Scaffd. / . Immunol., Vol. 5. 1976.
Morphologic and Antigenic Maturation of Lymphocytes in the Mouse Thymus In Vitro R. JUHLIN & G. V. ALM Department of Histology, Institute of Htiman Anatomy, Biomediatl Center, University of Uppsala, Uppsala, Sweden
Juhlin, R. & Aim, G. V. Morphologic and Antigenic Maturation of Lymphocytes in the Mouse Thymus In Vitro. Scatid. / . Immunol. 5, 497-503. 1976. A simple technique for organ culture of the thymus of 14-day-old moose embryos is described. It allowed a characteristic time-dependent development of the immature thymus into a lymphoid thymus with large numbers of nondividing small lymphocytes. Most of these cells carried the T-lymphocyte ajitigenic markers Thy-1 and TL, and all expressed H-2 antipens as determined in cytotoxicit/ assays. They probably developed from precursor cells without detectable Thy-1 and TL. This development appeared to be dependent on the thymic microenvironment, since it also occurred in serum-free organ cultures and in cultures with medium supplemented with serum from nude mice, R. Juhlin, Departmenl of Virology, Biomedical Center, Box 584, S-751 23 Uplisala, Sweden
Lymphoid precursor cells migrate into the thymus of mice both in fetal and adult life. They first appear in the embryonic epithelial thymus on day 11 or 12 of gestation (15, 18, 22). The properties of these precursor cells have not been established in detail, but, at least when in the bone marrow of young mice, they appear to be committed to lymphoid development but express little, if any, of the antigenic markers identifying T lymphocytes (11). The further development of lymphoid precursor cells within the thymus probably involves a series of events that includes cell proliferation and differentiation. Most of the more mature thymic lymphocytes are small nondividing cells witli a high density of antigens such as Thy-1 and TL. A small fraction of the mature lymphocytes has a lower density of Thy-1 and lack TL antigens; they represent the immunocompetent lymphocytes found in thc thymus (12, 23). The thymus is recognized as critical to the development of T-lymphocyte functions (3, 17-19), including the generation of immuno-
competent cells and possibly also maintenance of self-tolerance. A growing body of data suggests a considerable heterogeneity within thymic and peripheral T lymphocytes (4, 5, 7, 8). This raises the question of the developmental relation of the various antigenically and functionally defined subsets of T lymphocytes to the thymus. As recently reviewed (6, 25), there is dearly a possibility that lymphocytes within the thymus may develop along separate pathways and further that the different subsets of peripheral T lymphocytes may have special relations to these, perhaps representing separate developmental lineages. It would be desirable to have access to an in vitro system that would allow definition of the relation of the thymus to the development of T lymphocytes. The organ culture technique has previously been used to maintain the embryonic chicken (1, 24) and mouse thymus (2, 16, 20) in vitro. It allows the development of the thymus from a stage at which most nonepithelial cells are primitive lymphoid
498
R. Jtihlii! & G. V. Aim
precursor ceils, to a stage with large numbers of small lymphoc)tes. Consequently, the organcultured thymus may provide an experimental model in which questions such as the contribution of extra- and intra-thymic factors to the regulation of lymphopoietic activity and the developmental relation of T-lymphocyte subsets to the thymus could be approached. The present communication gives the details of the organ culture technique we have adopted for the thymus of l4-day-old mouse embryos and describes the characteristics of the in vitro growth of the thymus and the properties of the developing lymphocytes. Parts of the results have previously been presented in brief (10).
MATERIALS AND METHODS
same bridge in each culture. Cultures were established in at least duplicate. They were incubated at 37.0°C in gas-tight boxes with a water-saturated atmosphere of 5% CO2 in air. Quantitation of lymphoid cell numhers. The thymuses in organ culture were removed from the Millipore filters at the indicated time and transferred to small plastic homogenizers containing 0.15 ml of a mixture of equal volumes of Hanks's balanced salt solution and Dulbeeco's phosphate-buffered saline. After gentle homogenization of each thymus separate Iy the cell suspension was immediately tran^ ferred to a hemocj-tometer and the concentration of lymphoid cells determined. The result was expressed as the number of lymphoid cells per thymus, and these values were logarithmically transformed for the calculation of group means.
