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Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue
CELLULAR
IMMUNOLOGY
138,280-288 (199 1)
Interactions of Bone Marrow Cells from Young and Old Mice with Syngeneic and Allogeneic Thymic Tissue AYALASHARP,TANIAKUKULANSKY,AND
AMIELA GLOBERSON’
Department of Cell Biology, The Weizmann Institute of Science,Rehovot, 76100 Israel Received November I, 1990; acceptedJuly 3, 1991 Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in an in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy 1.2 and ARK/JThyl.1 seeded onto C57BL/6J IT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy I. mixed with C57BL/ Ka-Thyl.1 seeded onto C57BL/6J or C57BL/Ka IT), the Thyl+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thymocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice. 0 1991 Academic ores, Inc.
INTRODUCTION Cells differentiating in the thymus are selected so that only those manifesting selfMHC restriction, but not reactive against self-MHC determinants, are normally detectable in peripheral tissuesand in the circulation. At least two types of thymic stromal cells are believed to be involved in T cell selection: resident thymic epithelial cells (1, 2) and BM-derived’ dendritic cells, both of which constitutively express Class I and Class II MHC antigens (3-5). Epithelial cells have been found to mediate selection for self-MHC restriction, whereas medullary dendritic cells are involved in T cell ’ To whom correspondence should be addressed. 2 Abbreviations used: BM, bone marrow; FACS, fluorescence activated cell sorter; FT, fetal thymus.
280 0008~8749/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved
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tolerization (6-8). The mechanisms underlying positive and negative selection are still not adequately understood. Recent studies have revealed that MHC recognition can be expressed at an early phase of BM cell interaction with the thymic stroma and that it is associated with specific recognition of Class II MHC products (9). This was observed in an in vitro model in which cells from different lymphohemopoietic sources colonized lymphocytedepleted fetal thymic stroma and differentiated into thymocytes (10, 11). Using this organ culture system, an age-related decrease in the capacity of BM cells to colonize syngeneic FT explants was demonstrated (12). In light of these studies, it was of interest to determine whether any age-related changes occurred in MHC recognition of BM cells seeding the fetal thymus. We chose to employ the strategy of competitive reconstitution of FI’ explants by BM cells to determine whether the early manifestation of MHC-linked recognition was altered with age. Our results indicate an age-related decline in the syngeneic developmental preference of BM cells seeding the thymus and in the capacity of the thymic stroma to manifest tolerance to allogeneic MHC. MATERIALS
AND METHODS
Mice. Mice used throughout this study as source of 14-day-old fetal thymus explants included the Thy1 congenic strains C57BL/Ka (Thyl.1) and C57BL/6J (Thy1.2). Mating was limited to 16- 18 hr and the following day was considered as Day 0 of gestation. Thymus and BM cells were obtained from young (3 months) C57BL/Ka, C57BL/6J, AKR/J, or BALB/c mice, and old (24 months) C57BL/Ka, C57BL/6J, or BALB/c mice. The source of mice was as in our previous study (9). Mice with any overt pathological manifestations were excluded from the study. Cell preparation. Bone marrow cells were obtained from the femurs and tibias of donor mice. Thymus or BM cells were brought to form single cell suspensions, rinsed twice, and suspended in RPM1 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 1 mA4 Hepes, 5 X lo-’ M 2-mercaptoethanol, and antibiotics as described previously (11). Colonization of FT stroma. Fourteen-day-old fetal thymuses were depleted of their own thymocytes by treatment with 2-deoxyguanosine (10). They were then reconstituted with BM cells (6 X lo4 cells/explant, unless otherwise stated under Results) in hanging drops in Terasaki plates for 3 days and cultivated in organ cultures for 12 days. Thymic explants were subsequently recovered and single-cell suspensions were prepared for analysis ( 11). Colonization of FT stroma by sequential seedingof BM cells. Thymus explants were incubated with BM cells in hanging drops for 1 day. The explants were then removed, washed in organ culture medium, incubated with the cells of choice in hanging drops for 2 additional days, and transferred to organ cultures. Recolonization of FT stroma. Thymic explants were colonized by BM cells as described above. After 12 days of incubation, cell suspensions were prepared, washed, and counted, and samples were taken for flow cytometry. The rest of the cells were incubated for 3 days with freshly prepared FT, in hanging drops, and then transferred to organ cultures for 12 additional days. Analysis of cell phenotypes. Cell lines producing the monoclonal antibodies mouse anti-mouse Thy 1.1 (HO-22- 1) and Thy 1.2 (HO- 13-4) were obtained from the American Type Culture Collection (ATCC, USA). Fluorescein-conjugated goat anti-mouse IgG + IgA + IgM (H + L) was purchased from Zymed Laboratories Inc. (South San
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Francisco, CA). Cells were labeled by a two-step staining procedure and analyzed by fluorescence-activatedcell sorter (FACS 440, Becton-Dickinson, Mountain View, CA) as described (11). The data were analyzed by Consort 40 software on a microvax computer. Statistical analysis. Experiments were based on the seeding of cells onto at least 10 thymic lobes per experimental group. Cells harvested from these explants were then pooled for analysis. Results represent mean values f standard error (m + SE) of three to five repeated experiments, as specified in the Tables. Significance of differences was tested by Student’s t test. RESULTS MHC-Linked Syngeneic Preference of BM Cells for the Thymic Stroma In order to determine whether there were any age-related changes in the MHClinked colonization of thymic stroma by murine BM cells, FT explants were incubated with BM cells of young and old donors differing in MHC haplotypes. Chimeric thymuses containing syngeneic and allogeneic cells were constructed, and the number of thymocytes originating from each of the donors was determined. When young or old BM cells were seededonto syngeneic thymic stroma (C57BL/ 65) in a 1:1 mixture with young allogeneic BM cells (AKR/J), the Thy I+ cells which developed were mainly of the syngeneic type (Table 1A). The ratio of old syngeneic to young allogeneic BM-derived thymocytes was markedly lower than the ratio of young syngeneic to young allogeneic cells (P < 0.002). When the allogeneic BM cells originated from young and old BALB/c mice, we detected no significant differences between their capabilities to compete with young syngeneic cells (Table 1B). However, when old syngeneic cells were seededtogether with old allogeneic cells, the syngeneic advantage was not manifested (Table 1B). This indicated that the syngeneic preference which occurs in aging mice is significantly reduced compared with that in young mice.
