Proc. Nadl. Acad. Sci. USA Vol. 86, pp. 5547-5551, July 1989 Immunology Cell proliferation and thymocyte subset reconstitution in sublethally irradiated mice: Compared kinetics of endogenous and intrathymically transferred progenitors (radiation chimeras/thymus/bone marrow/bromodeoxyuridine) CLAUDE PENIT* AND SOPHIE EZINE Institut National de la Santd et de la Recherche MWdicale Unitd 25, Centre National de la Recherche Scientifique Unitd Associde 122, Immunologie Clinique, H6pital Necker, 161, rue de Stvres, 75743 Paris Cedex 15, France Communicated by Jean Dausset, March 28, 1989

ABSTRACT After sublethal (6 Gy) whole-body irradia- DNA synthetic labeling and cell-surface phenotype analysis, tion, the C57BL/Ba (Thy-1.1) murine thymus regenerated in we have been able to elucidate the early events leading to two waves, on days 3-10 and 25-32, separated by a severe sequential differentiation of thymocyte subsets (11). relapse. The second phase of depletion-reconstitution repro- Irradiation has been used widely to deplete the thymus; duced the first one, in a less synchronous manner. The deple- although its effects are not restricted to actively cycling cells, tion affected all cell subsets, but CD4+ CD8- cells decreased irradiation also leads to a sequential regeneration of thymo- later than immature cells. Cell proliferation, measured by cyte subsets (13, 14). Moreover, the irradiated thymus be- BrdUrd incorporation, started on day 3 after irradiation and comes highly receptive to exogenous bone marrow precur- concerned CD4- CD8-, CD4- CD8+, and CD4+ CD8+ cells, sors injected intravenously (i.v.) (6, 15-17) or intrathymically sequentially. CD4+ CD8- cells never represented a significant (i.t.) (6, 18, 19). Using congenic strains of mice, several percentage of cycling cells. When irradiation was immediately authors have described the comparative properties of bone followed by an intrathymic inJection of 105 C57BL/Ka (Thy- marrow and intrathymic precursor cells in this reconstitution 1.2) bone marrow cells, the relapse in thymus reconstitution system (20, 21). The intrathymic transfer technique with was no longer observed. Detected with anti-Thy-1.2 antibodies, sublethally irradiated recipient mice appeared the most useful donor cells started cycling on day 14 and showed only one wave for these studies. Using this experimental model, several of proliferation. In these chimeras, recipient thymocytes be- authors have compared the development of endogenous have exactly like thymocytes ofsolely irradiated mice. Intrathy- radioresistant precursors to that of exogenous bone marrow mically transferred CD4- CD8- thymocytes (105) showed the or thymic CD4- CD8- cells. Cell preparations could also be same proliferation kinetics as endogenous cells, with a peak in fractionated, mainly on the basis of cell-surface antigen number on day 10 but completely disappeared from the thymus can be sum- on days 14-21. These data reflect maturational differences expression (22-26). The main data established between intrathymic and bone marrow precursor cells and marized as follows. Radioresistant CD4- CD8- thymocytes suggest different radiosensitivities not linked to proliferative develop rapidly after a sublethal irradiation dose and give rise status. The resting state of the thymus immigrants was shown to all-thymocyte subsets. Exogenous CD4- CD8- thymo- by the absence of Thy-i acquisition by bone marrow cells cytes injected i.t. also give a transient reconstitution with all continuously labeled for 10 days with BrdUrd in vivo before thymocyte subsets, with kinetics apparently similar to the intrathymic transfer. When such labeled bone marrow cells initial endogenous ones. As compared to CD4- CD8- thy- were injected in the thymus, only the minor BrdUrd- subset mocytes, exogenous bone marrow cells give a delayed and gave rise to Thy-1+ cells. longer reconstitution. We have used the BrdUrd DNA synthetic labeling method All thymocyte subsets represent intermediary stages of the to correlate cell proliferation and thymocyte subset genera- T-cell