Mice. Mice of the CBA/J, BALB/c, and BALB/c -nu/nu or -nu/-t- (Gl Bomholtgard Ltd, Ry, Denmark) or A/Sn (Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden) strains were bred in our animal quarters. The appearance of vaginal plugs in mated females was considered day 0 of pregnancy. On day 14 of pregnancy the embryos were dissected out, usually 5-10 from each animal. The two thymic aniagen of each embryo were dissected out under the stereomicroscope, using sharpened watchmaker's forceps. Organ culture technique. The organ culture technique previously described in detail for the embr)'onic chicken thymus (I, 24) was used with minor modifications. The technique included the use of plastic 35 X 10-mm tissue culture dishes (Falcon Plastics, Oxnard, CaJif., USA), each equipped with a 60-mesh stainless steel bridge with a matching piece of Millipore filter, pore size 0.45 /nm (Millipore tilter Corp., Bedford, Mass., USA), situated in the gas-medium interphase. Two ml of RPMI l(i40 tissue culture medium (Flow, Irvine, Scotland) supplemented with penicillin (100 U/ml), streptomycin (100 /ig/ml), and heat-inactivated (56°C for 30 min) 10% fetal calf serum (FCS) (Flow, Irvine, Scotland) was added to each culture dish. Two randomly chosen thymic aniagen were placed about 5 mm apart on the
Incorporation oj \fH}thymidine. A total of 4 ;iCi pHJthymidine ([^'HjTdR), specific activity 1,8 Ci/mM (New England Nuclear, Boston, Mass., USA), in 0.2 ml was added to each organ culture. The pulsed thymuses were harvested 16 h later. Aliquots of thymic cell suspensions prepared as described above were processed with a Skatron multiple cell culture har\'ester (Skatron, LJerbyen, Norway) as described previously (9). The radioactivity of the glass fiber filters was measured in a liquid scintillation counter and expressed as counts per minute (cpm) per 10^ celis. Determination of cell size. For each time point, cell suspensions were pooled and thc mean diameter of 25 lymphocytes in MayGriinwald-Giemsa-stained smears was obtained by means of an ocular screw micrometer a.s previously described (9). Aiitisera. The anti-Thy-1.2 antiserum was produced by repeated weekly intraperitoneai injections of 10^ CBA thymocytes into AKR mice and used at a 1:40 dilution in the qrtotoxicity assay. The anti-TL antiserum, provided by Dr. E, A. Boyse through Dr. H. Wigzell, was produced in (C57BL/6 X A-TL-)Fi hybrids immunized with ASL-1 lymphomas and thus directed against TL 1, 2, 3. It was used at a 1:640 dilution.
Lymphocyte Maturation in Mowe Thymes
The anti-H-2'*^ antiserum was produced in (-57BL/6 mice by repeated intraperitoneal immunization with 2 X 10^ CBA spleen cells. It was used at a 1:40 dilution. Normal mouse sera at the same dilutions were used as controls. Cytotoxicity assay. The trypan blue dye exlusion test was used, with rabbit serum as the complement source. The assay was performed in triplicate, and in each case 100 cells were counted. The proportion of cells killed by antiserum is expressed by the cytotoxic index (CI): % dead cells with antiserum % dead cells with controi serum 100 - % dead cells with control serum
The antisera against Thy-1.2, TL, and H-2't were tested in the cytotoxicity assay in different dilutions on ceils derived from organ ailtures at both early and late culture times. The results showed that the proportions of killed cells were not increased at higher antiserum concentrations than those indicated above. lmmunoflitore.icence technique. The indirect technique described by Loor & Kindred (14) was used. Cells were incubated with the antiThy-1.2 antiserum described above or normal control AKR serum (dilution 1 :S0) and washed, and then fluorescein isothiocj'anateIabeled sheep anti-mouse immunoglobulin (SBL, Stockholm, Sweden) was applied. RESULTS Cell kinetics and cell sizes
The number of lymphoid cells per thymus, the incorporation of [^HjTdR per 10^ thymic cells, and the cell sizes showed marked alterations during the 15-day culture period (Fig. 1). The number of lymphocytes per thymus (Fig. I A) increased very rapidly during approximately the first 9 days of culture and thereafter more slowly, reaching 0.41 X 10^ on day 15. In another experiment, in which only the cell number per thymus was determined, it was 0.41 X 106 on day 16, 1.00 X 10* on day 24, and 0.46 X 10^ on day 32.
499
In contrast, the incorporation of {^H]TdR per 10*^ cells, indicating the proportion of proliferating cells, was very high on culture day 2 and then decreased by approximately 97% in another 2 days. A more gradual decline then followed from day 4 (65 X 10^ cpm) to day 15 (15 X 10^ cpm). The incorporation of [3H]TdR was not determined on culture day 0 because of the low numbers of cells present per thymus. The mean diameter of fixed and stained iymphoid cells in smears prepared from the thymuses at indicated culture times is shown in Fig. lC. The mean diameter of the cells decreased from 10.8 ^m on culture day 2 to 4.8 /(.m om culture day 15. Thus the results presented in Fig. 1 indicate that the cell numbers per thymus and the proportion of small, nondividing lymphocytes increased markedly during the culture period. Antigenic markers Cells derived from the thymus after various time periods in organ culture were analyzed for the presence of Thy-1.2, TL, and H-2it antigens by the cytotoxicity assay. The results v.iQ summarized in Fig. 2. Thy-1.2 (Pig. 2A). Cells prepared from the thymuses of 14-day-old CBA embryos were not killed by anti-Thy-1.2 antiserum and complement. Thy-1.2-positive cells were first detected on day 1 (CI 0.12); their proportion then increased until culture day 4, remained at a high level (CI 0.75-0.79) between days 4 and 8, and then decreased slightly. Cells prepared from eight separate thymuses of young CBA mice showed a mean CI of 0.86. We also examined the cells on culture days 0 and 2 for Thy-1.2 antigens, using indirect immunofluorescence. No clearly Thy-1.2-positive cells were found on day 0—that is, in the thymus of the l4-day embryo. The cells did show a very weak, diffuse membr.ine fluorescence, which may possibly indicate the presence of small quantities of antigen. On culture day 2, 30% of the cells showed a clearly positive, patchy membrane fluorescence. This number was equal to the proportion of cells
500
K, Juhlin & G. V. Aim Fig. 1. Time-dependent growth of the thymus of l-lday-old mouse embryos in organ culture (mean + SEM). A. Number of lymphocytes per thymus (log [No. cells per thymus]). B. [^H]thymidirie incorporation, expressed as cpm per 10^ lymphoid cells X lO*-^ Figures within parentheses in A and B represent number of thymuses assayed. C. Mean cell diameter (nm).