TABLE Colonization
1A
of Fetal Thymus Stroma” with Syngeneic and Allogeneic BM Cells Seeded either Alone or in a 1: 1 Mixture
Origin of donor cells
% Positive cellsb
Group
C57BL/6J Thyl.2
AKR/J Thyl.1
Syngeneic Thyl.2
A B C D E
Young Old Young Old
Young Young Young -
64 -t 3 50 f 3 Ok0 80 + 4 89 f 2
Allogeneic Thyl.1 18+ 1 22 f 1 92 f 1 Ok0 Ok0
RR’ 22d 3od 100 0 0
n Recipient Ff explants were of C57BL/6J origin. b Percentage positive cells was calculated as [(%Thyl+ - background)/( 100 - background)] X 100. ‘Relative reconstitution (RR) was calculated as RR(Thyl.1) = [(Thyl. l)/(Thyl .I + Thyl.2)] X 100. Results represent means f SE of four experiments. d The statistical significance of a Student’s t test comparing groups A and B was P < 0.002.
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IB
of Fetal Thymus Stroma” with Syngeneic and Allogeneic BM Cells Seeded either Alone or in a 1: 1 Mixture % Positive cells
Origin of donor cells
Group
C57BL/Ka Thyl.l
BALB/c Thy1.2
Syngeneic Thyl.l
Allogeneic Thyl.2
RR
A B C D E F
Young Young Old Young -
Young Old Old
69 f 3 63 f 5 47 k 2 89 f 2 Ok0 Ok0
11 f I 20 f 5 43 f 4 Ok0 88 f 2 87 It 3
86 76 52 100 0 0
Young Old
’ Recipient FT explants were of C57BL/Ka
origin. Other details were as in Table I A.
Competitive Reconstitution of FT with Limiting Doses of BM Cells Experiments involving competition between murine syngeneic and allogeneic BM cells on FT stroma colonization were routinely carried out using 60,000 BM cells per thymic lobe, as previously described (14). In order to determine whether the developmental advantage of syngeneic over allogeneic thymocyte progenitors was based solely on qualitative differences (e.g., a higher affinity to receptors on the FT), or whether there were any quantitative differences (i.e., different numbers of progenitors seeding the thymus or of additional cells which influence colonization of the stroma), we carried out competitive colonization assays using limiting doses of BM cells. Table 2 shows the results of colonization of FT stroma with young and old BM cells with a total dose of either 1 X 105, 1 X 104, or 1 X lo3 cells/lobe. These experiments show that seeding the FT with limiting doses of cells resulted in an increased contribution of allogeneic BM cells to the colonization of the ET. The developmental advantage of syngeneic over allogeneic cells was not manifested. These results indicate that the
TABLE 2 Colonization
of Fetal Thymus Stroma with Mixtures of Syngeneic and Allogeneic BM Cells Seeded in Saturating or Limiting Numbers
Competing groups Syngeneic Thyl.l C57BL/Ka C57BL/Ka C57BL/Ka C57BL/Ka C57BL/Ka C57BL/Ka
% Positive cells
Allogeneic Thy I .2 BALB/c, BALB/c, BALB/c, BALB/c, BALB/c, BALB/c,
young young young old old old
Nofe. Details as in Table 1B.
Number of cells seeded lo5 lo4 IO3 lo5 lo4 lo3
Syngeneic Thy1.l
Allogeneic Thy1.2
RR
66k 12 33+ 3 22f 7 67 + 14 39* 5 38k 3
8+ 6 37+ 6 33t 11 llf 8 36k 2 31t- 8
89 47 40 87 54 55
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syngeneic advantage observed in our model system cannot be based on a mere quantitative difference between the competing syngeneic and allogeneic progenitors. Competition between Syngeneic and Allogeneic Cells following Sequential Thymic Seeding The experiments which followed were designed to determine whether competition between syngeneic and allogeneic BM cells occurs in the initial phase of incubation with the FT explants. Cocultures of FT stroma with allogeneic BM cells were incubated for 1 day. The explants were then rinsed to remove any cells that did not seedthem, and then reincubated with syngeneic cells for 2 additional days. Additional cultures were prepared by seedingthe thymus first with syngeneic cells and then with allogeneic cells, and the FT explants were then transferred to organ cultures for a period of 12 days. We found that when allogeneic cells were used for the initial incubation with the FT explants, followed by syngeneic cells, there was no syngeneic advantage (Table 3, groups C and F). The difference in outcome was not statistically significant whether the allogeneic cells which were seededfirst were young or old. These results suggest that competition between syngeneic and allogeneic BM cells begins during the initial phase of seeding of the thymic explants and that no significant age-related changes are manifested at this stage. Effect of the Thymic Microenvironment on Allogeneic Cells Previous experiments have suggestedthat BM cells colonizing allogeneic thymic stroma were influenced by the thymic microenvironment, such that in a subsequent competitive reconstitution of the FT, the BM-derived thymocytes manifested an advantage over cells syngeneic to that stroma (9). To find out whether this phenomenon affected old BM-derived thymocytes to the same extent as young, we performed sequential transfers of cells to FT explants. First we reconstituted FT explants with allogeneic BM cells in organ cultures for 12 days. Subsequently,we usedthe thymocytes TABLE 3 Colonization
of FT Stroma” by Sequential Seeding of Syngeneic (Syn) and Allogeneic (Allo) BM Cells % Positive cells’
Cells seeded b Group A B C D E F
Day 1 Syn, Allo, Syn, Allo, Syn, Allo, Sm Allo,
young young young young young old young old
Days 2-3 Syn, Allo, Allo, Syn, Syn, Allo, Allo, Syn,
young young young young young old old ww-
Al10
Syn
6lk 70+ 23k
7 8 9
71-r- 7 65k 5 38 f 10
14f 17f 52f
RR’
5 9 12
79 80 31d
12f 6 16k 5 392 10
86 80 49d
a Receipient FT was of C57BL/6J or C57BL/Ka origin. b Syngeneic BM cells were of C57BL/Ka (Thy 1.1) origin, and allogeneic BM cells were from either young (3 months) or old (24 months) BALB/c mice (Thy1.2). c Results represent means + SE values of five independent experiments. Other details as in Table 1A. d Differences between C and F were not statistically significant.