differentiation pathway, which can be followed in tion in irradiated (6 Gy) C57BL/Ba (Thy-1.1) mice. The particular by CD4 and CD8 surface expression (1, 2). In the results were compared to those obtained when these mice fetus (3-5) as well as in the adult (6), all thymocytes develop were injected i.t. with 105 bone marrow cells or CD4- CD8- from exogenously produced precursors that colonize the thymocytes from C57BL/Ka (Thy-1.2) mice. Thus, we de- organ. In the adult steady-state thymus, the study of the termined the detailed kinetics of endogenous (radioresistant) properties ofrecently immigrated precursors and ofthe early cells and the phenotype of their progeny, and we compared events leading to differentiation is rendered difficult by the the results to those obtained for injected precursors. The asynchronous coexistence ofsuccessive steps and by the fact results show that radioresistant endogenous cells give a that these precursors represent a very minor subset. biphasic reconstitution in both simply irradiated and i.t. Cell proliferation is an essential event in T-cell differenti- injected mice, and that i.t. injected bone marrow cells or ation (7, 8), and it is clear that cell activation is the first in a CD4- CD8- thymocytes give a single reconstitution wave. cascade of events driving thymocyte precursors (CD4- The discontinuous endogenous reconstitution was strikingly CD8-) to cortical thymocytes (CD4+ CD8+) and to mature illustrated by the evolution of the absolute number of CD4+ medullary cells (single positive, essentially CD4+ CD8-) CD8- mature cells. Finally, we confirmed the resting state of (9-12). Treatment of mice by specific cell-proliferation in- thymus seeding bone marrow precursor cells by showing the hibitors leads to transient cessation of cell generation and of absence of intrathymic development of continuously Brd- differentation. Thymus regeneration then takes place by a Urd-labeled bone marrow cells. The implications of these synchronous development of progenitors. By combining results for the physiology of bone marrow and intrathymic precursor cells are discussed. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: i.t., intrathymically. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5547 Downloaded by guest on September 26, 2021 5548 Immunology: Penit and Ezine Proc. Natl. Acad. Sci. USA 86 (1989) MATERIALS AND METHODS Total Mice. C57BL/Ba (Thy-1.1) and C57BL/Ka (Thy-1.2) mice C04+8+ bred in our own facilities were used. They were 6-8 weeks old (recipients) and 4 weeks old (donors). Irradiation and Intrathymic Transfer. Mice received a single 6-Gy (600-rad) irradiation dose for spontaneous regen- C04+8- eration studies. C57BL/Ba mice received the same dose and E CD4-8- 3 hr later were anesthetized with ether. A longitudinal C04-8+ incision of the sternum was made with scissors, and 105 I- C57BL/Ka cells (standard dose) were injected in both thy- . mus lobes in 2 Al of minimal essential medium (GIBCO). Bone marrow cells were obtained by flushing the marrow from tibia and femur. Donor CD4- CD8- thymocytes were prepared by negative selection as described (12). Proliferative Status and Thymocyte Phenotype Determina- tion. At various times after irradiation with or without ae intrathymic transfer, mice were injected with BrdUrd (Sigma) (two injections of 0.25 mg 4 hr apart) and killed 1 hr later. Total thymocyte counts were carefully determined. Cell suspensions were submitted to surface labeling with biotinylated anti-Lyt-2 antibody (CD8, clone 53-6.7) (27) and

