Immunology 1979 37 817
Lactate dehydrogenase isoenzyme pattern of the pre-thymic precursor cell
J. PLUM, S. RINGOIR* & MAGDA DE SMEDT Department of Medical Microbiology, University ofGhent, Department of Medicine, Division ofNephrology, University ofGhent, Ghent, Belgiwn
Acceptedfor publication I February 1979
Summary. The thymic cell precursor was obtained from thymic rudiments of 14 day old mouse embryos of strain NMRI. The LDH isoenzyme pattern of this cell population showed a higher LDH-5 activity than the cortical and medullary thymocytes. These results indicate that during the T-cell development, a shift in the pattern occurs towards the LDH-1 end of the spectrum, as described for other murine tissues and cells. As cortical thymocytes contain less LDH-5 activity than the medullary thymocytes, however, the outcome of the latter cells from the former is in conflict with the shift toward the LDH-I end of the spectrum. On the contrary a relationship whereby cortical and medullary thymocytes are independently derived from the thymic cell percursor, is consistent with the shift toward LDH-1.
ment a gradual shift occurs, so that enzyme activity is progressively transferred toward the LDH-l end of the spectrum (Markert & Ursprung, 1962). A conflicting result was obtained by analysing the LDH isoenzyme distribution of thymocyte subpopulations. Thymocytes can be divided into cortical and medullary thymocytes and it is generally accepted that the medullary thymocytes are derived from the cortical ones. These two cell populations can be distinguished according to their LDH isoenzyme pattern, since the cortical thymocytes exhibit significantly higher percentage activity in LDH-l, 2 and 3 than the medullary thymocytes (Plum, Ringoir, De Smedt & Dhont, 1978; Plum, Monteyne, Dhont & De Smedt, 1978). -This means that this time a shift of the LDH pattern toward the LDH-5 end of the spectrum occurs during T-cell development within the thymus. The aim of the present investigation was to confirm this unique shift in the opposite direction by analysing the isoenzyme pattern of the pre-thymic precursor cell. This cell population is at an earlier stage of T-cell development than those studied up to now. The prethymic precursor cell has been obtained from thymic rudiments of 14 day old mouse embryos. A higher activity in LDH-I in this cell population should unequivocally stress the shift toward LDH-5 during T cell development in the thymus.
INTRODUCTION LDH (L-lactate: NAD + oxidoreductase, EC 1.1.1.27) is a tetrameric enzyme composed of two sub-units. When combined, these two sub-units result in a total of five different tetrameric isoenzymes: LDH- I, 2, 3, 4 and 5. The isoenzyme system has been most widely used in studies of tissue differentiation. In mice, embryonic tissues first exhibit a predominance of LDH-5, and then with continuing developCorrespondence: Dr J. Plum, Laboratorium voor Bacteriologie en Virologie, Rijksuniversiteit Gent, Akademisch Ziekenhuis, Blok A, De Pintelaan 135, B-9000 Gent, Belgium. 00 19-2805/79/0800-08 17$02.00 ©) 1979 Blackwell Scientific Publications
METHODS AND MATERIALS
Animals Mice of strain NMRI were used. Gestational stage was 817
J. Plum, S. Ringoir & Magda De Smedt
818
determined by noting the appearance of vaginal plugs
(day 0). Cell suspensions Suspensions of thymus cells were prepared by removing the thymuses of 14 day old embryos and disrupting these organs mechanically by gentle pressure from a polysterene pestle in cold RPMI 1640 medium buffered with HEPES. ......
Assayfor Thy-1.2 antigen The cell suspensions obtained from the thymic rudiments were assayed for Thy-1.2 antigen using a cytotoxic assay with anti-Thy-1.2 antiserum (Searle, England) and absorbed guinea-pig serum complement as described by Golub (1971). The test was also performed on thymocytes from aAult mice as control. Cell counts and staining All cell counts were done by a haemocytometer. Viability was determined by eosin exclusion. The cells were sedimented on grease-free slides with the use of a cytocentrifuge and stained with May-GrunwaldGiemsa.
n~~~~~~1
'..
Determination of LDH isoenzyme pattern
The pre-thymic precursor cells, obtained from the thymic rudiments were suspended in Tris buffer at a concentration of 2 x 107 cells/ml. These cells were frozen and thawed three times consecutively and spun at 2000 g for 10 min. The LDH isoenzymes of the supernatant were determined by agar gel electrophoresis as described (Plum & Ringoir, 1975; Plum & Ringoir, 1977).
