential cytotoxic assay to measure the levels of high 0 (highly sensitive) and low 0 (less sensitive) cells at various times after culture suggested a marked difference in the survival characteristics of antigenically defined subsets. The results of many experiments are summarized in Fig. 7. The ‘high 0’ subpopulation (comprising SS% of all thymocytes) appeared to die rapidly in culture.

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a more stable residue of S-1070 usually survived after GO hr at 37”C, these may have represented a small component of the minor population also killed in the cytotoxic assay (see Table 1 and text above). The ‘high 8’ cells therefore accounted for most of the total thymocyte loss curve. High-8 cells clearly differed from peripheral lymphocytes in their extreme lability. In contrast, the low-0 subpopulation (comprising only 15% of all thymocytes) retained viability, their survival resembling peripheral lymphocytes. In some individual experiments there was an actual net increase in low-0 cells over the first 10 hr of culture. Althoiigli

Conzparison of the Survival

of Low-HZ

and High-HZ

Subpopulations

The immunofluorescent assay for the level of HZ alloantigen on the thymocyte surface was used in two experiments as an additional marker for the survival kinetics of the major and minor subpopulations. As shown in Fig. 8, the major thymocyte subset (low H2) showed extensive cell death in culture, whereas the minor subset (high H2) showed better survival. The results were generally within the range of those obtained using the “high 8 - low 0” cytotoxic assay, except that the minor subset showed a reduced survival at later times. Comparison of the Survival

of TL - ve and TL + ve Subpopulations

The use of imunofluorescent anti-TL antisera to define the major and the minor thymocyte subsets provided a more clear-cut marker than other antisera since the cells were either positive or negative. Two experiments using A/J mouse thymocytes (Fig. 9) showed that the major, TL-positive population was extremely labile in culture, whereas the minor, TL-negative subset showed excellent survival. A net increase in TL - ve cells was, in fact, obtained over the early stages of culture, a result occasionally seen with CBA mice using the differential cytotoxic assay to define the minor subset. The results were in general agreement with those of Figs. 7 and 8. Because the basic findings were the same, all subsequent studies on antigenically defined subsets were limited to the anti-B and complement differential cytotoxic assay.

TIME

IN CULTURE

[HOURSI

FIG. 8. Comparison of the survival kinetics of low-H2 and high-H2 cells in total thymus cells from CBA mice. The results were obtained from viable cell immunofluorescent assay on the cells surviving in culture, as discussed in text. The the means of two separate experiments, each involving single assays on triplicate each time point.

cultures of counts and results are cultures at

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TIME IN CULTURE [HOURS1

FIG. 9. Comparison of survival kinetics of TL-positive and TL-negative cells in cultures of thymocytes from A/J mice. The results were obtained from viable cell counts and immunofluorescent assay on the cells surviving in cultures, as discussed in text. The results are the means of two separate experiments, each involving duplicate assays on duplicate cultures of each time point.

The Efect of Culture High-0 Cells

Conditions

and Physiological

State on the Survival

of

To check if the extreme lability of the major, high-e thymus subset was an artifact of certain culture conditions, or of the physiological state of the donor mice, the following parameters were studied : (a) The presence of FCS was found to be essential for maintaining the relative viability of both high-e and low-B cells at the levels shown in Fig. 7. In the absence of FCS both cell types died more rapidly. Still higher FCS levels did not improve viability. (b) The use of mouse sera, rather than FCS, had relatively little effect on the results. (c) The omission of 2-mercaptoethanol (included in the medium under standard conditions) caused a marginal drop in survival of low-0 cells after 48 hr, but did not affect high-e survival. (d) The use of RPM1 medium instead of MEM, or HEPES buffer instead of bicarbonate, gave no difference in the survival of either category of cells. (e) The use of cellophane membranes instead of Nuclepore membranes in the Marbrook vessels, or the use of plastic culture trays instead of Marbrook vessels, did not alter the survival curves. (f) In the standard medium containing 10% FCS, cultures of low cell density (0.5 X lo6 cells/ml) survived better than those of higher cell density, presumably due to limitations of nutrients or other factors in the medium. (g) ‘Conditioning’ the culture medium, by placing slices of normal or of X-irradiated (2 days post-800 rads) thymus or spleen in the outer chamber of the Marbrook vessel 12 hr before and during thymocyte culture failed to significantly improTe the survival of the high-e thymocytes.