biotinylated anti-L3T4 (CD4, clone GK 1.5) (28), both re- C o vealed with Texas Red-streptavidin (Amersham). The cells were then centrifuged onto slides with a cytofuge (Shandon, London) and treated for BrdUrd detection with the mono- 0 5 10 15 20 25 30 35 40 45 50 clonal antibody 76-7 (a gift of T. Ternynk, Institut Pasteur, Time after irradiation (days) Paris) and purified goat F(ab')2 fragments of anti-mouse IgG conjugated to fluorescein (Immunotech, Luminy, France). FIG. 1. Thymocyte subset reconstitution and total cell prolifer- For i.t. injected mice, the same labeling protocol was per- ation after 6-Gy irradiation. C57BL/Ba mice were irradiated (6 Gy) formed for donor type (Thy-1.2) and recipient type (Thy-1.1) on day 0. On indicated times, BrdUrd (250 ,g) was injected twice at thymocytes with the 30H12 (29) and 19XE5 (a gift of R. a 4-hr interval. One hour later, thymocyte suspensions were made and processed for CD4/CD8 and BrdUrd detection. The phenotype Nowinsky) antibodies, respectively. of total thymocytes was determined, and the absolute numbers of Stained cytofuge preparations were analyzed under a Leitz each subset were calculated (Upper). The percentage of BrdUrd+ Orthoplan microscope. Because of the inability to analyze cells was also determined (Lower). The phenotype of BrdUrd+ cells more than two fluorochromes on each cell, BrdUrd+ cells is shown in Table 1. Each point is the mean of three experiments, were scored as positive or negative for CD4, CD8, or both, each with two mice. and the phenotypes (CD4+ CD8+, CD4+ CD8-, CD4- CD8+, and CD4- CD8-) were calculated by the subtraction method. cells per thymus in this series). These overall results were For a better homogeneity ofthe results, the same method was complemented by determination of cell phenotypes in the applied to total cells (BrdUrd+ and BrdUrd-), although it was irradiated thymus, and the absolute numbers of each subset possible in this case to use a direct double-staining technique were calculated (Fig. 1). CD4+ CD8+ cells were strongly for surface antigens. As already noted in our previous reports affected by irradiation and dropped to an insignificant number (9-12), the precision of the subtraction method was good (1.7 ± 1.7% of thymocytes) on day 5. CD4- CD8- cells enough mainly because ofthe stability ofthe stained cytofuge decreased less, as already observed after antimitotic drug preparations and because it was restricted to antigens ex- administration (11), and increased again much faster, reaching pressed at high level on the surface of the cells. half their normal count on day 5. After a maximum count on Intrathymic Transfer of Continuously BrdUrd-Labeled day 7, they progressively decreased until day 25 and then Cells. C57BL/Ka mice received BrdUrd in drinking water for reincreased to normal values. CD4- CD8+ cells also were 7 days (first experiment) and 10 days (second experiment). significantly affected by irradiation, with a minimum number Labeled bone marrow cells (2 x 106) were injected i.t. in on day 3 and a rapid increase on day 5; then their number irradiated C57BL/Ba recipients. At various times after trans- evolved like that of CD4- CD8- cells. As shown below, this fer, Thy-1.2 antigen expression was scored on BrdUrd- evolution reflected mostly the proliferative capacity of both labeled and unlabeled cells. immature CD4- CD8- and CD4- CD8+ cell subsets. CD4+ CD8- cell number showed very different and dra- RESULTS matic changes. These cells initially showed the highest re- Regeneration of 6-Gy-Irradiated Thymus. The total number sistance to irradiation, representing >50o of all thymocytes of thymocytes dramatically decreased after irradiation, began on day 3, but then decreased to an insignificant number on to increase on day 5, and reached a plateau on days 10-14. day 7. After this decline, they reincreased but showed a However, this reconstitution was not stable because the size second decrease on day 28, reproducing much more dramat- ofthe thymus decreased after day 15, reaching a minimum on ically the evolution of total thymocyte counts after a 4-day days 21-25 before returning to a high and constant value after interval. These relative changes of thymocyte subsets have days 28-30 (Fig. 1). Thus, thymic reconstitution was not a been partially observed by others (13, 14), mainly during the continuous process but proceeded in two growth phases initial period (days 3-10). separated by a transitory but severe relapse. The biphasic Using the BrdUrd DNA labeling method, we attempted to pattern of thymus reconstitution after