killed by antibody and complement. Control thymic lymphocytes of 1-month-old CBA mice had a CI of 0.97 and the same proportion of Thy-1.2-positive cells in the immunofluorescence test. TL (Fig, 2B), For tlie study of the TL antigens CBA X A embryos were used. The time-dependent changes in the frequency of TLpositive cells showed, with minor exceptions, the same pattern as for Thy-1.2. The differences for the TL were that the cytotoxic indices were comparatively low (maximal CI 0.65) and did not show the peak on culture days 4-8 noted for Thy-1.2. Cells prepared from five separate thymuses of 1-month-oid C3A X A hybrids showed a mean CI of 0.63- Cells derived from organ-cultured CBA thymuses harvested on culture days 6-8 (three experiments) showed a CI of 0.00. H-2^ (Fig. 2C). As with the Thy-1.2 and TL antigens, the proportion of cells killed by anti-H-2'^ antiserum increased during the cul-
1.D
Serum-free or mouse-serum-supplemented culttires
To investigate whether the fetal calf serum supplement used throughout this study was essential for the development of lymphocytes with the Thy-1.2 antigen, we cultured CBA thymuses for 7 days in serum-free medium or in medium supplemented with serum (0.5%) from normal or nude BALB/c mice. The results of the cytotoxicity assays, summarized in Table I, indicate that Thy-1.2-positive cells develop with the same frequency in all types of cultures. DISCUSSION We could demonstrate marked time-dependent changes in the lymphopoiesis in the 14-day
•
A
B
Q.8
/
0.6
0,6
0.4
0.2
ture period. However, they showed a CI of 0.39 already on culture day 0, and this increased to 0.99 on culture day 8.
O.U
OJ
/
0 12
IB
0
^
Ij
12
Fig. 2. Time-dependent development of antigenic markers on lymphotytes in the embryonic mouse thymus in organ culture. Proportion of cells killed by antiserum and complement expressed as cytotoxic index ( Q ) . A. Thy-1.2. B. TL. C H-2k.
Lymphocyte Maturation in Mouse Thymus
embryonic mouse thymus in organ culture. These changes included an initial increase in the number of lymphoq'tes per thymus during approximately the first 9 culture days, followed by a phase of more slow growth. In contrast, during the entire culture period of 15 days, the proportion of proliferating cells in the thymuses decreased, most markedly so during the first 4 days. The mean size of the cells decreased gradually, so that at the termination of the cultures most of the lymphocytes were small nonproliferating cells. These results are similar to those previously described for the embryonic chicken thymus in organ culture (9). These data suggest a morphological maturation into small lymphocytes. This could either be due to a time-dependent maturation of the lymphocytes per se or a consequence of a maturation of the thymic microenvironment-—for example, differentiation of the thymic epithelial cells. We have previously suggested, on the basis of results obtained in the embryonic chicken thymus organ culture system, that the number of lymphocytes per imit thymus volume may limit lymphopoietic activity and force cells to develop into small nonproliferating lymphocytes (9). Using the cytotoxicity assay, we also analyzed the time-dependent changes in the proportion of lymphocytes expressing the antigens Thy1.2, TL, and 'H-l^. The finding that a large proportion of the lymphocytes derived from the thymus in organ culture could be killed by anti-Thy-1.2 or anti-TL antiserum suggests that most of the cells generated in the thymus in vitro had T-cell characteristics. The possibility that they passively acquired Thy-1.2 antigen is less likely because the cells in fused AKR and CBA thymuses in organ culture showed the expected proportions of cells carrying Thy-1.1 and Thy-1.2 antigens, respectively (unpublished observation). Although most of the cells carried Thy-1.2 antigen in the thymus in organ culture, their frequencies were usually lower than in the thymus of young mice. The considerable proportion of apparently negative cells could be either very immature cells or cells that had lost
Table I. Development of Thy-I .2-positive serum-fre:; thymic organ cultures and in supplemented with fetal calf serum (FCS) or serum from nu/nu and nu/-+- BALB/c mice
501
cells in cultures (10%) (0.5%)
Serum supplement BALB/c-nu/nii
BALB/c-nu/-H
FCS
None
0.90*
0.95
I). 94
0.93
* Cytotoxic index with anti-Thy-1.2 antiserum and complement.
most or all of these antigens during their differentiation. The latter alternative is quite possible since the mature cells may accumulate in the organ-cultured thymus but will leave the thymus in vivo. We found that essentially all cells were Thy1.2- and TL-negative in the cytotoxicity assay cm culture day 0—that is, in the thymus of the 14-day mouse embryo. These findings are similar to those of others (21, 26). However, Loor et al. (13) found, with an immunofluorescence technique, that the thymus of approximately 14-day-old mouse embryos contained Thy-1- and TL-positive cells. We were not able to confirm this for Thy-1.2 using a similar technique but our own reagents. These discrepant results may be due to, for example, differences between mouse strains, the determination of the age of the embryos, and, perhaps, the antisera used. We would therefore conclude that Thy-1and TL-negative lymphoid precursor cells develop into more mature cells with these antigenic markers in the embryonic thymus in organ culture. It could be argued that the observed morphologic and antigenic maturation was a consequence of the supplementary fetal calf serum in the organ culture medium, because such serum has been reported to contain thymic hormone (A. L. Goldstein, personal communication). We obtained some evidence against the possibility that such serum-derived thymic hormone was necessary for the development of T lymphocytes in the thymus in vitro by showing that Thy-1.2-positive lymphocytes
502
R. Juhlin & G. V, Aim
were present in the same high frequency in serum-free cultures and in cultures supplemented with an optimal concentration (0.5%) of serum from nude mice. This suggests that the thymic microenvironment per se is sufficient for at least some differentiation of T lymphocj-tes. The present study was undertaken to clarify the extent of cell proliferation and differentiation of lymphocytes in the adopted organ culture system, The results demonstrate a development of lymphocytes which continued in the cultures for more than 30 days. These cells were mainly small lymphocj'tes, possessing Tcell characteristics. We have also shown that these cells are mitogen-reactive and immunocompetent (10), and we are now further using organ cultures of the mouse thymus to study the functional maturation of the various subsets of T lymphocytes.
