Eur. J. Immunol. 1991.21: 109-113
Ronald Palacios and Jacqueline Samaridis Basel Institute for Immunologyv, Basel
Pro-T lymphocyte development
109
Thymus colonization in the developing mouse embryo We have directly followed the formation of and the thymus colonization by pro-T lymphocytes in the developing C57BL/6 mouse embryo by using the monoclonal antibody JORO 37-5 specific for pro-T lymphocytes, immunofluorescence staining and flow fluorocytometry or microscopy analysis. The results show that JORO 37-5+ cells begin to appear in the liver at day 9 of gestation. These JORO 37-5+ cells migrate to and colonize the thymus 1 day later, where they expand vigorously during the next 4-5 days and, subsequently, switch off expression of JORO 37-5 as they further differentiate into mature thymocytes.
1 Introduction In the developing mouse embryo, hematopoietic pluripotent stem cells are first formed in the 7-8-day yolk sac, probably from cells that migrate out of particular regions of the primitive streak [l-31. Although stem cells in the yolk sac differentiate into first-generation erythrocytes, some of them divide and form further stem cells which, at the time of initiation of circulation (day 9), reach the bloodstream [2] and seed the liver, BM and spleen, where they differentiate into various hematopoietic cell types [2, 41. In postnatal life, the stem cell pool, probably derived from the stem cells in these embryonic sites, reside in the BM and are responsible for the maintenance of hemopoiesis during the life span of the mouse [5-71. Over the last 8 years, a great deal of knowledge has been accumulated on the sequence and timing of expression of theTcR genes, phenotypic characteristics and production of growth factors and expression of their corresponding receptors by the developing thymocytes (for reviews see [8, 91. However,very little is known about the formation of the very early T cell progenitors (pro-T lymphocytes), their migration to and colonization of the thymus because of the lack of specific cell surface markers for these cells. Elsewhere [lo], we describe the generation and characterization of rat mAb specific for mouse pro-T lymphocytes. In the present study we have used one of these antibodies, JORO 37-5, to directly follow the formation of and the thymus colonization by these cells in the developing embryo.
2 Materials and methods All embryos were obtained from C57BL/6 females, mated with C57BL/6 males, which were raised in the animal facility of our Institute. The age of the embryos was determined by scoring the day of the appearance of vaginal plugs, which is taken as day 0. Further assessment of the age
of gestation was based on the following criteria; 8 days, a well-developed ectoplacental cone and early membrane formation; 9 days, a well-developed yolk sac, a conspicuous heart and no liver pigment; 10 days, early pigmentation in the liver; 11 to 12 days, a well-pigmented liver and the absence of a differentiated thymus; 13 to 18 days, the relative size and development of the fetus. As there was at times variation in the individual development of the embryos within a single pregnancy, the estimates in some instances may be in error by 1/2 to 1 day. Cell suspensions were prepared from liver and thymus, free of erythrocytes, as previously described [ 11, 121. Immunofluorescence staining and FCM analysis was carried out as described [12], using a FACScan instrument (Becton Dickinson, Mountain View, CA) with purified JORO 37-5 mAb [lo] and FITC-anti-rat IgG-specific antibody (Jackson Immunoresearch), as second-step reagent. Dead cells were excluded from the analysis by using propidium iodide. Fluorescence emitted by single viable cells was detected with logarithmic amplification. The data collected from 2.5 x lo4-5 x lo4 cells were analyzed with Consort 30 Software. Embryo sections (4-6 pm) were prepared and acetone-fixed on glass slides and stored at - 20°C until use. Immunofluorescence staining of the slides (four previously selected slides per embryo) was carried out using a previously determined optimal concentration of purified JORO 37-5 mAb in staining buffer (PBS 2% BSA 0.1% sodium azide), slides were then incubated at 20°C for 1 h. Following two washes with buffer, FITC-anti-rat IgG-specific antibody (final dilution 1 : 200) was added and the slides were incubated at 4°C for 1 h. Following three washes with buffer (each for 10 min), the slides were mounted under glass cover slips using 50 mM Tris (pH 8.6) containing gelvatol and analyzed, both under phase contrast and green fluorescence with an Axiophot Zeiss microscope. In all cases the entire embryo was scanned for the presence of cells that specifically bound the JORO 37-5 mAb. JORO 37-5+ cells usually displayed a ring-dotted pattern of fluorescence, and sometimes a patchy pattern on one pole of the cells was also observed.
+
+
[I 87351 The Basel Institute was founded and is supported by E Hoffmann-La Roche Ltd., Basel, Switzerland.