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which were obtained in these cultures for an additional reconstitution, in competition with either syngeneic BM cells or syngeneic BM-derived thymocytes that had developed in ET explants. After the first colonization, syngeneic MHC preference occurred, irrespective of whether the reconstituing cells were from young or old allogeneic mice (Table 4, groups 1A and 1B). This preference was abolished following the second colonization (groups 2A and 2B). Moreover, the young allogeneic BM-derived thymocytes reconstituted the FT to a significantly greater extent than did the young syngeneic BMderived thymocytes (P < 0.03). No significant differences were seen when old allogeneic BM-derived thymocytes competed with young syngeneic BM-derived thymocytes (group 2A vs group 2B). Both old and young allogeneic BM-derived thymocytes exhibited a considerable advantage in competition with freshly drawn syngeneic BM cells (groups 2F and 2G, respectively). These results indicate that both young and old BM cells are influenced by the thymic microenvironment, and that the old cells are less markedly affected than the young cells. Syngeneic MHC Pwference qf Thvmocytes In view of the loss of syngeneic advantage which we observed when FT was reconstituted competitively by BM-derived thymocytes, we asked whether syngeneic MHC recognition of thymocytes was similar to that of BM cells. Therefore, mixtures of syngeneic and allogeneic thymocytes, or combinations of syngeneic and allogeneic BM cells and thymocytes, were seeded onto FT explants, and the proportions of Thy l+ cells developing under these conditions were determined and compared with Thyl+ cells which developed from similar mixtures of syngeneic and allogeneic BM cells. As shown in Table 5, the proportions of cells developing from a mixture of syngeneic and allogeneic thymocytes were the same as those arising from a mixture of syngeneic and allogeneic BM cells (groups A vs C and B vs D, respectively). When the syngeneic cells derived from the BM and the allogeneic cells from the thymus, syngeneic preference was either not expressed (e.g., in the case of the young mice, group E) or it was not significant (e.g., in the case of the old mice, group F). When the syngeneic cells originated from the thymus, they manifested a more pronounced advantage when competing with the allogeneic BM cells (groups G and H). Thus, an MHC-linked syngeneic preference was shown to occur both in thymic cells and in BM cells. However, when thymocytes were mixed with BM cells, other factors (e.g., affinity to stroma, number of precursors) seemed to influence the ratios of developing cells in such a way as to yield a higher proportion of ceils of thymic origin. DISCUSSION Our study focused on age-related changes in the interactions of BM cells with allogeneic thymic stroma. It was found that syngeneic MHC preference, manifested upon colonization of the FT by BM cells, is also expressed in BM cells originating from old mice. This preference is, however, reduced and in certain cases abolished in aging. Our experiments on allogeneic cells were based mainly on BALB/c (H-2d) vs C57BL/ 6 (H-2b) mice. In some experiments we also employed AKR/d (H-2k) mice. Since the AKR are known to be prone to leukemia, we used them only as young donors. The fact that the results obtained were similar to those in experiments on BALB/c mice
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TABLE 4 Effect of the Thymic Microenvironment
on Young and Old BM-Derived
Donor cells Group
Syngeneic
First colonization IA 1B 1c ID 1E Second colonization 2A 2B 2c 2D 2E
Thymocytes”
% Positive cellsb
Allogeneic
Syngeneic
Old Young Old Young
63 + 5 69 + 3 Ok0 Ok0 89 f 2
20 f 5 11 + 1 87 + 3 88 + 2 Ok0
76d 86d 0 0 100
51+5 30 f 8 Ok0 Ok0 91 f 1
37 f 7 60 + 7 85 +4 91 f3 Ok0
58’ 33’ 0 0 100
10 + 4 422
85 f 5 89 f 4
10 4
Allogeneic
RR’
BM cells Young Young Young BM-derived Young Young Young
thymocytes Old Young Old Young -
Syngeneic BM cells + BM-derived thymocytes 2F 2G
Young Young
Old Young
’ Recipient FT and reconstituting cells were as in Table 1B. b Results represent mean + SE values of 3-6 experiments. ’ Calculated as in Table 1A. d Differences in results between 1A and 1B were not statistically significant. e The statistical significance of a Student’s t test comparing 2A and 2B was P < 0.03.