ACKNOWLEDGEMENTS We are grateful for the excellent technical assistance of Mrs. Marianne Carlsson and Miss Eva Hansson. This study was supported by grants from the Swedish Medical Research Council and the Medical faculty of the University of Uppsala.
UKFERENCES l.Alm, G. V. & Sallstrom, J. F. The /'« vitro maturation of the embr>'onic chicken thymus. I. Development of an organ culture system. Acta palh. mhrohiol. scand. Sect. A 80, 778, 1972. 2. Ball, W. D. & Aiierbach, R. In viiro formation of iymphocj'tes from embryonic thymus. Exp. Cell Res. 20, 245. I960. 3. Burnet, M. F. pp. 57—73, 359-379 in Cellular Immuntilogy. Cambridge University Press, Melbourne, 1969. 4. Cantor, H. & Boyse, E. A. Characterization of subclasses of T lymphocytes at different stages of tbymus-dependent differentiation, pp. 77-82 in van Bekkum, D. W. (ed.) The Biological Afiiviiy of Thymic Hormones. Kooyker Scientific Publications, Rotterdam, 1975. 5. Cantor. H., Simpson. E., Sato, V. L., Fathmaii. C. G. & Herzenber^, L. A. Characterization o:' subpopulations of T-lymphocytes. L Separation
and functional studies of peripheral T-cells binding different amounts of fluorescent antiThy-1.2 (Theta) antibody using a fluorescenceactivated cell sorter (FACS). Cell. Immunol. 75. 180, 1975. 6. Droeye, W. & Zucker, R. Lymphocyte subpopulations in tbe thymus. Transplant. Rev. 25, 3, 1975. 7. Dyminski, J. W., Forbes, J., Gebliardt, B., Nakao. Y., Konda, S. & Smith, R. T. Relationsbip between structure and function of human and mouse thymus cell subpopuiations. pp. 35—47 in Brent, L. & Holborow, J. (eds.) Progress in Immunology II, Vol. 3. North-Holland Publishing Co., Amsterdam & Oxford, 1974. 8. Fatbnian, C. G., Small, M., Herzenberg, L. A. & Weissman, J. L. Tbymus cell maturation. IL Differentiation of three "mature" subclasses in viro. Cell. Immunol. 15, 109, 1975. 9. Juhlin, R. & Aim. G. V. Analysis of time dependent cbanges of lymphopoietic activity in organ cultures of embryonic chicken thymus. Cell Tiss. Kinet. fl, 567, 197'5. 10. Juhlin, R., Tufveson, G., Steineck, G. & Aim. G. V. Growth and differentiation of lymphocytes in the mou.se thymus in vitro (abstract). ScanJ. J. Immunol. 5, 447, 1976. n . K o m u r o , K., Goldstein, G. Ik Boyse, B. A. Thymus-repopulating capacity of cells that can be induced to differentiate to T cells in vitro, J, Immunol. 115. 195, 1975. 12. Leckband, E. & Boyse, E. A. Immunocompetent cells among mouse thymocytes: a minor population. Science 372, 1258, 1971. 13. Loor, F., Block, N. & Little, J. R. Dynamics of the TL antigens on thymus and leukemia cells. Cell. Immunol, 77. 351, 1975. 14. Loor, F. & Kindred, B. Differentiation of Tcell precursors in nude mice demonstrated by immunofluorescence of T-cell membrane markers. / . exp. Med. 138, 1044, 1973. 15. Mandel, T. Differentiation of epithelial cells in the mouse tbymus. Z. Zellforsch. 106, 498, 1970. 16. Mandel, T. & Russel, P. J. Differentiation of foetal mouse tbymus. Ultrastructure of organ cultures and of subcapsular grafts. Immunology 21, 659, 1971. l7.Metcalf, D. The Thymus. Springer, Berlin, 1966. 18. Metcalf, D. & Moore, M. A. S. pp. 224-242 in Haemopoietic Cells. North Holland Publishing Co., Amsterdam, 1971. 19. Miller, J. F. A. P. & Osoba, D. Current concepts of the immunological function of the thymus. Physiol. Rev. 47. 4?>1, 1967. 20. Owen, J. J. T. Ontogeny of the immune system, pp. 163-173 in Brent, L. & Holborow, J. (eds.) Progress in Immunology II, Vol. 5. Nortb-H(»Iland Publishing Co., Amsterdam & Oxford, 1974. 21. Owen, J. J. T. & Raff, M. C. Studies on tlio differentiation of tbymus-derived lymphocytes. /, exp. Med. 132, 1216, 1970.