irradiation has already correlate the variations of thymocyte subsets with cell pro- been noted by Huiskamp et al. (13). The mean thymocyte liferation. Fig. 1 Lower shows the labeling index of total count was 5 x 107 on day 10 after irradiation, was only 6.3 x thymocytes observed 1 hr after the second of two BrdUrd 106 on day 25, and returned to 5 x 107 on day 35. These injections performed 4 hr apart. This injection protocol numbers were lower than in the control thymuses (nearly 108 allows detection of all DNA-synthesizing cells. Cell prolif- Downloaded by guest on September 26, 2021 Immunology: Penit and Ezine Proc. Natl. Acad. Sci. USA 86 (1989) 5549 eration began between days 3 and 5, when >60% of thymo- cytes were cycling, showing a transient synchronous wave. iO8r The labeling index returned to a normal value (10%) from day 14 to day 25 and then showed a second transient increase on day 28. These proliferation waves corresponded to the two total cell count increases observed in Fig. 1 Upper. The phenotype of cycling cells was determined, and the results E 0l-7 are presented in Table 1. When compared with the normal C4 thymus, cycling cells were initially highly enriched in CD4- t0 CD8- and CD4- CD8' cells (days 3-5). Then the percentage E ofCD4' CD8' cells increased, yielding an overall phenotype of cycling cells similar to that of the normal thymus on day 1016I A 6Gy+5x105 BMIT 7 (with, however, a supranormal percentage of CD4- CD8' 6Gy+1O5 BMIT cells). Then the percentage of CD4- CD8- cells decreased 6G3y only (2.3% of labeled cells on day 14, compared with 10%o in the normal thymus) until day 25; at day 28, it was again higher than in the normal thymus. During this second proliferation wave, the percentage ofCD4- CD8' cells increased, and that 0 10 20 30 40 50 of CD41 CD8' cells decreased. At all times, cycling CD4' Time after irradiation (days) CD8- cells represented an insignificant number, showing that, by contrast with the other subsets, their changes were FIG. 2. Total thymocyte number in 6-Gy-irradiated and bone capacity. These results marrow cell i.t. reconstituted (BMIT) mice. C57BL/Ba mice were not related to their own proliferation simply irradiated (e) or irradiated and i.t. injected with 105 (o) or 5 show that the irradiated thymus is the site of complex X 105 (A) bone marrow cells. Total thymocytes were counted at the dynamic events leading to two phases of growth, separated indicated times. Each point is the mean ofthree experiments ± SEM, by a gap during weeks 3 and 4. This evolution of the each with two mice. endogenous cell subsets was compared to that of injected cells in the transfer experiments described below. the growth of donor type cells derived from the injected bone Regeneration of 6-Gy-Irradiated and Bone Marrow-Recon- marrow. stituted Thymus. Irradiated mice received bone marrow cells BrdUrd labeling was also performed and analyzed for both injected directly into the thymus 3 hr after irradiation. Fig. 2 host and donor cells (Fig. 3B). The initial reconstitution was shows the evolution of total thymocyte counts in simply entirely due to host cells, which showed a proliferative be- irradiated mice, as compared with those in mice that had been havior similar to that of thymocytes of noninjected mice, with i.t. reconstituted by 105 or 5 x 10 bone marrow cells. The a proliferative wave on day 6 and a second, less pronounced total number observed in the plateau region was not very wave on day 28; during the intermediary period, the labeling different, but the initial phase ofthe reconstitution seemed to index ofThy-1.1 cells was lower than normal (2.7% on day 21) be slightly delayed in i.t. injected animals, probably because but was always significant. Proliferation of Thy-1.2 (donor) of the stress of the injection procedure. The only striking cells was not detected before day 14, when it showed a high but difference was the absence of a decrease in thymocyte unique peak, returning to the 10%o normal thymocyte index on number between 3 and 4 weeks in i.t. injected animals, the