indicates that the observations are not limited to the H-2d/H-2b combination, as reported by us previously on the BIO.F congenic mice (9). When IT colonization was carried out with limiting numbers of cells, syngeneic preference was not manifested. This could be due to the availability of an excessof sites for colonization, such that no competition occurred between colonizing cells and clonal seedingof the explants may have taken place. The fact that under such conditions the same reconstitution was obtained with syngeneic and allogeneic cells suggeststhat both strains possessthe same numbers of T cell progenitors and a similar potential for clonal development within the individual thymic explant. Alternatively, if cells in the BM other than T cell progenitors are responsible for the syngeneic preference (e.g., macrophages, dendrytic cells, or mature lymphocytes), then seeding them in limiting numbers could also result in abolishing their influence on the cells developing in the FT. In this respect, it should be noted that the proportion of ThylI cells increasesin the FT seededwith limiting numbers of BM cells. The phenotype of these cells has not yet been characterized and it is possible that they might have an influence on the process of FT colonization. The strategy of sequential incubation revealed that the first day of coculture was critical in determining the type of cells which subsequently developed in the PT. Prior
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THYMUS
TABLE 5 Competition
between Syngeneic and Allogeneic Thymocytes and BM Cells on Fetal Thymus Colonization Source of cellsh
Group’
% Positive cells‘
Syngeneic
Allogeneic
Syngeneic
BM, young BM, young Thymus, young Thymus, young BM, young BM, young Thymus, young Thymus, young
BM, young BM, old Thymus, young Thymus, old Thymus, young Thymus, old BM, young BM, old
60% 5 51+- 9 7li: 6 84+ 6 36+- 9 41* I1 172 4 942 1
Allogeneic 18k 12-+ 28k 15f 33a 26k 5* 1*
9 7 8 7 15 6 2 0
RR‘ II 83 72 85 52 61 94 99
’ Young (3 months) and old (24 months) mice were used. h Cells from syngeneic (C57BL/Ka) mice and young or old allogeneic (BALB/c) mice were mixed (I: 1) to provide for a total of 60,000 cells/m. ’ Results represent means f SE of 3-5 experiments. Other details as in Table 1A.
of allogeneic cells for even a single day was sufficient to reduce or abolish the syngeneic advantage in the old mice, or to promote the advantage of the allogeneic cells in the case of young mice. This may be based on the fact that, following the initial seeding, irreversible cell-cell interactions occur between the BM cells and the thymic stroma. If this is the case, then the syngeneic advantage may not be manifested, because after the first day of colonization the allogeneic cells already occupy a larger number of “niches” on the stroma, thus inhibiting subsequent colonization by syngeneic cells. This is in line with the idea that the number of “niches” in the stroma is limited (13). Alternatively, the allogeneic BM cells (possibly macrophages, dendritic cells, or mature T cells) may induce changes in the fetal thymus, such that tolerance is established and any further syngeneic advantage is abolished. Both of these possibilities are currently being investigated in our laboratory. Whatever the mechanism underlying this phenomenon, it should be noted that in these experiments no significant differences were found between young and old allogeneic cells. Data from recolonization of FT explants showed that the syngeneic advantage was abolished when BM-derived thymocytes were reseeded onto secondary FT explants. It is possible that during the first colonization, competition between syngeneic and allogeneic cells was on the level of binding to a homing marker associated with the MHC. An advantage in such binding results in the occupation of a larger number of “niches” within the fetal thymus and therefore a development of more cells. During the second colonization, however, BM-derived thymocytes of allogeneic origin could have been altered, or cells capable of binding to the MHC of thymic epithelium could have been positively selected, analogous to the suggested model of positive selection of cells expressing receptors which can bind the MHC expressed by the thymic epithelium (2, 14- 18). In such a case, competition at the recolonization would be on the homing receptors, regardless of the MHC determinants. Alternatively, cells other than thymocytes, developing in the fetal thymus during the first colonization, might participate in inducing tolerance during the second colonization. Indeed, there are loincubation
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30% Thyll cells in the FT after organ culture and these may influence the outcome of the recolonization. Irrespective of the mechanism, significant differences were noted between young and old allogeneic BM-derived thymocytes in the recolonization experiments. Thus, the old allogeneic BM-derived thymocytes showed no significant disadvantage when competing with young syngeneic BM-derived cells, whereas young allogeneic BMderived thymocytes gained an advantageover young syngeneicBM-derived thymocytes. The syngeneic advantage was revealed when freshly drawn thymocytes were used, with a pattern of colonization similar to that of syngeneic and allogeneic BM cells. However, when allogeneic BM-derived thymocytes were seededwith freshly drawn syngeneic BM cells, the allogeneic thymocytes were practically the sole source of reconstituting cells. Moreover, when syngeneic BM cells competed with freshly drawn allogeneic thymus cells, the syngeneic advantage was not manifested, due perhaps to the presence of a larger number of T cell progenitors in the thymocyte population, or to a higher affinity of the thymocytes to the thymic stroma. In conclusion, the mechanisms underlying the phenomenon of syngeneicpreference are not yet clear and until the phenotypes of all cells recovered from the FT are accounted for, aswell asthe effectsof mature lymphocytes, macrophages,and dendrytic cells on colonization of the IT’, our conclusions remain tentative. Whatever the mechanisms of syngeneic preference, our data on the early stagesof interaction between BM or thymus cells with the thymus point at a significant agerelated decreasein the MHC-linked recognition of cells from old mice during their interaction with allogeneic thymic stroma. The basis of this aging phenomenon will require further elucidation. ACKNOWLEDGMENTS Supported by the SANDOZ Foundation for Gerontological Research and the U.S.-Israel Binational Science Foundation. A.G. is an incumbent of the Harriet and Harold Brady Chair for Cancer Research. The technical assistance of Mrs. Hana Stup is greatly appreciated.