Lymphocyte Maturation in Mouse Thymus 22. Owen, J. J. T. St Ritter, M. A. Tissue interaction in tbe development of thymus lympbo
Morphologic and Antigenic Maturation of Lymphocytes in the Mouse Thymus In Vitro R. JUHLIN & G. V. ALM Department of Histology, Institute of Htiman Anatomy, Biomediatl Center, University of Uppsala, Uppsala, Sweden
Juhlin, R. & Aim, G. V. Morphologic and Antigenic Maturation of Lymphocytes in the Mouse Thymus In Vitro. Scatid. / . Immunol. 5, 497-503. 1976. A simple technique for organ culture of the thymus of 14-day-old moose embryos is described. It allowed a characteristic time-dependent development of the immature thymus into a lymphoid thymus with large numbers of nondividing small lymphocytes. Most of these cells carried the T-lymphocyte ajitigenic markers Thy-1 and TL, and all expressed H-2 antipens as determined in cytotoxicit/ assays. They probably developed from precursor cells without detectable Thy-1 and TL. This development appeared to be dependent on the thymic microenvironment, since it also occurred in serum-free organ cultures and in cultures with medium supplemented with serum from nude mice, R. Juhlin, Departmenl of Virology, Biomedical Center, Box 584, S-751 23 Uplisala, Sweden
Lymphoid precursor cells migrate into the thymus of mice both in fetal and adult life. They first appear in the embryonic epithelial thymus on day 11 or 12 of gestation (15, 18, 22). The properties of these precursor cells have not been established in detail, but, at least when in the bone marrow of young mice, they appear to be committed to lymphoid development but express little, if any, of the antigenic markers identifying T lymphocytes (11). The further development of lymphoid precursor cells within the thymus probably involves a series of events that includes cell proliferation and differentiation. Most of the more mature thymic lymphocytes are small nondividing cells witli a high density of antigens such as Thy-1 and TL. A small fraction of the mature lymphocytes has a lower density of Thy-1 and lack TL antigens; they represent the immunocompetent lymphocytes found in thc thymus (12, 23). The thymus is recognized as critical to the development of T-lymphocyte functions (3, 17-19), including the generation of immuno-
competent cells and possibly also maintenance of self-tolerance. A growing body of data suggests a considerable heterogeneity within thymic and peripheral T lymphocytes (4, 5, 7, 8). This raises the question of the developmental relation of the various antigenically and functionally defined subsets of T lymphocytes to the thymus. As recently reviewed (6, 25), there is dearly a possibility that lymphocytes within the thymus may develop along separate pathways and further that the different subsets of peripheral T lymphocytes may have special relations to these, perhaps representing separate developmental lineages. It would be desirable to have access to an in vitro system that would allow definition of the relation of the thymus to the development of T lymphocytes. The organ culture technique has previously been used to maintain the embryonic chicken (1, 24) and mouse thymus (2, 16, 20) in vitro. It allows the development of the thymus from a stage at which most nonepithelial cells are primitive lymphoid
498
R. Jtihlii! & G. V. Aim
precursor ceils, to a stage with large numbers of small lymphoc)tes. Consequently, the organcultured thymus may provide an experimental model in which questions such as the contribution of extra- and intra-thymic factors to the regulation of lymphopoietic activity and the developmental relation of T-lymphocyte subsets to the thymus could be approached. The present communication gives the details of the organ culture technique we have adopted for the thymus of l4-day-old mouse embryos and describes the characteristics of the in vitro growth of the thymus and the properties of the developing lymphocytes. Parts of the results have previously been presented in brief (10).
MATERIALS AND METHODS
same bridge in each culture. Cultures were established in at least duplicate. They were incubated at 37.0°C in gas-tight boxes with a water-saturated atmosphere of 5% CO2 in air. Quantitation of lymphoid cell numhers. The thymuses in organ culture were removed from the Millipore filters at the indicated time and transferred to small plastic homogenizers containing 0.15 ml of a mixture of equal volumes of Hanks's balanced salt solution and Dulbeeco's phosphate-buffered saline. After gentle homogenization of each thymus separate Iy the cell suspension was immediately tran^ ferred to a hemocj-tometer and the concentration of lymphoid cells determined. The result was expressed as the number of lymphoid cells per thymus, and these values were logarithmically transformed for the calculation of group means.