Figure 1. Cells obtained from thymic rudiments (Experiment 3). These cells are large basophilic (magnification l0Ox 100).
different experiments, the pattern exhibited by the pre-thymic cell precursor obtained from the thymus shows a similar pattern, with activity in the LDH-2, LDH-3, LDH-4 and LDH-5 bands.
RESULTS
Cells obtained from thymic rudiments The cells that were released into suspension were large basophilic cells as seen in Fig. 1. These cells were Thy-1.2 antigen negative. No Thy-1.2 antigen-bearing cells or contaminating erythrocytes were seen. The viability of the cells was at least 96%. Ninety-nine per cent of the thymocytes from normal adult mice were found to bear Thy- 1.2 antigen.
The LDH isoenzyme pattern of the cells The LDH isoenzyme pattern of the cells for the different experiments are summarized in Table 1. For the
DISCUSSION The thymic cells showed distinct properties of the pre-thymic cell precursor: the cells were Thy-1 negative (Raff, 1971) and large basophilic blast-like cells (Moore & Owen, 1967); moreover at this stage, the mouse embryo contained only these cell precursors in the thymus (Stutmati 1977). As seen in Table 1, the pattern of this cell population as compared with the cortical thymocytes showed a higher percentage of LDH-5 activity. Therefore, the assumption that during T-cell development a unique shift toward LDH-5 end occurred is not confirmed.
LDH isoenzyme pattern of the pre-thymic precursor cell
819
Table 1. LDH isoenzyme pattern of pre-thymic cell precursors in thymus
Cell population
LDH-1
Percentage activity LDH-3 LDH-4
LDH-2
LDH-5
Thymocytes obtained from thymic rudiments of 14 day old embryos (pre-thymic cell precursor) Experiment 1 1-8 33-2 69-0 2 0-8 7-6 36-3 55-3 3 0-9 4-1 30-4 64 5 4 1-0 6-0 29-0 640 5 1-0 9-6 34-6 548 Thymocytes obtained from thymuses of normal adult mice 8-10 weeks of age (mainly cortical thymocytes)* 6-7+0-7 13-7+0-7 17-5+0-7 27-3+0-6 34-7+1-4 Thymocytes obtained from thymuses of adult mice 8-10 weeks of age, 96 h after in vivo cortisone treatment (enriched in medullary thymocytes)* 1-2+0-3 3-1+1-5 10-7+2-8 31-5+3-8 53-3+6-4 * Data included from Plum, Ringoir, De Smedt & Dhont (1978), these values represent the mean ( ± SE) obtained from six experiments.
This LDH pattern is of great value, because it provides biochemical information on the differentiation of cells. Indeed, whereas the LDH pattern varies to a great extent, according to the cell population itself, Markert & Ursprung (1962) described that in mice, for a given cell population the less mature precursors contain at least as much or even more LDH-5 activity than the mature cell population. This phenomenon is confirmed in our analysis of the thymic cell precursor with regard to the cortical thymocyte. Next to the analysis of the early T-cell development, the further differentiation steps of the T cells within the thymus can be discussed, taking into account the previous data on the LDH pattern of cortical and medullary thymocytes (Table 1). This is important, because the relationship between medullary and cortical thymocytes is still a matter of discussion. It has been accepted for years that the medullary thymocytes are the sole progeny of the cortical thymocytes. This was based particularly on the appearance of immunological competence, as estimated by the capacity to mediate the graft v. host reaction, the mixed lymphocyte response or cell-mediated lympholysis. In ontogenic studies with thymus from mouse embryo and newborn mice, it was shown that these immunological competences were absent and were acquired only after 3 weeks of age (Katz, 1977). Thus a lack of immunological competence as judged by these parameters precedes the immune competence. The thymocytes can be