REFERENCES 1. Jordan, R. K., Robinson, J. H., Hopkinson, N. A., House, K. C., and Bently, A. L., Nature 314, 454, 1985. 2. Ron, Y., Lo, D., and Sprent, J., J. Immunol. 137, 1764, 1986. 3. Steinman, R. M., and Nussenzweig, M. C., Immunol. Rev. 53, 127, 1980. 4. Longo, D. L., and Schwartz, R. H., Nature 287, 44, 1980. 5. Benson, M. T., Buckley, E. J., Jenkinson, E. J., and Owen, J. J. T., Immunology 60, 593, 1987. 6. Ready, A. R., Jenkinson, E. J., Kingston, R., and Owen, J. J. T., Nature 310, 231, 1984. 7. Jenkinson, E. J., Jhittay, P., Kingston, R., and Owen, J. J. T., Transplantation 39, 331, 1985. 8. Lo, D., and Sprent, J., Nature 319, 672, 1986. 9. Eren, R., Abel, L., and Globerson, A., Eur. J. Immunol. 19, 2087, 1989. 10. Jenkinson, E. J., Franchi, L. L., Kingston, R., and Owen, J. J. T., Eur. J. Immunol. 12, 583, 1982. 11. Eren, R., Zharhary, D., Abel, L., and Globerson, A., Cell. Immunol. 108, 76, 1987. 12. Eren, R., Zharhary, D., Abel, L., and Globerson, A., Cell. Immunol. 112,449, 1988. 13. Shortman, K., Egerton, M., Spangrude, G. J., and Scollay, R., Semin. Immunol. 2, 3, 1990. 14. Bevan, M., and Fink, P., Immunol. Rev. 42, 3, 1978. 15. Zinkernagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Klein, P. A., and Klein, J., J. Exp. Med. 147,882, 1978. 16. Zinkernagel, R. M., Callahan, G. N., Klein, J., and Dennert, G., Nature 271, 251, 1978. 17. Singer, A., Hathcock, K. S., and Hodes, R. J., J. Immunol. 126, 1327, 1982. 18. Marrack, P., Lo, D., Brinster, R., Palmiter, R., Burkly, L., Flavell, R. H., and Kappler, J., Cell 53,627, 1988.
IMMUNOLOGY
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Interactions of Bone Marrow Cells from Young and Old Mice with Syngeneic and Allogeneic Thymic Tissue AYALASHARP,TANIAKUKULANSKY,AND
AMIELA GLOBERSON’
Department of Cell Biology, The Weizmann Institute of Science,Rehovot, 76100 Israel Received November I, 1990; acceptedJuly 3, 1991 Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in an in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy 1.2 and ARK/JThyl.1 seeded onto C57BL/6J IT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy I. mixed with C57BL/ Ka-Thyl.1 seeded onto C57BL/6J or C57BL/Ka IT), the Thyl+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thymocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice. 0 1991 Academic ores, Inc.
INTRODUCTION Cells differentiating in the thymus are selected so that only those manifesting selfMHC restriction, but not reactive against self-MHC determinants, are normally detectable in peripheral tissuesand in the circulation. At least two types of thymic stromal cells are believed to be involved in T cell selection: resident thymic epithelial cells (1, 2) and BM-derived’ dendritic cells, both of which constitutively express Class I and Class II MHC antigens (3-5). Epithelial cells have been found to mediate selection for self-MHC restriction, whereas medullary dendritic cells are involved in T cell ’ To whom correspondence should be addressed. 2 Abbreviations used: BM, bone marrow; FACS, fluorescence activated cell sorter; FT, fetal thymus.
280 0008~8749/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved
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tolerization (6-8). The mechanisms underlying positive and negative selection are still not adequately understood. Recent studies have revealed that MHC recognition can be expressed at an early phase of BM cell interaction with the thymic stroma and that it is associated with specific recognition of Class II MHC products (9). This was observed in an in vitro model in which cells from different lymphohemopoietic sources colonized lymphocytedepleted fetal thymic stroma and differentiated into thymocytes (10, 11). Using this organ culture system, an age-related decrease in the capacity of BM cells to colonize syngeneic FT explants was demonstrated (12). In light of these studies, it was of interest to determine whether any age-related changes occurred in MHC recognition of BM cells seeding the fetal thymus. We chose to employ the strategy of competitive reconstitution of FI’ explants by BM cells to determine whether the early manifestation of MHC-linked recognition was altered with age. Our results indicate an age-related decline in the syngeneic developmental preference of BM cells seeding the thymus and in the capacity of the thymic stroma to manifest tolerance to allogeneic MHC. MATERIALS
AND METHODS
Mice. Mice used throughout this study as source of 14-day-old fetal thymus explants included the Thy1 congenic strains C57BL/Ka (Thyl.1) and C57BL/6J (Thy1.2). Mating was limited to 16- 18 hr and the following day was considered as Day 0 of gestation. Thymus and BM cells were obtained from young (3 months) C57BL/Ka, C57BL/6J, AKR/J, or BALB/c mice, and old (24 months) C57BL/Ka, C57BL/6J, or BALB/c mice. The source of mice was as in our previous study (9). Mice with any overt pathological manifestations were excluded from the study. Cell preparation. Bone marrow cells were obtained from the femurs and tibias of donor mice. Thymus or BM cells were brought to form single cell suspensions, rinsed twice, and suspended in RPM1 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 1 mA4 Hepes, 5 X lo-’ M 2-mercaptoethanol, and antibiotics as described previously (11). Colonization of FT stroma. Fourteen-day-old fetal thymuses were depleted of their own thymocytes by treatment with 2-deoxyguanosine (10). They were then reconstituted with BM cells (6 X lo4 cells/explant, unless otherwise stated under Results) in hanging drops in Terasaki plates for 3 days and cultivated in organ cultures for 12 days. Thymic explants were subsequently recovered and single-cell suspensions were prepared for analysis ( 11). Colonization of FT stroma by sequential seedingof BM cells. Thymus explants were incubated with BM cells in hanging drops for 1 day. The explants were then removed, washed in organ culture medium, incubated with the cells of choice in hanging drops for 2 additional days, and transferred to organ cultures. Recolonization of FT stroma. Thymic explants were colonized by BM cells as described above. After 12 days of incubation, cell suspensions were prepared, washed, and counted, and samples were taken for flow cytometry. The rest of the cells were incubated for 3 days with freshly prepared FT, in hanging drops, and then transferred to organ cultures for 12 additional days. Analysis of cell phenotypes. Cell lines producing the monoclonal antibodies mouse anti-mouse Thy 1.1 (HO-22- 1) and Thy 1.2 (HO- 13-4) were obtained from the American Type Culture Collection (ATCC, USA). Fluorescein-conjugated goat anti-mouse IgG + IgA + IgM (H + L) was purchased from Zymed Laboratories Inc. (South San
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Francisco, CA). Cells were labeled by a two-step staining procedure and analyzed by fluorescence-activatedcell sorter (FACS 440, Becton-Dickinson, Mountain View, CA) as described (11). The data were analyzed by Consort 40 software on a microvax computer. Statistical analysis. Experiments were based on the seeding of cells onto at least 10 thymic lobes per experimental group. Cells harvested from these explants were then pooled for analysis. Results represent mean values f standard error (m + SE) of three to five repeated experiments, as specified in the Tables. Significance of differences was tested by Student’s t test. RESULTS MHC-Linked Syngeneic Preference of BM Cells for the Thymic Stroma In order to determine whether there were any age-related changes in the MHClinked colonization of thymic stroma by murine BM cells, FT explants were incubated with BM cells of young and old donors differing in MHC haplotypes. Chimeric thymuses containing syngeneic and allogeneic cells were constructed, and the number of thymocytes originating from each of the donors was determined. When young or old BM cells were seededonto syngeneic thymic stroma (C57BL/ 65) in a 1:1 mixture with young allogeneic BM cells (AKR/J), the Thy I+ cells which developed were mainly of the syngeneic type (Table 1A). The ratio of old syngeneic to young allogeneic BM-derived thymocytes was markedly lower than the ratio of young syngeneic to young allogeneic cells (P < 0.002). When the allogeneic BM cells originated from young and old BALB/c mice, we detected no significant differences between their capabilities to compete with young syngeneic cells (Table 1B). However, when old syngeneic cells were seededtogether with old allogeneic cells, the syngeneic advantage was not manifested (Table 1B). This indicated that the syngeneic preference which occurs in aging mice is significantly reduced compared with that in young mice.