day 28 and progressively decreasing thereafter; thus, i.t. thymus reconstitution thus appearing continuous. marrow cells abolished the gap observed in Donor-type (Thy-1.2) and host-type (Thy-1.1) cells were transferred bone in irradiated animals injected i.t. with 105 the reconstitution of simply irradiated thymus, presumably by evaluated (Fig. 3A) a irradiation. The bone marrow cells. The evolution ofthe number of host-type replacing cell population killed by prolifer- thymocytes reproduced closely that of total thymocytes in ation of these transferred cells appeared superimposed on the noninjected animals. By contrast, donor-type cells were first evolution of endogenous cells, which was not modified by the detectable at 14 days and then increased exponentially until transfer. As shown in the subsequent experiment, the behavior 3 weeks, when they reached a plateau before decreasing. of i.t. injected CD4- CD8- thymocytes was very different. ofthe relapse at 3-4 weeks was due to Reconstitution with CD4- CD8- Thymocytes. Irradiated (6 Thus, the suppression Gy) C57BL/Ba mice received 105 CD4- CD8- thymocytes The Table 1. Phenotype of DNA-synthesizing thymocytes in isolated by negative selection from C57BL/Ka donors. postirradiation reconstitution total number of thymocytes evolved exactly as in irradiated noninjected mice (data not shown). The data on donor- and Time,* host-type cell numbers and proliferation rate are shown in days CD4+ CD8+ CD4- CD8+ CD4+ CD8- CD4- CD8- Fig. 3, in comparison with those ofthe bone marrow transfer 3 3.8 2.1 30.7 ± 9 5 ± 4.6 60.4 ± 6.6 experiments described above (see the legend to Fig. 3). 5 1.7 1.7 59.5 ± 10 3.3 ± 2 37.1 ± 8.6 Proliferation of Thy-1.2 cells was detected shortly after 7 81.9 ± 3.2 8.2 ± 3 1.05 ± 0.5 9.0 ± 1.9 transfer, and the curves of BrdUrd-labeling indexes of both 10 81.6 ± 0.6 11.3 ± 1 2.5 ± 1.7 4.5 ± 0.1 donor- and host-type cells were superimposable. However, 14 91.2 ± 1.4 6.3 ± 1.3 0.1 ± 0.1 2.3 ± 0.3 the number of donor-type cells remained much lower than 18 90 ±1.7 4 ± 0.4 1.3 ± 0.4 4.5 ± 1.6 that of recipient ones, probably because of the limited 21 84.9 ± 0.8 8.3 ± 0.9 0.4 ± 0.4 6.3 ± 0.2 number of cells injected. The percentage of Thy-1.2 thymo- 25 73.9 ± 0.5 12.4 ± 2.6 0.8 ± 0.6 13.1 ± 2 cytes never exceeded 10%o. Thy-1.2 cell proliferation showed 28 69.4 ± 3.4 13.8 ± 1.7 0.2 ± 0.2 16.4 ± 1.7 a single peak around day 10, and then the number of donor- 31 84.7 ± 2.9 5.1 ± 2.3 0.15 ± 0.15 10 ± 0.8 type cells rapidly decreased. On day 21, no more Thy-1.2+ 48 87.9 ± 0.3 4.3 ± 1.9 0.3 ± 0.3 7.4 ± 1.3 cells were found in the thymus. Bone Marrow Cells. The phenotype of BrdUrd+ cells observed after two injections of Resting State of Thymus-Colonizing the nucleoside was determined as indicated in Materials and Meth- The interval observed between intrathymic injection and ods and the legend to Fig. 1. Data are the mean of three independent proliferation of bone marrow cells has been observed by experiments, each with two mice, ± SEM. many authors. It does not seem to be dependent on the *After irradiation. number of cells injected or on the irradiation dose. In an Downloaded by guest on September 26, 2021 5550 Immunology: Penit and Ezine Proc. Natl. Acad. Sci. USA 86 (1989) day 3). They gave rise to all-thymocyte subsets but could not 1080 compensate for the absence of CD41 CD8- cell generation, which transiently disappeared from the thymus around day 7. New CD4' CD8- cells appeared again around day 10, 3 days E after CD4' CD8' cell emergence. These findings confirm the results reported by Tomooka et al. (14) and are coherent with 107h SoC the generation of helper-like thymocytes from double- positive cells, as shown by our kinetic studies in the normal .) 0 thymus (9, 11). E During this period, CD4- CD8' cell changes paralleled 106 those ofCD4- CD8- cells, confirming that most ofthem were F a between CD4- CD8- and ~0. transitory subset, intermediate CD CD4' CD8' cells (11, 30-32) and not mature cytotoxic T 0 cells. Although slightly delayed and more dramatic, the -0 sequence of events described above reproduces the one 105I observed after treatment of mice with antimitotic drugs (11). The decrease in thymus size observed thereafter is accom- panied by a relative depletion of CD4- CD8- thymocytes, which showed a reduced mitotic index. 