divided into cortical thymocytes, with no immune competence as judged by these parameters and medullary thymocytes with these capacities. Therefore, it seems logical to assume that the cortical thymocytes precede the medullary thymocytes. Some data are in conffict with this hypothesis. First, Shortman & Jackson (1974) were able to show by means of studies in cell kinetics that both cell populations have distinct cell kinetics, whereby for each thymocyte subpopulation a continuous renewal was possible and also these authors failed to show a transition from cortical to medullary thymocytes. Second, Papiernik & Bach (1977), by means of an hetero-antiserum to cortisone resistant thymocytes, i.e. medullary thymocytes, showed that this cell population appears during ontogeny before the cortisone sensitive or cortical thymocytes. These data strongly favour the hypothesis that medullary thymocytes are not derived from the cortical. In addition, other authors have already mentioned that the peripheral T cells consist of at least a small subpopulation, which is directly derived from the cortical thymocytes, as judged by their electrophoretic mobility (Droege, 1976) or alkaline phosphatase activity as far as the guinea-pig is concerned (Soppi, Ruuskanen & Kouvalainen, 1977). These findings also argue for the fact that cortical thymocytes do not have to transform into medullary thymocytes to leave the thymus. Two separate path-
J. Plum, S. Ringoir & Magda De Smedt
820
ways for cortical and medullary thymocytes with separate outcome of both cell populations is also possible. In our analysis of the LDH distribution of the thymocytes a further argument is found for the latter hypothesis. This is based on two findings: first, in this study the thymus cell precursor has a high percentage activity in LDH-5. This is consistent with the theory that during the development of cells, progressive shift toward the LDH-1 end is found. Second, when the LDH pattern of the cortical and medullary thymocytes is considered (Table 1), the LDH shift toward LDH-1 fits only with the hypothesis of two separate cell lines within the thymus. Indeed, only in this situation a shift toward the LDH-I end of the spectrum is possible for both cell lines, but to different extents. Further application of this enzyme system is to look whether there are within the T-cell populations, subpopulations with a high LDH activity, because these cells should be derived from the cortical cell line. In view of the tremendous heterogeneity of the T cell, which is discovered more and more to have new and unsuspected functional capacities, i.e. regulatory functions, it is not unreasonable to assume that the cortical cell line might correlate with one of these functions which have not been investigated systematically in earlier studies of thymus ontogeny.
REFERENCES DROEGE W. (1976) 'Early T cells' and 'Late T cells': suggestive evidence for two T cell lineages with separate developmental pathways. Europ. J. Immunol. 6, 763.
GOLUB E.S. (1971) Brain-associated ) antigen: reactivity of rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 2,353. KATZ D.H. (1977) Lymphocyte Differentiation, Recognition, and Regulation, p. 127. Academic Press, New York. MARKERT C.L. & URSPRUNG H. (1962) The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Develop. Biol. 5,363. MooRE M.A.S. & OWEN J.J.T. (1967) Experimental studies on the development of the thymus. J. exp. Med. 126, 715. PAPIERNIK M. & BACH J.F. (1977) Thymocyte subpopulations in young and adult mice. II. Study of steroid-resistant populations by means of a specific heteroantiserum. Europ. J. Immunol. 7, 800. PLuM J. & RINGoIR S. (1975) A characterization of human B and T lymphocytes by their lactate dehydrogenase isoenzyme pattern. Europ. J. Immunol. 5, 871. PLuM J. & RINGOIR S. (1977) Lactate dehydrogenase isoenzyme pattern as a measure of cellular differentiation in lymphocytic cells. J. reticuloendothel. Soc. 21, 225. PLUM J., RINGOIR S., DE SMEDT M. & DHONT E. (1978) Lactate dehydrogenase isoenzymatic analysis of murine lymphocyte populations: a parameter of cellular differentiation. Cell. Immunol. 36, 37. PLUM J., MONTEYNE R., DHoNT E. & DE SMEDT M. (1978) Lactate dehydrogenase isoenzyme pattern: Marker for differentiation of lymphocytes. Scand. J. Immunol. 7,515. RAPF M.C. (1971) Surface antigenic marker for distinguish T and B lymphocytes in mice. Transplant. Rev. 6, 52. SHORTMAN K. & JACKSON H. (1974) The differentiation of T lymphocytes. 1. Proliferation kinetics and interrelationship of subpopulations of mouse thymus cells. Cell. Immunol. 12, 230. SoPPi E., RUUSKANEN 0. & KOUVALANEN (1977) Alkaline phosphatase in the differentiation of guinea pig T lymphocytes. Acta Path. Microbiol. Scand. Sect. C, 85,367. SrUTMAN 0. (1977) Two main features of T cell development: thymus traffic and postthymic maturation. In: Contemporary Topics in Immunobiology, vol. 7 (Ed. by 0. Stutman), p. 9. Plenum Press, New York.