TABLE Colonization
1A
of Fetal Thymus Stroma” with Syngeneic and Allogeneic BM Cells Seeded either Alone or in a 1: 1 Mixture
Origin of donor cells
% Positive cellsb
Group
C57BL/6J Thyl.2
AKR/J Thyl.1
Syngeneic Thyl.2
A B C D E
Young Old Young Old
Young Young Young -
64 -t 3 50 f 3 Ok0 80 + 4 89 f 2
Allogeneic Thyl.1 18+ 1 22 f 1 92 f 1 Ok0 Ok0
RR’ 22d 3od 100 0 0
n Recipient Ff explants were of C57BL/6J origin. b Percentage positive cells was calculated as [(%Thyl+ - background)/( 100 - background)] X 100. ‘Relative reconstitution (RR) was calculated as RR(Thyl.1) = [(Thyl. l)/(Thyl .I + Thyl.2)] X 100. Results represent means f SE of four experiments. d The statistical significance of a Student’s t test comparing groups A and B was P < 0.002.
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IB
of Fetal Thymus Stroma” with Syngeneic and Allogeneic BM Cells Seeded either Alone or in a 1: 1 Mixture % Positive cells
Origin of donor cells
Group
C57BL/Ka Thyl.l
BALB/c Thy1.2
Syngeneic Thyl.l
Allogeneic Thyl.2
RR
A B C D E F
Young Young Old Young -
Young Old Old
69 f 3 63 f 5 47 k 2 89 f 2 Ok0 Ok0
11 f I 20 f 5 43 f 4 Ok0 88 f 2 87 It 3
86 76 52 100 0 0
Young Old
’ Recipient FT explants were of C57BL/Ka
origin. Other details were as in Table I A.
Competitive Reconstitution of FT with Limiting Doses of BM Cells Experiments involving competition between murine syngeneic and allogeneic BM cells on FT stroma colonization were routinely carried out using 60,000 BM cells per thymic lobe, as previously described (14). In order to determine whether the developmental advantage of syngeneic over allogeneic thymocyte progenitors was based solely on qualitative differences (e.g., a higher affinity to receptors on the FT), or whether there were any quantitative differences (i.e., different numbers of progenitors seeding the thymus or of additional cells which influence colonization of the stroma), we carried out competitive colonization assays using limiting doses of BM cells. Table 2 shows the results of colonization of FT stroma with young and old BM cells with a total dose of either 1 X 105, 1 X 104, or 1 X lo3 cells/lobe. These experiments show that seeding the FT with limiting doses of cells resulted in an increased contribution of allogeneic BM cells to the colonization of the ET. The developmental advantage of syngeneic over allogeneic cells was not manifested. These results indicate that the
TABLE 2 Colonization
of Fetal Thymus Stroma with Mixtures of Syngeneic and Allogeneic BM Cells Seeded in Saturating or Limiting Numbers
Competing groups Syngeneic Thyl.l C57BL/Ka C57BL/Ka C57BL/Ka C57BL/Ka C57BL/Ka C57BL/Ka
% Positive cells
Allogeneic Thy I .2 BALB/c, BALB/c, BALB/c, BALB/c, BALB/c, BALB/c,
young young young old old old
Nofe. Details as in Table 1B.