0o During the fourth week, a second regeneration phase was observed, with again a severe depletion of CD41 CD8- cells. B However, the phenotype changes preceding this disappear- r-nI ance were less dramatic than on days 3-5: in particular, CD4' c-O CD8' cells were still detectable. This suggests that a decrease in the CD4' CD8' cells to a number around 5 x 106 is sufficient to virtually abolish CD4+ CD8- cell generation. > 25 The second regeneration-proliferation phase was much less 0 E's acute than the first, probably because it was also much less .1L synchronous. The cells involved in this second wave might be U I I -I I- A either radioresistant intrathymic precursor cells that need a 0 10 20 30 40 50 70 very long time (3 weeks) before being able to proliferate, or, Time after irradiation and I.T. transfer (days) alternatively, they might be bone marrow precursor cells. The second hypothesis appears more likely because of the FIG. 3. Evolution ofabsolute number and mitotic rate ofhost and known properties of intrathymic precursor cells, which, as donor thymocytes after intrathymic adoptive transfer. Irradiated (6 suggested by our recent studies (12), appear to be completely Gy) C57BL/Ba (Thy-1.1) mice were injected i.t. 3 hr later with 10i renewed in 10 Another reason to the bone marrow cells (e, o) or CD4- CD8- thymocytes (A, A). At nearly days. prefer successive times, BrdUrd was injected as indicated in methods, and second hypothesis stems from the results described in Fig. thymocytes were subjected to Thy-1.1, Thy-1.2, and BrdUrd detec- 3A, which show that i.t. injected bone marrow cells give only tion. (A) Total number of host (Thy-1.1; *, *) and donor (Thy-1.2, o, one wave of proliferation. However, the interval between A) thymocytes. (B) BrdUrd-labeling index ofthymocytes (same sym- injection of bone marrow cells and proliferation of their bols as in A). Each point represents the mean ofthree determinations. progeny was shorter than between the irradiation and the endogenous second wave as clearly observed in Fig. 3A. attempt to study the events preceding proliferation, we Thus, we should hypothesize that bone marrow precursor labeled bone marrow cells by administering BrdUrd in drink- cells, derived from radioresistant (resting) stem cells, reside ing water to C57BL/Ka mice for 10 days. At this time, bone in the bone marrow itself or elsewhere in the body for 2-3 marrow cells were harvested, and >95% were found to be weeks before going into the thymus, where they again stay BrdUrd'. This labeling percentage was similar to that found resting for 10-14 days before entering in cycle. This hypoth- in thymocytes (12) but higher than in spleen (55% of total but esis might explain why bone marrow cells giving progeny in only 18% of Thy-1+ splenocytes) and lymph node cells (25% the irradiated thymus were found unlabeled in our experi- of total and 15% of Thy-1+ LN cells). The BrdUrd label was ment described in the last part ofResults. Our recent studies used as a marker to trace bone marrow cells that were on CD4- CD8- thymocytes suggested a continuous inflow of injected i.t. into irradiated C57BL/Ba mice. Thymuses were precursor cells in the normal thymus and showed a daily harvested on different days after transfer, and labeled cells entry in cycle ofnew cells (12). A transient interruption ofthis were counted and scored for Thy-1.1 and interleukin 2 continuous process probably explains the gap observed in receptor (IL-2R) expression. The initial aim of this experi- thymic reconstitution, as confirmed by the decrease in CD4- ment was not attained, as most labeled injected cells rapidly CD8- cell count at this time. Such a gap is not observed after disappeared and none became Il-2R' or Thy-1+ (data not colchicine or hydroxyurea injection (12), and this suggests shown). Despite this behavior of labeled bone marrow cells, that, contrary to these antimitotic agents, the action of Thy-1.2+ (donor) cells developed in the thymus, following irradiation is not restricted to proliferating cells. It has been kinetics identical to those found after transfer of normal, suggested that irradiation damages thymus stromal cells (33). non-BrdUrd-labeled bone marrow cells. The initial and very In the present case, however, the capacity of stromal thymic rare Thy-1.2+ cells found were always unlabeled. cells to induce precursor cell proliferation appeared pre- served, as shown by the rapid entry in the cycle of endoge- DISCUSSION nous or injected CD4- CD8- cells as well as, later, that of Thymus reconstitution after sublethal irradiation is biphasic. injected bone marrow cells. This observation already made by others (13) must be dis- The interval between endogenous and injected bone mar- cussed in reference to the properties ofintrathymic and bone row cell proliferation (8 days) also requires discussion. As marrow precursor cells. Radioresistant thymocytes, mainly proposed by Goldschneider et al. (18), the delay might be of the CD4- CD8- phenotype, were responsible for the first explained by the existence of a limited number of "niches" wave of reconstitution, which started relatively rapidly (on for precursor cell activation in the thymic stroma. Endoge- Downloaded by guest on September 26, 2021 Immunology: Penit and Ezine Proc. Natl. Acad. Sci. USA 86 (1989) 5551 nous radioresistant precursor cells might lodge in these resistance is unknown, but it may be related not only to niches, and injected cells might have to wait for free space to intrinsic stages of the cells but also to a protective role of the be activated. Although tempting, this hypothesis is not com- bone marrow and thymus microenvironments. These data patible with the fact that CD4- CD8- thymocytes injected i.t. emphasize the role of the thymus environment in triggering proliferate much faster than bone marrow cells (21) and also precursor cell proliferation, as it appears clear that the differ- that the second wave of endogenous cell proliferation after 4 ences observed in the kinetic properties ofthe subsets studied weeks is not delayed in the presence ofdonor cells (compare are related to the duration of their intrathymic residence. Fig. 2 and Fig. 3A). Finally, it appears that the kinetics of proliferation of We thank Florence Vasseur for expert technical assistance and intrathymic precursor cells, injected exogenous bone marrow Michele Pitte for typing. This work was supported by Institut cells, and endogenous bone marrow cells are different, with National de la Sante et de la Recherche Medicale, Centre National peaks observed at 3-5 days, 14 days, and 28 days after de la Recherche Scientifique, and Ligue Nationale Contre le Cancer. irradiation, respectively. This is likely to be due to intrinsi- cally different stages of these cells in the differentiation 1. Scollay, R., Bartlett, P. & Shortman, K. (1984) Immunol. Rev. process, as schematized in Fig. 4 and summarized below. 82, 79-103. (i) Stage 1: bone marrow stem cells. These cells are radiore- 2. Mathieson, B. J. & Fowlkes, B. J. (1984) Immunol. Rev. 82, sistant unless they are in active cycle and give rise to precur- 141-173. sors of unknown radiosensitivity. These precursors are prob- 3. Moore, M. A. S. & Owen, J. J. T. (1967) J. Exp. Med. 126, in the T-cell lineage. It is known that 715-723. ably not really committed 4. Le Douarin, N. & Jotereau, F. (1975) J. Exp. Med. 142, 17-40. bone marrow stem cells are partially radioresistant (34). 5. Fontaine-Perus, J. C., Calman, F. M., Kaplan, C. & Le (ii) Stage 2: migrating precursors. After leaving the bone Douarin, N. (1981) J. Immunol. 126, 2310-2316. marrow, these cells circulate and are radiosensitive. Their 6. Scollay, R., Smith, S. & Stauffer, V. (1986) Immunol. Rev. 91, absence in irradiated animals explains the gap in thymus 129-157. reconstitution. The total time between production of precur- 7. Rothenberg, E. & Lugo, J. P. (1985) Dev. Biol. 112, 1-17. sor cells and thymus seeding is 2 weeks. It is in fact not known 8. Shortman, K. & Jackson, H. (1974) Cell. Immunol. 