Mice. Mice of the CBA/J, BALB/c, and BALB/c -nu/nu or -nu/-t- (Gl Bomholtgard Ltd, Ry, Denmark) or A/Sn (Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden) strains were bred in our animal quarters. The appearance of vaginal plugs in mated females was considered day 0 of pregnancy. On day 14 of pregnancy the embryos were dissected out, usually 5-10 from each animal. The two thymic aniagen of each embryo were dissected out under the stereomicroscope, using sharpened watchmaker's forceps. Organ culture technique. The organ culture technique previously described in detail for the embr)'onic chicken thymus (I, 24) was used with minor modifications. The technique included the use of plastic 35 X 10-mm tissue culture dishes (Falcon Plastics, Oxnard, CaJif., USA), each equipped with a 60-mesh stainless steel bridge with a matching piece of Millipore filter, pore size 0.45 /nm (Millipore tilter Corp., Bedford, Mass., USA), situated in the gas-medium interphase. Two ml of RPMI l(i40 tissue culture medium (Flow, Irvine, Scotland) supplemented with penicillin (100 U/ml), streptomycin (100 /ig/ml), and heat-inactivated (56°C for 30 min) 10% fetal calf serum (FCS) (Flow, Irvine, Scotland) was added to each culture dish. Two randomly chosen thymic aniagen were placed about 5 mm apart on the
Incorporation oj \fH}thymidine. A total of 4 ;iCi pHJthymidine ([^'HjTdR), specific activity 1,8 Ci/mM (New England Nuclear, Boston, Mass., USA), in 0.2 ml was added to each organ culture. The pulsed thymuses were harvested 16 h later. Aliquots of thymic cell suspensions prepared as described above were processed with a Skatron multiple cell culture har\'ester (Skatron, LJerbyen, Norway) as described previously (9). The radioactivity of the glass fiber filters was measured in a liquid scintillation counter and expressed as counts per minute (cpm) per 10^ celis. Determination of cell size. For each time point, cell suspensions were pooled and thc mean diameter of 25 lymphocytes in MayGriinwald-Giemsa-stained smears was obtained by means of an ocular screw micrometer a.s previously described (9). Aiitisera. The anti-Thy-1.2 antiserum was produced by repeated weekly intraperitoneai injections of 10^ CBA thymocytes into AKR mice and used at a 1:40 dilution in the qrtotoxicity assay. The anti-TL antiserum, provided by Dr. E, A. Boyse through Dr. H. Wigzell, was produced in (C57BL/6 X A-TL-)Fi hybrids immunized with ASL-1 lymphomas and thus directed against TL 1, 2, 3. It was used at a 1:640 dilution.
Lymphocyte Maturation in Mowe Thymes
The anti-H-2'*^ antiserum was produced in (-57BL/6 mice by repeated intraperitoneal immunization with 2 X 10^ CBA spleen cells. It was used at a 1:40 dilution. Normal mouse sera at the same dilutions were used as controls. Cytotoxicity assay. The trypan blue dye exlusion test was used, with rabbit serum as the complement source. The assay was performed in triplicate, and in each case 100 cells were counted. The proportion of cells killed by antiserum is expressed by the cytotoxic index (CI): % dead cells with antiserum % dead cells with controi serum 100 - % dead cells with control serum
The antisera against Thy-1.2, TL, and H-2't were tested in the cytotoxicity assay in different dilutions on ceils derived from organ ailtures at both early and late culture times. The results showed that the proportions of killed cells were not increased at higher antiserum concentrations than those indicated above. lmmunoflitore.icence technique. The indirect technique described by Loor & Kindred (14) was used. Cells were incubated with the antiThy-1.2 antiserum described above or normal control AKR serum (dilution 1 :S0) and washed, and then fluorescein isothiocj'anateIabeled sheep anti-mouse immunoglobulin (SBL, Stockholm, Sweden) was applied. RESULTS Cell kinetics and cell sizes
The number of lymphoid cells per thymus, the incorporation of [^HjTdR per 10^ thymic cells, and the cell sizes showed marked alterations during the 15-day culture period (Fig. 1). The number of lymphocytes per thymus (Fig. I A) increased very rapidly during approximately the first 9 days of culture and thereafter more slowly, reaching 0.41 X 10^ on day 15. In another experiment, in which only the cell number per thymus was determined, it was 0.41 X 106 on day 16, 1.00 X 10* on day 24, and 0.46 X 10^ on day 32.
499
In contrast, the incorporation of {^H]TdR per 10*^ cells, indicating the proportion of proliferating cells, was very high on culture day 2 and then decreased by approximately 97% in another 2 days. A more gradual decline then followed from day 4 (65 X 10^ cpm) to day 15 (15 X 10^ cpm). The incorporation of [3H]TdR was not determined on culture day 0 because of the low numbers of cells present per thymus. The mean diameter of fixed and stained iymphoid cells in smears prepared from the thymuses at indicated culture times is shown in Fig. lC. The mean diameter of the cells decreased from 10.8 ^m on culture day 2 to 4.8 /(.m om culture day 15. Thus the results presented in Fig. 1 indicate that the cell numbers per thymus and the proportion of small, nondividing lymphocytes increased markedly during the culture period. Antigenic markers Cells derived from the thymus after various time periods in organ culture were analyzed for the presence of Thy-1.2, TL, and H-2it antigens by the cytotoxicity assay. The results v.iQ summarized in Fig. 2. Thy-1.2 (Pig. 2A). Cells prepared from the thymuses of 14-day-old CBA embryos were not killed by anti-Thy-1.2 antiserum and complement. Thy-1.2-positive cells were first detected on day 1 (CI 0.12); their proportion then increased until culture day 4, remained at a high level (CI 0.75-0.79) between days 4 and 8, and then decreased slightly. Cells prepared from eight separate thymuses of young CBA mice showed a mean CI of 0.86. We also examined the cells on culture days 0 and 2 for Thy-1.2 antigens, using indirect immunofluorescence. No clearly Thy-1.2-positive cells were found on day 0—that is, in the thymus of the l4-day embryo. The cells did show a very weak, diffuse membr.ine fluorescence, which may possibly indicate the presence of small quantities of antigen. On culture day 2, 30% of the cells showed a clearly positive, patchy membrane fluorescence. This number was equal to the proportion of cells
500
K, Juhlin & G. V. Aim Fig. 1. Time-dependent growth of the thymus of l-lday-old mouse embryos in organ culture (mean + SEM). A. Number of lymphocytes per thymus (log [No. cells per thymus]). B. [^H]thymidirie incorporation, expressed as cpm per 10^ lymphoid cells X lO*-^ Figures within parentheses in A and B represent number of thymuses assayed. C. Mean cell diameter (nm).