Number of cells seeded lo5 lo4 IO3 lo5 lo4 lo3
Syngeneic Thy1.l
Allogeneic Thy1.2
RR
66k 12 33+ 3 22f 7 67 + 14 39* 5 38k 3
8+ 6 37+ 6 33t 11 llf 8 36k 2 31t- 8
89 47 40 87 54 55
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syngeneic advantage observed in our model system cannot be based on a mere quantitative difference between the competing syngeneic and allogeneic progenitors. Competition between Syngeneic and Allogeneic Cells following Sequential Thymic Seeding The experiments which followed were designed to determine whether competition between syngeneic and allogeneic BM cells occurs in the initial phase of incubation with the FT explants. Cocultures of FT stroma with allogeneic BM cells were incubated for 1 day. The explants were then rinsed to remove any cells that did not seedthem, and then reincubated with syngeneic cells for 2 additional days. Additional cultures were prepared by seedingthe thymus first with syngeneic cells and then with allogeneic cells, and the FT explants were then transferred to organ cultures for a period of 12 days. We found that when allogeneic cells were used for the initial incubation with the FT explants, followed by syngeneic cells, there was no syngeneic advantage (Table 3, groups C and F). The difference in outcome was not statistically significant whether the allogeneic cells which were seededfirst were young or old. These results suggest that competition between syngeneic and allogeneic BM cells begins during the initial phase of seeding of the thymic explants and that no significant age-related changes are manifested at this stage. Effect of the Thymic Microenvironment on Allogeneic Cells Previous experiments have suggestedthat BM cells colonizing allogeneic thymic stroma were influenced by the thymic microenvironment, such that in a subsequent competitive reconstitution of the FT, the BM-derived thymocytes manifested an advantage over cells syngeneic to that stroma (9). To find out whether this phenomenon affected old BM-derived thymocytes to the same extent as young, we performed sequential transfers of cells to FT explants. First we reconstituted FT explants with allogeneic BM cells in organ cultures for 12 days. Subsequently,we usedthe thymocytes TABLE 3 Colonization
of FT Stroma” by Sequential Seeding of Syngeneic (Syn) and Allogeneic (Allo) BM Cells % Positive cells’
Cells seeded b Group A B C D E F
Day 1 Syn, Allo, Syn, Allo, Syn, Allo, Sm Allo,
young young young young young old young old
Days 2-3 Syn, Allo, Allo, Syn, Syn, Allo, Allo, Syn,
young young young young young old old ww-
Al10
Syn
6lk 70+ 23k
7 8 9
71-r- 7 65k 5 38 f 10
14f 17f 52f
RR’
5 9 12
79 80 31d
12f 6 16k 5 392 10
86 80 49d
a Receipient FT was of C57BL/6J or C57BL/Ka origin. b Syngeneic BM cells were of C57BL/Ka (Thy 1.1) origin, and allogeneic BM cells were from either young (3 months) or old (24 months) BALB/c mice (Thy1.2). c Results represent means + SE values of five independent experiments. Other details as in Table 1A. d Differences between C and F were not statistically significant.
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which were obtained in these cultures for an additional reconstitution, in competition with either syngeneic BM cells or syngeneic BM-derived thymocytes that had developed in ET explants. After the first colonization, syngeneic MHC preference occurred, irrespective of whether the reconstituing cells were from young or old allogeneic mice (Table 4, groups 1A and 1B). This preference was abolished following the second colonization (groups 2A and 2B). Moreover, the young allogeneic BM-derived thymocytes reconstituted the FT to a significantly greater extent than did the young syngeneic BMderived thymocytes (P < 0.03). No significant differences were seen when old allogeneic BM-derived thymocytes competed with young syngeneic BM-derived thymocytes (group 2A vs group 2B). Both old and young allogeneic BM-derived thymocytes exhibited a considerable advantage in competition with freshly drawn syngeneic BM cells (groups 2F and 2G, respectively). These results indicate that both young and old BM cells are influenced by the thymic microenvironment, and that the old cells are less markedly affected than the young cells. Syngeneic MHC Pwference qf Thvmocytes In view of the loss of syngeneic advantage which we observed when FT was reconstituted competitively by BM-derived thymocytes, we asked whether syngeneic MHC recognition of thymocytes was similar to that of BM cells. Therefore, mixtures of syngeneic and allogeneic thymocytes, or combinations of syngeneic and allogeneic BM cells and thymocytes, were seeded onto FT explants, and the proportions of Thy l+ cells developing under these conditions were determined and compared with Thyl+ cells which developed from similar mixtures of syngeneic and allogeneic BM cells. As shown in Table 5, the proportions of cells developing from a mixture of syngeneic and allogeneic thymocytes were the same as those arising from a mixture of syngeneic and allogeneic BM cells (groups A vs C and B vs D, respectively). When the syngeneic cells derived from the BM and the allogeneic cells from the thymus, syngeneic preference was either not expressed (e.g., in the case of the young mice, group E) or it was not significant (e.g., in the case of the old mice, group F). When the syngeneic cells originated from the thymus, they manifested a more pronounced advantage when competing with the allogeneic BM cells (groups G and H). Thus, an MHC-linked syngeneic preference was shown to occur both in thymic cells and in BM cells. However, when thymocytes were mixed with BM cells, other factors (e.g., affinity to stroma, number of precursors) seemed to influence the ratios of developing cells in such a way as to yield a higher proportion of ceils of thymic origin. DISCUSSION Our study focused on age-related changes in the interactions of BM cells with allogeneic thymic stroma. It was found that syngeneic MHC preference, manifested upon colonization of the FT by BM cells, is also expressed in BM cells originating from old mice. This preference is, however, reduced and in certain cases abolished in aging. Our experiments on allogeneic cells were based mainly on BALB/c (H-2d) vs C57BL/ 6 (H-2b) mice. In some experiments we also employed AKR/d (H-2k) mice. Since the AKR are known to be prone to leukemia, we used them only as young donors. The fact that the results obtained were similar to those in experiments on BALB/c mice
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TABLE 4 Effect of the Thymic Microenvironment
on Young and Old BM-Derived
Donor cells Group
Syngeneic
First colonization IA 1B 1c ID 1E Second colonization 2A 2B 2c 2D 2E
Thymocytes”
% Positive cellsb
Allogeneic
Syngeneic
Old Young Old Young
63 + 5 69 + 3 Ok0 Ok0 89 f 2
20 f 5 11 + 1 87 + 3 88 + 2 Ok0
76d 86d 0 0 100
51+5 30 f 8 Ok0 Ok0 91 f 1
37 f 7 60 + 7 85 +4 91 f3 Ok0
58’ 33’ 0 0 100
10 + 4 422
85 f 5 89 f 4
10 4
Allogeneic
RR’
BM cells Young Young Young BM-derived Young Young Young
thymocytes Old Young Old Young -
Syngeneic BM cells + BM-derived thymocytes 2F 2G
Young Young
Old Young
’ Recipient FT and reconstituting cells were as in Table 1B. b Results represent mean + SE values of 3-6 experiments. ’ Calculated as in Table 1A. d Differences in results between 1A and 1B were not statistically significant. e The statistical significance of a Student’s t test comparing 2A and 2B was P < 0.03.