12, 230- if this radiosensitive state corresponds to intramedullary or 246. circulating precursor cells. Thymus homing cells are present 9. Penit, C. (1986) J. Immunol. 137, 2115-2121. the bone marrow 10. Penit, C. (1987) Immunol. Res. 6, 271-278. outside of (23). 11. Penit, C. & Vasseur, F. (1988) J. Immunol. 140, 3315-3323. (iii) Stage 3: intrathymic precursor cells. After thymus 12. Penit, C., Vasseur, F. & Papiernik, M. (1988) Eur. J. Immunol. seeding, precursor cells undergo a series ofprocesses leading 18, 1343-1350. to proliferation. The total duration ofthese processes is again 13. Huiskamp, R. & van Ewijk, W. (1985) J. Immunol. 134, 2 weeks. At this stage, the cells are radiosensitive until a time 2161-2169. corresponding to nearly 3 days before effective DNA syn- 14. Tomooka, S., Matsuzaki, G., Kishihara, K., Takaka, K., thesis, when they are again radioresistant. Yoshikai, Y., Taniguchi, K., Himeno, K. & Nomoto, K. (1987) (iv) Stage 4: cycling thymocyte precursor cells (radiosensi- J. Immunol. 139, 3986-3990. tive). The reason for successive phases ofradiosensitivity and 15. Kadish, J. L. & Basch, R. (1976) J. Exp. Med. 143, 1082-1099. 16. Boersma, W., Betel, I., Daculsi, R. & Van der Westen, G...... (C) (1981) Cell Tissue Kinetics 14, 179-196. 0 r Production I(C) BM :....: 0 .: 17. Ceredig, R. & MacDonald, H. R. (1982) J. Immunol. 128, 614-619. 18. Goldschneider, I., Komshlies, K. L. & Greiner, D. L. (1986) J. 5 Exp. Med. 163, 1-17. Migration PERIPHERY 19. Hirokawa, K., Utsuyama, M., Katsura, Y. & Sado, T. (1988) (spleen ?) Arch. Pathol. Lab. Med. 112, 13-21. 10 I 20. Ezine, S., Weissman, I. L. & Rouse, R. V. (1984) Nature (London) 309, 629-631...... b 21. Fowlkes, B. J., Edison, L., Mathieson, B. J. & Chused, T. M. 4-.. B) Thymus (b) (D 4-8- '. (1985) J. Exp. Med. 162, 802-822. 15 _ seeding 22. Spangrude, G. J., Muller-Sieburg, C. E., Heinfeld, S. & Weiss- man, I. L. (1988) J. Exp. Med. 167, 1671-1683. E 23. Katsura, Y., Kina, T., Takaoki, Y. & Mishikawa, S. (1988) Eur. 20- J. Immunol. 18, 889-895. 24. Crispe, I. N., Moore, M. W., Husmann, L. A., Smith, L., Activation (a) 0 4-8- THYMUS I Bevan, M. J. & Shimonkevitz, R. P. (1987) Nature (London) 329, 336-339. 25 F 25. Shimonkevitz, R. P., Husmann, L. A., Bevan, M. J. & Crispe, Proliferation I. N. (1987) Nature (London) 329, 157-159. Differentiation 44-8+'. 26. Hyman, R., Lesley, J., Schulte, R. & Trotter, J. (1986) Cell. 30 '.4+8+'. 0 Resting Immunol. 101, 320-327. Cycling 27. Ledbetter, J. A. & Seaman, W. E. (1982) Immunol. Res. 68, Maturation 4+8-' 0 197-218. I ) Radio sensitive 28. Dialynas, D. P., Wilde, D. B., Marrack, P., Pierres, A. & 35L ...... *.. Fitch, F. W. (1983) Immunol. Rev. 74, 29-56. 29. Ledbetter, J. A. & Herzenberg, L. A. (1979) Immunol. Rev. 47, FIG. 4. Timing of thymocyte development from bone marrow 63-90. precursor cells to mature helper T cells. This timing is deduced from 30. Paterson, B. J. & Williams, A. F. (1987) J. Exp. Med. 166, the delays (vertical bars) observed between irradiation and early (A) 1603-1608. and late (C) wave of endogenous cell proliferation and also between 31. Shorman, K., Wilson, A., Egerton, M., Pearse, M. & Scollay, intrathymic injection and proliferation of bone marrow cells (B) or R. (1988) Cell. Immunol. 113, 462-479. CD4- CD8- thymocytes (A). The early spontaneous regeneration 32. MacDonald, H. R., Budd, R. C. & Howe, R. C. (1988) Eur. J. starts on step (a) as the development of i.t. injected CD4- CD8- Immunol. 18, 519-523. thymocytes. Injected bone marrow cells correspond to step (b). Cells 33. Adkins, B., Gaudour, D., Strober, S. & Weissman, I. (1988) J. at the earliest step, (c), are responsible for the second wave of Immunol. 140, 3373-3379. spontaneous regeneration. (See details in the text.) 34. Till, J. E. & McCulloch, E. A. (1961) Radiat. Res. 14,213-222. Downloaded by guest on September 26, 2021