killed by antibody and complement. Control thymic lymphocytes of 1-month-old CBA mice had a CI of 0.97 and the same proportion of Thy-1.2-positive cells in the immunofluorescence test. TL (Fig, 2B), For tlie study of the TL antigens CBA X A embryos were used. The time-dependent changes in the frequency of TLpositive cells showed, with minor exceptions, the same pattern as for Thy-1.2. The differences for the TL were that the cytotoxic indices were comparatively low (maximal CI 0.65) and did not show the peak on culture days 4-8 noted for Thy-1.2. Cells prepared from five separate thymuses of 1-month-oid C3A X A hybrids showed a mean CI of 0.63- Cells derived from organ-cultured CBA thymuses harvested on culture days 6-8 (three experiments) showed a CI of 0.00. H-2^ (Fig. 2C). As with the Thy-1.2 and TL antigens, the proportion of cells killed by anti-H-2'^ antiserum increased during the cul-
1.D
Serum-free or mouse-serum-supplemented culttires
To investigate whether the fetal calf serum supplement used throughout this study was essential for the development of lymphocytes with the Thy-1.2 antigen, we cultured CBA thymuses for 7 days in serum-free medium or in medium supplemented with serum (0.5%) from normal or nude BALB/c mice. The results of the cytotoxicity assays, summarized in Table I, indicate that Thy-1.2-positive cells develop with the same frequency in all types of cultures. DISCUSSION We could demonstrate marked time-dependent changes in the lymphopoiesis in the 14-day
•
A
B
Q.8
/
0.6
0,6
0.4
0.2
ture period. However, they showed a CI of 0.39 already on culture day 0, and this increased to 0.99 on culture day 8.
O.U
OJ
/
0 12
IB
0
^
Ij
12
Fig. 2. Time-dependent development of antigenic markers on lymphotytes in the embryonic mouse thymus in organ culture. Proportion of cells killed by antiserum and complement expressed as cytotoxic index ( Q ) . A. Thy-1.2. B. TL. C H-2k.
Lymphocyte Maturation in Mouse Thymus
embryonic mouse thymus in organ culture. These changes included an initial increase in the number of lymphoq'tes per thymus during approximately the first 9 culture days, followed by a phase of more slow growth. In contrast, during the entire culture period of 15 days, the proportion of proliferating cells in the thymuses decreased, most markedly so during the first 4 days. The mean size of the cells decreased gradually, so that at the termination of the cultures most of the lymphocytes were small nonproliferating cells. These results are similar to those previously described for the embryonic chicken thymus in organ culture (9). These data suggest a morphological maturation into small lymphocytes. This could either be due to a time-dependent maturation of the lymphocytes per se or a consequence of a maturation of the thymic microenvironment-—for example, differentiation of the thymic epithelial cells. We have previously suggested, on the basis of results obtained in the embryonic chicken thymus organ culture system, that the number of lymphocytes per imit thymus volume may limit lymphopoietic activity and force cells to develop into small nonproliferating lymphocytes (9). Using the cytotoxicity assay, we also analyzed the time-dependent changes in the proportion of lymphocytes expressing the antigens Thy1.2, TL, and 'H-l^. The finding that a large proportion of the lymphocytes derived from the thymus in organ culture could be killed by anti-Thy-1.2 or anti-TL antiserum suggests that most of the cells generated in the thymus in vitro had T-cell characteristics. The possibility that they passively acquired Thy-1.2 antigen is less likely because the cells in fused AKR and CBA thymuses in organ culture showed the expected proportions of cells carrying Thy-1.1 and Thy-1.2 antigens, respectively (unpublished observation). Although most of the cells carried Thy-1.2 antigen in the thymus in organ culture, their frequencies were usually lower than in the thymus of young mice. The considerable proportion of apparently negative cells could be either very immature cells or cells that had lost
Table I. Development of Thy-I .2-positive serum-fre:; thymic organ cultures and in supplemented with fetal calf serum (FCS) or serum from nu/nu and nu/-+- BALB/c mice
501
cells in cultures (10%) (0.5%)
Serum supplement BALB/c-nu/nii
BALB/c-nu/-H
FCS
None
0.90*
0.95
I). 94
0.93
* Cytotoxic index with anti-Thy-1.2 antiserum and complement.
most or all of these antigens during their differentiation. The latter alternative is quite possible since the mature cells may accumulate in the organ-cultured thymus but will leave the thymus in vivo. We found that essentially all cells were Thy1.2- and TL-negative in the cytotoxicity assay cm culture day 0—that is, in the thymus of the 14-day mouse embryo. These findings are similar to those of others (21, 26). However, Loor et al. (13) found, with an immunofluorescence technique, that the thymus of approximately 14-day-old mouse embryos contained Thy-1- and TL-positive cells. We were not able to confirm this for Thy-1.2 using a similar technique but our own reagents. These discrepant results may be due to, for example, differences between mouse strains, the determination of the age of the embryos, and, perhaps, the antisera used. We would therefore conclude that Thy-1and TL-negative lymphoid precursor cells develop into more mature cells with these antigenic markers in the embryonic thymus in organ culture. It could be argued that the observed morphologic and antigenic maturation was a consequence of the supplementary fetal calf serum in the organ culture medium, because such serum has been reported to contain thymic hormone (A. L. Goldstein, personal communication). We obtained some evidence against the possibility that such serum-derived thymic hormone was necessary for the development of T lymphocytes in the thymus in vitro by showing that Thy-1.2-positive lymphocytes
502
R. Juhlin & G. V, Aim
were present in the same high frequency in serum-free cultures and in cultures supplemented with an optimal concentration (0.5%) of serum from nude mice. This suggests that the thymic microenvironment per se is sufficient for at least some differentiation of T lymphocj-tes. The present study was undertaken to clarify the extent of cell proliferation and differentiation of lymphocytes in the adopted organ culture system, The results demonstrate a development of lymphocytes which continued in the cultures for more than 30 days. These cells were mainly small lymphocj'tes, possessing Tcell characteristics. We have also shown that these cells are mitogen-reactive and immunocompetent (10), and we are now further using organ cultures of the mouse thymus to study the functional maturation of the various subsets of T lymphocytes.