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3 s zg ::c s;: Zb zl-

NEW BORN

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-8 a-

-.

-1

0.5~24h ALL CELL5

.A

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,48h 24-48h

5 s 2

2

0 0

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TIME IN CULTURE [HOURSI

FIG. 10. Effect of intrathymic age on the survival kinetics of high-0 thymocytes. Continuous ijz &IO thymidine administration was used to label cells of differing intrathymic age and the survival kinetics of different groups of high-6 cells calculated as given in detail in the text. Calculations are based on duplicate assays of viable cells and on counts of labelled cells in single smears from triplicate cultures for each time point. Three mice were injected with [‘H]TdR per group and the thymus glands were pooled for each series. The results are the means of two separate experiments.

(h) Adrenalectomy of mice 1-2 weeks prior to isolation of thymocytes for culture failed to improve the survival of the high-8 cells, indicating that the extreme lability over the 72 hr of culture was not due to prior corticosteroid-mediated stress effects. (i) The high-0 subpopulation of young mouse thymus (4- to 7-day-old, 2-weekold, and 4-week-old) showed a marked decline in culture similar to the thymocytes from adult animals. The Efiect of Intrathynzic

Cell Aye on Swvival

of High-9 Cells in Culture

To further define the subset of thymocytes most subject to death in culture, the effect of length of prior residence of cells in the thymus was studied. The maximum intrathymic lifespan of most thymocytes is 3 days. It was possible by repeated injections of thymidine to produce a continuous labelling effect in three separate groups of mice, and so produce three groups of labelled cells : (i) new born in the thymus (30 min pulse, dividing cells, only 11% of the population) ; (ii) those born O-24 hr earlier (24 hr of labelling, 39% of the population) ; (iii) those born O-48 hr earlier (48 hr of labelling, 63% of the population). In the latter situation the unlabelled cells (37% of the population) were older than 48 hr. By difference, it was therefore possible to divide cells into four different categories by intrathymic age. Thymus cells from three such groups of mice, previously subject to the three different length labelling protocols, were cultured for various periods. The cells were recovered from culture, damaged cells were removed by a neutral pH density cut (25), and cell counts and radioautographic assessment of labelled cells were performed. In this way it was possible differentially to follow the survival of cells of different intrathtymic age. This analysis was limited to the major high-8 category that dies in culture, by making a small correction based on the known lower labelling index of low-8 cells under these conditions (20), and the known good culture survival kinetics of low-8 cells from this study (Fig. S), The results of this analysis are shown in Fig. 10.

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The results showed the relative survival of high-0 cells in culture varied with intrathymic age. Cells labelled itt V&O with a 30-min pulse, which would mark the larger progenitor cells in division at this time in the intact animal, survived well in culture, or at least maintained their numbers by division. This would agree with the earlier evidence that large cells maintained cell numbers in culture (Fig. 5). Newborn high-0 small lymphocytes (those labelled by 24 hr of thymidine with the 30-min dividing population subtracted) survived better than high-0 cells in general. In contrast high-t9 small thymocytes older than 24 hr survived less well than most cells. There appeared to be some heterogeneity within the high-0 subset, since cells older than 45 hr appeared to show better survival than those 24-48 hr of age. However, this could result from artifically classifying some of the minor subset in with the major, high-8 group, a possibility with the differential cytotoxic assay (see Table 1 and earlier results). In general, it was clear that the older high-8 cells died more rapidly than newborn high-8 cells.