indicates that the observations are not limited to the H-2d/H-2b combination, as reported by us previously on the BIO.F congenic mice (9). When IT colonization was carried out with limiting numbers of cells, syngeneic preference was not manifested. This could be due to the availability of an excessof sites for colonization, such that no competition occurred between colonizing cells and clonal seedingof the explants may have taken place. The fact that under such conditions the same reconstitution was obtained with syngeneic and allogeneic cells suggeststhat both strains possessthe same numbers of T cell progenitors and a similar potential for clonal development within the individual thymic explant. Alternatively, if cells in the BM other than T cell progenitors are responsible for the syngeneic preference (e.g., macrophages, dendrytic cells, or mature lymphocytes), then seeding them in limiting numbers could also result in abolishing their influence on the cells developing in the FT. In this respect, it should be noted that the proportion of ThylI cells increasesin the FT seededwith limiting numbers of BM cells. The phenotype of these cells has not yet been characterized and it is possible that they might have an influence on the process of FT colonization. The strategy of sequential incubation revealed that the first day of coculture was critical in determining the type of cells which subsequently developed in the PT. Prior
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TABLE 5 Competition
between Syngeneic and Allogeneic Thymocytes and BM Cells on Fetal Thymus Colonization Source of cellsh
Group’
% Positive cells‘
Syngeneic
Allogeneic
Syngeneic
BM, young BM, young Thymus, young Thymus, young BM, young BM, young Thymus, young Thymus, young
BM, young BM, old Thymus, young Thymus, old Thymus, young Thymus, old BM, young BM, old
60% 5 51+- 9 7li: 6 84+ 6 36+- 9 41* I1 172 4 942 1
Allogeneic 18k 12-+ 28k 15f 33a 26k 5* 1*
9 7 8 7 15 6 2 0
RR‘ II 83 72 85 52 61 94 99
’ Young (3 months) and old (24 months) mice were used. h Cells from syngeneic (C57BL/Ka) mice and young or old allogeneic (BALB/c) mice were mixed (I: 1) to provide for a total of 60,000 cells/m. ’ Results represent means f SE of 3-5 experiments. Other details as in Table 1A.
of allogeneic cells for even a single day was sufficient to reduce or abolish the syngeneic advantage in the old mice, or to promote the advantage of the allogeneic cells in the case of young mice. This may be based on the fact that, following the initial seeding, irreversible cell-cell interactions occur between the BM cells and the thymic stroma. If this is the case, then the syngeneic advantage may not be manifested, because after the first day of colonization the allogeneic cells already occupy a larger number of “niches” on the stroma, thus inhibiting subsequent colonization by syngeneic cells. This is in line with the idea that the number of “niches” in the stroma is limited (13). Alternatively, the allogeneic BM cells (possibly macrophages, dendritic cells, or mature T cells) may induce changes in the fetal thymus, such that tolerance is established and any further syngeneic advantage is abolished. Both of these possibilities are currently being investigated in our laboratory. Whatever the mechanism underlying this phenomenon, it should be noted that in these experiments no significant differences were found between young and old allogeneic cells. Data from recolonization of FT explants showed that the syngeneic advantage was abolished when BM-derived thymocytes were reseeded onto secondary FT explants. It is possible that during the first colonization, competition between syngeneic and allogeneic cells was on the level of binding to a homing marker associated with the MHC. An advantage in such binding results in the occupation of a larger number of “niches” within the fetal thymus and therefore a development of more cells. During the second colonization, however, BM-derived thymocytes of allogeneic origin could have been altered, or cells capable of binding to the MHC of thymic epithelium could have been positively selected, analogous to the suggested model of positive selection of cells expressing receptors which can bind the MHC expressed by the thymic epithelium (2, 14- 18). In such a case, competition at the recolonization would be on the homing receptors, regardless of the MHC determinants. Alternatively, cells other than thymocytes, developing in the fetal thymus during the first colonization, might participate in inducing tolerance during the second colonization. Indeed, there are loincubation
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30% Thyll cells in the FT after organ culture and these may influence the outcome of the recolonization. Irrespective of the mechanism, significant differences were noted between young and old allogeneic BM-derived thymocytes in the recolonization experiments. Thus, the old allogeneic BM-derived thymocytes showed no significant disadvantage when competing with young syngeneic BM-derived cells, whereas young allogeneic BMderived thymocytes gained an advantageover young syngeneicBM-derived thymocytes. The syngeneic advantage was revealed when freshly drawn thymocytes were used, with a pattern of colonization similar to that of syngeneic and allogeneic BM cells. However, when allogeneic BM-derived thymocytes were seededwith freshly drawn syngeneic BM cells, the allogeneic thymocytes were practically the sole source of reconstituting cells. Moreover, when syngeneic BM cells competed with freshly drawn allogeneic thymus cells, the syngeneic advantage was not manifested, due perhaps to the presence of a larger number of T cell progenitors in the thymocyte population, or to a higher affinity of the thymocytes to the thymic stroma. In conclusion, the mechanisms underlying the phenomenon of syngeneicpreference are not yet clear and until the phenotypes of all cells recovered from the FT are accounted for, aswell asthe effectsof mature lymphocytes, macrophages,and dendrytic cells on colonization of the IT’, our conclusions remain tentative. Whatever the mechanisms of syngeneic preference, our data on the early stagesof interaction between BM or thymus cells with the thymus point at a significant agerelated decreasein the MHC-linked recognition of cells from old mice during their interaction with allogeneic thymic stroma. The basis of this aging phenomenon will require further elucidation. ACKNOWLEDGMENTS Supported by the SANDOZ Foundation for Gerontological Research and the U.S.-Israel Binational Science Foundation. A.G. is an incumbent of the Harriet and Harold Brady Chair for Cancer Research. The technical assistance of Mrs. Hana Stup is greatly appreciated.
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