Lactate dehydrogenase isoenzyme pattern of the pre-thymic precursor cell
J. PLUM, S. RINGOIR* & MAGDA DE SMEDT Department of Medical Microbiology, University ofGhent, Department of Medicine, Division ofNephrology, University ofGhent, Ghent, Belgiwn
Acceptedfor publication I February 1979
Summary. The thymic cell precursor was obtained from thymic rudiments of 14 day old mouse embryos of strain NMRI. The LDH isoenzyme pattern of this cell population showed a higher LDH-5 activity than the cortical and medullary thymocytes. These results indicate that during the T-cell development, a shift in the pattern occurs towards the LDH-1 end of the spectrum, as described for other murine tissues and cells. As cortical thymocytes contain less LDH-5 activity than the medullary thymocytes, however, the outcome of the latter cells from the former is in conflict with the shift toward the LDH-I end of the spectrum. On the contrary a relationship whereby cortical and medullary thymocytes are independently derived from the thymic cell percursor, is consistent with the shift toward LDH-1.
ment a gradual shift occurs, so that enzyme activity is progressively transferred toward the LDH-l end of the spectrum (Markert & Ursprung, 1962). A conflicting result was obtained by analysing the LDH isoenzyme distribution of thymocyte subpopulations. Thymocytes can be divided into cortical and medullary thymocytes and it is generally accepted that the medullary thymocytes are derived from the cortical ones. These two cell populations can be distinguished according to their LDH isoenzyme pattern, since the cortical thymocytes exhibit significantly higher percentage activity in LDH-l, 2 and 3 than the medullary thymocytes (Plum, Ringoir, De Smedt & Dhont, 1978; Plum, Monteyne, Dhont & De Smedt, 1978). -This means that this time a shift of the LDH pattern toward the LDH-5 end of the spectrum occurs during T-cell development within the thymus. The aim of the present investigation was to confirm this unique shift in the opposite direction by analysing the isoenzyme pattern of the pre-thymic precursor cell. This cell population is at an earlier stage of T-cell development than those studied up to now. The prethymic precursor cell has been obtained from thymic rudiments of 14 day old mouse embryos. A higher activity in LDH-I in this cell population should unequivocally stress the shift toward LDH-5 during T cell development in the thymus.
INTRODUCTION LDH (L-lactate: NAD + oxidoreductase, EC 1.1.1.27) is a tetrameric enzyme composed of two sub-units. When combined, these two sub-units result in a total of five different tetrameric isoenzymes: LDH- I, 2, 3, 4 and 5. The isoenzyme system has been most widely used in studies of tissue differentiation. In mice, embryonic tissues first exhibit a predominance of LDH-5, and then with continuing developCorrespondence: Dr J. Plum, Laboratorium voor Bacteriologie en Virologie, Rijksuniversiteit Gent, Akademisch Ziekenhuis, Blok A, De Pintelaan 135, B-9000 Gent, Belgium. 00 19-2805/79/0800-08 17$02.00 ©) 1979 Blackwell Scientific Publications
METHODS AND MATERIALS
Animals Mice of strain NMRI were used. Gestational stage was 817
J. Plum, S. Ringoir & Magda De Smedt
818
determined by noting the appearance of vaginal plugs
(day 0). Cell suspensions Suspensions of thymus cells were prepared by removing the thymuses of 14 day old embryos and disrupting these organs mechanically by gentle pressure from a polysterene pestle in cold RPMI 1640 medium buffered with HEPES. ......
Assayfor Thy-1.2 antigen The cell suspensions obtained from the thymic rudiments were assayed for Thy-1.2 antigen using a cytotoxic assay with anti-Thy-1.2 antiserum (Searle, England) and absorbed guinea-pig serum complement as described by Golub (1971). The test was also performed on thymocytes from aAult mice as control. Cell counts and staining All cell counts were done by a haemocytometer. Viability was determined by eosin exclusion. The cells were sedimented on grease-free slides with the use of a cytocentrifuge and stained with May-GrunwaldGiemsa.
n~~~~~~1
'..
Determination of LDH isoenzyme pattern
The pre-thymic precursor cells, obtained from the thymic rudiments were suspended in Tris buffer at a concentration of 2 x 107 cells/ml. These cells were frozen and thawed three times consecutively and spun at 2000 g for 10 min. The LDH isoenzymes of the supernatant were determined by agar gel electrophoresis as described (Plum & Ringoir, 1975; Plum & Ringoir, 1977).