The previous results showed that viable high-8 cells were lost rapidly in culture, this loss being largely accounted for by an accumulation of recoverable dead cells. At the same time, in the earlier stages of culture, there was relatively good survival of low-0 cells, with a net increase sometimes recorded. It was necessary to check if this maintenance of low-0 cell level could arise by a small proportion of the major high-0 cells being transformed into low-8 cells, as the conventional models of T-cell differentiation would suggest. To test this possibility, high-0 cells from thymocyte populations were killed in bulk by the differential anti-0 cytotoxic procedure, the cells were washed. and all damaged cells were removed by low ionic strength

s;_, ISOLATED LOW 8

IO

20

30

40

50

60

TIME IN CULTURE [HOURS]

FIG. 11. The survival kinetics of isolated low-8 cells. All ‘high-0 cells were differentially killed by limited anti-0 and complement treatment, and all damaged cells were removed. The remaining resistant cells, termed ‘low-~,’ were cultured at the level of 10 X 10” viable cells/ml. The results are from a single experiment in which thymus cells from three mice were used, and duplicate cultures at each time point were assayed in duplicate. In other experiments very similar curves were obtained when the number of isolated low-8 cells cultured (1-2 X 10’) was the same as the number of low-8 cells normally present in the cultures of the unseparated population.

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CORTISONE~RESISTANT CELLS

I

I

I

IO

20

30

1'IME IN CULTURE[HOURS] FIG. 12. The survival in culture ofI co rtisone-resistant thymocytes. The 4% resistant thymocytes were recovered from six mice 48 hr after injection of cortisone acetate. The thymocytes were cultured in triplicate at a level of 10 X 10’ viable cells/culture, and duplicate counts of surviving viable cells were made at the times stated. The results are pooled data from two experiments.

aggregation (26). These isolated low-0 cells were then cultured, and their survival was compared with the previous results from cultures of unfractionated thymus on which low-8 assays were carried out at various time points. As shown in Fig. 11, the separated low-8 cells behaved in essentially the same way as the low-8 cells in unfractionated thymus (Fig. 7)) or of the high-H2 cells in unfractionated thymus (Fig. S), despite the absence of high-0 elements and the prior incubation and separation procedures. Thus, the enhanced survival of the low-8 cell population was not due to continuous replenishment by transformation of some high-0 cells into low-8 cells. Survival

of Cortisone-Resistant

Cells

The cortisone-resistant subpopulation, as discussed previously, is composed largely of low-e, high-H2, TL -ve cells, although it does not exactly correspond to the antigenically defined minor thymocyte subpopulation. On the basis of the previous results, these cells might be expected to be relatively stable in culture, the cells subject to rapid death being eliminated by pretreatment of the animals with steroids. The survival in culture of the 4% of thymocytes remaining 48 hr after steroid administration was therefore tested. As illustrated in Fig. 12, cortisoneresistant cells did show good survival in culture. Since the major thymocyte population had largely been eliminated, this provides further evidence that the stability of the minor subset was an inherent property of these cells, and not due to continuous

replenishment

from the cortisone-sensitive,

dominant

thymocyte

type.

DISCUSSION These studies show that certain properties of thymocyte subpopulations can be determined by following in detail, using a careful balance-sheet approach, the population kinetics of thymus cells in a simple cell culture system. Under these conditions stem cell entry is prevented and thymus architecture is destroyed, and