ACKNOWLEDGEMENTS We are grateful for the excellent technical assistance of Mrs. Marianne Carlsson and Miss Eva Hansson. This study was supported by grants from the Swedish Medical Research Council and the Medical faculty of the University of Uppsala.
UKFERENCES l.Alm, G. V. & Sallstrom, J. F. The /'« vitro maturation of the embr>'onic chicken thymus. I. Development of an organ culture system. Acta palh. mhrohiol. scand. Sect. A 80, 778, 1972. 2. Ball, W. D. & Aiierbach, R. In viiro formation of iymphocj'tes from embryonic thymus. Exp. Cell Res. 20, 245. I960. 3. Burnet, M. F. pp. 57—73, 359-379 in Cellular Immuntilogy. Cambridge University Press, Melbourne, 1969. 4. Cantor, H. & Boyse, E. A. Characterization of subclasses of T lymphocytes at different stages of tbymus-dependent differentiation, pp. 77-82 in van Bekkum, D. W. (ed.) The Biological Afiiviiy of Thymic Hormones. Kooyker Scientific Publications, Rotterdam, 1975. 5. Cantor. H., Simpson. E., Sato, V. L., Fathmaii. C. G. & Herzenber^, L. A. Characterization o:' subpopulations of T-lymphocytes. L Separation
and functional studies of peripheral T-cells binding different amounts of fluorescent antiThy-1.2 (Theta) antibody using a fluorescenceactivated cell sorter (FACS). Cell. Immunol. 75. 180, 1975. 6. Droeye, W. & Zucker, R. Lymphocyte subpopulations in tbe thymus. Transplant. Rev. 25, 3, 1975. 7. Dyminski, J. W., Forbes, J., Gebliardt, B., Nakao. Y., Konda, S. & Smith, R. T. Relationsbip between structure and function of human and mouse thymus cell subpopuiations. pp. 35—47 in Brent, L. & Holborow, J. (eds.) Progress in Immunology II, Vol. 3. North-Holland Publishing Co., Amsterdam & Oxford, 1974. 8. Fatbnian, C. G., Small, M., Herzenberg, L. A. & Weissman, J. L. Tbymus cell maturation. IL Differentiation of three "mature" subclasses in viro. Cell. Immunol. 15, 109, 1975. 9. Juhlin, R. & Aim. G. V. Analysis of time dependent cbanges of lymphopoietic activity in organ cultures of embryonic chicken thymus. Cell Tiss. Kinet. fl, 567, 197'5. 10. Juhlin, R., Tufveson, G., Steineck, G. & Aim. G. V. Growth and differentiation of lymphocytes in the mou.se thymus in vitro (abstract). ScanJ. J. Immunol. 5, 447, 1976. n . K o m u r o , K., Goldstein, G. Ik Boyse, B. A. Thymus-repopulating capacity of cells that can be induced to differentiate to T cells in vitro, J, Immunol. 115. 195, 1975. 12. Leckband, E. & Boyse, E. A. Immunocompetent cells among mouse thymocytes: a minor population. Science 372, 1258, 1971. 13. Loor, F., Block, N. & Little, J. R. Dynamics of the TL antigens on thymus and leukemia cells. Cell. Immunol, 77. 351, 1975. 14. Loor, F. & Kindred, B. Differentiation of Tcell precursors in nude mice demonstrated by immunofluorescence of T-cell membrane markers. / . exp. Med. 138, 1044, 1973. 15. Mandel, T. Differentiation of epithelial cells in the mouse tbymus. Z. Zellforsch. 106, 498, 1970. 16. Mandel, T. & Russel, P. J. Differentiation of foetal mouse tbymus. Ultrastructure of organ cultures and of subcapsular grafts. Immunology 21, 659, 1971. l7.Metcalf, D. The Thymus. Springer, Berlin, 1966. 18. Metcalf, D. & Moore, M. A. S. pp. 224-242 in Haemopoietic Cells. North Holland Publishing Co., Amsterdam, 1971. 19. Miller, J. F. A. P. & Osoba, D. Current concepts of the immunological function of the thymus. Physiol. Rev. 47. 4?>1, 1967. 20. Owen, J. J. T. Ontogeny of the immune system, pp. 163-173 in Brent, L. & Holborow, J. (eds.) Progress in Immunology II, Vol. 5. Nortb-H(»Iland Publishing Co., Amsterdam & Oxford, 1974. 21. Owen, J. J. T. & Raff, M. C. Studies on tlio differentiation of tbymus-derived lymphocytes. /, exp. Med. 132, 1216, 1970.
Lymphocyte Maturation in Mouse Thymus 22. Owen, J. J. T. St Ritter, M. A. Tissue interaction in tbe development of thymus lympbo