Figure 1. Cells obtained from thymic rudiments (Experiment 3). These cells are large basophilic (magnification l0Ox 100).
different experiments, the pattern exhibited by the pre-thymic cell precursor obtained from the thymus shows a similar pattern, with activity in the LDH-2, LDH-3, LDH-4 and LDH-5 bands.
RESULTS
Cells obtained from thymic rudiments The cells that were released into suspension were large basophilic cells as seen in Fig. 1. These cells were Thy-1.2 antigen negative. No Thy-1.2 antigen-bearing cells or contaminating erythrocytes were seen. The viability of the cells was at least 96%. Ninety-nine per cent of the thymocytes from normal adult mice were found to bear Thy- 1.2 antigen.
The LDH isoenzyme pattern of the cells The LDH isoenzyme pattern of the cells for the different experiments are summarized in Table 1. For the
DISCUSSION The thymic cells showed distinct properties of the pre-thymic cell precursor: the cells were Thy-1 negative (Raff, 1971) and large basophilic blast-like cells (Moore & Owen, 1967); moreover at this stage, the mouse embryo contained only these cell precursors in the thymus (Stutmati 1977). As seen in Table 1, the pattern of this cell population as compared with the cortical thymocytes showed a higher percentage of LDH-5 activity. Therefore, the assumption that during T-cell development a unique shift toward LDH-5 end occurred is not confirmed.
LDH isoenzyme pattern of the pre-thymic precursor cell
819
Table 1. LDH isoenzyme pattern of pre-thymic cell precursors in thymus
Cell population
LDH-1
Percentage activity LDH-3 LDH-4
LDH-2
LDH-5
Thymocytes obtained from thymic rudiments of 14 day old embryos (pre-thymic cell precursor) Experiment 1 1-8 33-2 69-0 2 0-8 7-6 36-3 55-3 3 0-9 4-1 30-4 64 5 4 1-0 6-0 29-0 640 5 1-0 9-6 34-6 548 Thymocytes obtained from thymuses of normal adult mice 8-10 weeks of age (mainly cortical thymocytes)* 6-7+0-7 13-7+0-7 17-5+0-7 27-3+0-6 34-7+1-4 Thymocytes obtained from thymuses of adult mice 8-10 weeks of age, 96 h after in vivo cortisone treatment (enriched in medullary thymocytes)* 1-2+0-3 3-1+1-5 10-7+2-8 31-5+3-8 53-3+6-4 * Data included from Plum, Ringoir, De Smedt & Dhont (1978), these values represent the mean ( ± SE) obtained from six experiments.
This LDH pattern is of great value, because it provides biochemical information on the differentiation of cells. Indeed, whereas the LDH pattern varies to a great extent, according to the cell population itself, Markert & Ursprung (1962) described that in mice, for a given cell population the less mature precursors contain at least as much or even more LDH-5 activity than the mature cell population. This phenomenon is confirmed in our analysis of the thymic cell precursor with regard to the cortical thymocyte. Next to the analysis of the early T-cell development, the further differentiation steps of the T cells within the thymus can be discussed, taking into account the previous data on the LDH pattern of cortical and medullary thymocytes (Table 1). This is important, because the relationship between medullary and cortical thymocytes is still a matter of discussion. It has been accepted for years that the medullary thymocytes are the sole progeny of the cortical thymocytes. This was based particularly on the appearance of immunological competence, as estimated by the capacity to mediate the graft v. host reaction, the mixed lymphocyte response or cell-mediated lympholysis. In ontogenic studies with thymus from mouse embryo and newborn mice, it was shown that these immunological competences were absent and were acquired only after 3 weeks of age (Katz, 1977). Thus a lack of immunological competence as judged by these parameters precedes the immune competence. The thymocytes can be