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because of these factors the system is useful for only a relatively short period. It is limited to investigation of the already formed major thymocyte subpopulations, and probably only to the terminal stages of any developmental pathway. This is in contrast to studies of the induction of new T-lymphocytes by the action of humoral factors (30-31) or studies of the development and growth of embryonic thymus explants in long-term culture (32). At the early stages of our culture system about the normal proportion of cells are dividing and occasionally net increases in certain subpopulations are observed. In a forthcoming paper we will examine in detail the net increase in functional T-cells which can be obtained in short-term culture (Hopper, Ryden, and Shortman, in preparation). However, the main emphasis in the present report is on events after this early phase of cell division falls off. One characteristic of this culture system is a continuing decrease in cell size, demonstrated by sedimentation velocity separation. This is a characteristic of cell differentiation and maturation in the intact organ. This is followed by a steady loss of viable cells. Cell death is in fact the predominant feature of the system, despite optimisation of culture conditions and the search for a wide range of factors that might prolong cell survival. Obviously this cell death could still be an artifact of the culture conditions. However. the specificity of the phenomenon suggests it is an inherent property of the cells involved, and this feature also may be a model of normal if2 V%O events. The loss of viable cells in culture occurs in two phases, separable both in time and by the experimental conditions. A very rapid loss over the first 2 hr, markedly enhanced by low cell and low serum concentrations, reflects the exceptional lability of most thymocytes. This rapid loss is characterised by total disintegration of cells rather than an accumulation of recoverable dead thymocytes. Such a phenomenon could explain why dead cells fail to accumulate in sufficient quantities to be detected histologically, if cell death is a normal and frequent event in the thymus as some models propose. The disintegrating cells are probably related to the ‘labile thymocytes’ previously described in this laboratory (20). A similar rapid loss of thymocytes, at low cell and protein concentrations, has been described by Rockwell and Sibatani (33)) and this has since been shown to be caused by total cell disintegration (personal communication). This early death phenomenon can be largely but not completely eliminated from the culture system by optimising serum and cell concentrations. In order to hare a more stable starting point to follow later events, we have often allowed any rapid cell death to occur in a preliminary incubation, and taken 2 hr of culture as the starting point for further study. The loss of viable thymocytes over the second phase, from 2 to 72 hr of culture, occurs even under optimised culture conditions, and is not reduced by altering a number of variables, including the presence of thymus-conditioned medium, and the use of young or adrenalectomised animals. The survival of thymocytes is much lower than for cells from spleen or lymph nodes, which serve as ‘controls’ for the culture medium itself. The fact that all lymphocytes show some death in culture weakens the argument that death is ‘programmed.’ However, the differential behaviour of different thymocyte subpopulations points strongly to reduced survival rate being an inherent property of a defined class of cells. Rapid death in culture is largely a feature of the subpopulation characterised as high 0, low H2, TLpositive, and cortisone sensitive. Within this subpopulation it is the small rather than the large cells that die first, with the most labile subset of small cells being

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those of more than 24-hr intrathymic age after the last division. Failure to survive cannot be ascribed simply to ‘immaturity’ since the least mature, high 0, large dividing cells, and the less mature, newborn, high-8 small cells, both survive relatively well. Rather, this property is acquired by a subset of high-8 small thymocytes during their intrathymic development. In marked contrast to the high-8 cells, the minor subpopulation characterized as low 8, high H2, and TL-negative, and containing cortisone-resistant cells, survives as well in culture as peripheral lymphocytes. In fact, an increase in these cells is frequently noted over the first 10 hr. If this minor subset, resembling peripheral T-cells, was continuously produced from the major, high-8 cells, as conventional models of T-cell development assume, such a finding might be expected. In such a case the individual cells would not necessarily be stable, but the whole subpopulation would be maintained by continuous replenishment. This is made unlikely by the finding that the drop in the major, high-8 cells is accounted for, not by transformation into low-0 cells, but by accumulation of dead cells. The possibility is eliminated for the minor subset defined by the cytotoxic assay by the direct experiments showing that the isolated low-8 cells were maintained well if cultured in the absence of the major, high-B thymocytes. In addition, the minor subset of cortisone-resistant cells also survives well in culture, in the absence of the major population of cortisone sensitive cells. Overall these observations point to the relative independence of the major (high 0) and minor (low 0) subpopulations in thymus. This supports our previous models of relatively independent lineages (20-21) rather than the conventional view of ‘immature’ small high-8 cells as precursors of the immunologically active low-0 elements. In addition, the fact that high-0 cells acquire the property of extreme lability in culture durin g their development supports (but does not prove) the concept that these cells are destined to die in the intact animal, either in the thymus or soon after export. ACKNOWLEDGMENTS The authors are grateful to Mrs. S. Dunn and Miss A. Ryden for excellent technical assistance and to Dr. T. Mandel for his assistance with the TL assays. Dr. K. Hopper was a recipient of a postdoctoral Fellowship from the Australian Cancer Society. The work was supported by the National Health and Medical Research Council, Australia, by the Whitehall Foundation, U.S.A., and the Warman Foundation, Australia.

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Sci. USA 68, 2734, 1971. 31. Basch, R. S., and Goldstein, G., Pror. Kat. Acad. Sri. USA, in press. 32. Mandel, T. E., and Kennedy, M. M., submitted for publication, 1976. 33. Rockwell, G. ‘4., and Sibatani, A. Proc-. Amt. Bioclzcljl. SOC. 8, 96, 197j.