divided into cortical thymocytes, with no immune competence as judged by these parameters and medullary thymocytes with these capacities. Therefore, it seems logical to assume that the cortical thymocytes precede the medullary thymocytes. Some data are in conffict with this hypothesis. First, Shortman & Jackson (1974) were able to show by means of studies in cell kinetics that both cell populations have distinct cell kinetics, whereby for each thymocyte subpopulation a continuous renewal was possible and also these authors failed to show a transition from cortical to medullary thymocytes. Second, Papiernik & Bach (1977), by means of an hetero-antiserum to cortisone resistant thymocytes, i.e. medullary thymocytes, showed that this cell population appears during ontogeny before the cortisone sensitive or cortical thymocytes. These data strongly favour the hypothesis that medullary thymocytes are not derived from the cortical. In addition, other authors have already mentioned that the peripheral T cells consist of at least a small subpopulation, which is directly derived from the cortical thymocytes, as judged by their electrophoretic mobility (Droege, 1976) or alkaline phosphatase activity as far as the guinea-pig is concerned (Soppi, Ruuskanen & Kouvalainen, 1977). These findings also argue for the fact that cortical thymocytes do not have to transform into medullary thymocytes to leave the thymus. Two separate path-
J. Plum, S. Ringoir & Magda De Smedt
820
ways for cortical and medullary thymocytes with separate outcome of both cell populations is also possible. In our analysis of the LDH distribution of the thymocytes a further argument is found for the latter hypothesis. This is based on two findings: first, in this study the thymus cell precursor has a high percentage activity in LDH-5. This is consistent with the theory that during the development of cells, progressive shift toward the LDH-1 end is found. Second, when the LDH pattern of the cortical and medullary thymocytes is considered (Table 1), the LDH shift toward LDH-1 fits only with the hypothesis of two separate cell lines within the thymus. Indeed, only in this situation a shift toward the LDH-I end of the spectrum is possible for both cell lines, but to different extents. Further application of this enzyme system is to look whether there are within the T-cell populations, subpopulations with a high LDH activity, because these cells should be derived from the cortical cell line. In view of the tremendous heterogeneity of the T cell, which is discovered more and more to have new and unsuspected functional capacities, i.e. regulatory functions, it is not unreasonable to assume that the cortical cell line might correlate with one of these functions which have not been investigated systematically in earlier studies of thymus ontogeny.
REFERENCES DROEGE W. (1976) 'Early T cells' and 'Late T cells': suggestive evidence for two T cell lineages with separate developmental pathways. Europ. J. Immunol. 6, 763.
GOLUB E.S. (1971) Brain-associated ) antigen: reactivity of rabbit anti-mouse brain with mouse lymphoid cells. Cell. Immunol. 2,353. KATZ D.H. (1977) Lymphocyte Differentiation, Recognition, and Regulation, p. 127. Academic Press, New York. MARKERT C.L. & URSPRUNG H. (1962) The ontogeny of isozyme patterns of lactate dehydrogenase in the mouse. Develop. Biol. 5,363. MooRE M.A.S. & OWEN J.J.T. (1967) Experimental studies on the development of the thymus. J. exp. Med. 126, 715. PAPIERNIK M. & BACH J.F. (1977) Thymocyte subpopulations in young and adult mice. II. Study of steroid-resistant populations by means of a specific heteroantiserum. Europ. J. Immunol. 7, 800. PLuM J. & RINGoIR S. (1975) A characterization of human B and T lymphocytes by their lactate dehydrogenase isoenzyme pattern. Europ. J. Immunol. 5, 871. PLuM J. & RINGOIR S. (1977) Lactate dehydrogenase isoenzyme pattern as a measure of cellular differentiation in lymphocytic cells. J. reticuloendothel. Soc. 21, 225. PLUM J., RINGOIR S., DE SMEDT M. & DHONT E. (1978) Lactate dehydrogenase isoenzymatic analysis of murine lymphocyte populations: a parameter of cellular differentiation. Cell. Immunol. 36, 37. PLUM J., MONTEYNE R., DHoNT E. & DE SMEDT M. (1978) Lactate dehydrogenase isoenzyme pattern: Marker for differentiation of lymphocytes. Scand. J. Immunol. 7,515. RAPF M.C. (1971) Surface antigenic marker for distinguish T and B lymphocytes in mice. Transplant. Rev. 6, 52. SHORTMAN K. & JACKSON H. (1974) The differentiation of T lymphocytes. 1. Proliferation kinetics and interrelationship of subpopulations of mouse thymus cells. Cell. Immunol. 12, 230. SoPPi E., RUUSKANEN 0. & KOUVALANEN (1977) Alkaline phosphatase in the differentiation of guinea pig T lymphocytes. Acta Path. Microbiol. Scand. Sect. C, 85,367. SrUTMAN 0. (1977) Two main features of T cell development: thymus traffic and postthymic maturation. In: Contemporary Topics in Immunobiology, vol. 7 (Ed. by 0. Stutman), p. 9. Plenum Press, New York.