Correspondence: Ronald PaIacios, Basel Institute for Immunology Grenzacherstrasse 487. CH-4058 Basel, Switzerland 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
3 Results and discussion The JORO 37-5 mAb was obtained from a fusion of LN cells of a rat immunized with the mouse BM pro-T 0014-2980/91/0101-0109$3.50+ .25/0
110
R. Palacios and J. Samaridis
Eur. J. Immunol. 1991. 21: lo!-113
lymphocyte clone C4-77 [ 101. This mAb binds to all pro-T lymphocyte clones from BM or fetal thymus tested, but it does not react with cell lines representing later stages of T cell development, B lymphocyte lineage cells (from pro-B to plasma cells), myeloid cells or fibroblasts. The two embryonic stem cell lines tested were also negative. JORO 37-5+ cells, purified by cell sorter from the BM of 3-week-old normal mice or from 14-15-day fetal liver, gave rise to mature T lymphocytes, but not to B lymphocytes, following their transfer into sublethally irradiated scid mice [lo] and unpublished results). In young C57BL/6 mice, which were exposed to 600 rad of y-rays, JORO 37-5+ cells enter the thymus 2 days post-irradiation, where they expand during the next 4 days. and subsequently differentiate into JORO 37-5- thymocytes. At day 8 post-irradiation, JORO 37-5+ cells constitute < 1% of thymocytes [ 101. We have now studied the formation of and the thymus colonization by JORO 37-5+ cells in the developing C57BL/6 mouse embryo. By immunofluorescence staining and FCM analysis of cell suspensions from C57BL/6 embryos we could determine the frequency of JORO 37-5+ cells in the liver and thymus from day 14 of gestation onwards. Fig. 1 shows that in the liver about 3% to 5% of mononuclear cells are JORO 37-5+ at day 14 and 15.Their number then declines to < 1% at birth. In the thymus, most cells are JORO 37-5+ at day 14. Subsequently, the frequency progressively declines to < 1% of the thymocytes at birth (Fig. 1). As we could not obtain sufficient numbers of cells from embryos before day 14, we searched for JORO 37-5+ cells by immunofluorescence staining and fluorescence microscopy of tissue sections from day 9 to day 16 C57BL/6 embryos. Scanning of the entire mouse embryos at all ages studied showed that cells specifically staining with JORO 37-5 were only present in the liver and thymus. The results of these studies are illustrated with some examples in Fig. 2 (liver) and Fig. 3 (thymus). Very few JORO 37-5+ cells were found in the liver primordium at day 9 of gestation; these cells increased both in numbers and brightness at day 10. They were often found in clusters (Fig. 2). Embryo liver sections in which
the JORO 37-5 mAb was omitted in the staining procedure showed no positive cells (control, Fig. 2). Very few JORO 37-5+ cells were first detected in the day-10 thymus anlage; these JORO 37-5+ thymocytes underwent an impressive expansion at days 13-15 of gestation, but their frequency declined from day 16 of gestation (Fig. 3). Staining without inclusion of the JORO 37-5 mAb showed n o positive cells (control, Fig. 3). The same type of results were obtained in three independent experiments. The timing of thymus colonization by JORO 37-5+ that we find here is in good agreement with that reported by Moore and Owen [ 131. As JORO 37-5+ cells were found in the d a y 4 liver primordium (Fig. 2 ) it was important to test for the presence of these cells in day-8-9 yolk sac. No detectable JORO 37-5+ cells were found in this tissue. Taken together, our results suggest the following sequence of events. Circulating yolk sac-derived hemopoietic stem cells seed the liver, where some differentiate into JORO 37-5+ cells from day 9 of gestation. The JORO 37-5+ cells migrate to and colonize the thymus 1 day later, where they vigorously expand during the next 4-5 days and subsequently switch off expression of JORO 37-5 as they differentiate. Interestingly, there seems to be a correlation, both between the time at which JORO 37-5+ thymocytes vigorously expand and the initiation of rearrangement and expression of TcR genes and between the time when the developing thymocytes switch off expression of JORO 37-5 and the beginning of expression of TcR complexes, and CD8 and CD4 molecules, on the cell membrane. Thus, previous studies have shown that at day 14 of gestation, the time of massive expansion of JORO 37-5+ thymocytes, the developing thymocytes start of rearrangeTcR genes (6, y. f3). At day 16-17 of gestation, when most thymocytes have become JORO 37-5-, thymocytes bearing y/6 and a/f3TcR heterodimers, CD8 and CD4 surface markers begin to appear [8, 91. Our previous in vivo studies with purified JORO 37-5+ cells from either BM of young mice or 14-15-day fetal liver show their restricted functional potential to develop along the T lymphocyte pathway ([lo] and unpublished results). We have thus far not been able to obtain sufficient numbers of purified JORO 37-5+ cells from day-9 liver to directly assess their functional potential. Hence, we cannot exclude the possibility that the JORO 37-5+ cells in the day4 embryonic liver have a less restricted functional potential than those in the BM of young mice. In any case, it will be most interesting to try now to understand the events which lead to the differentiation of stem cells into the first JORO 37-5+ cells.
loo]
U I
1'4 1'5 1's 117
Days of gestation
1-
116 Newborn
Days of gestation
Fipure 1 . The freouencv o f JORO 37-5+ cells in mononuclear cell " suspensions from liver and thymus of C57BL/6 mouse embryos (from day 14 of gestation until birth) was determined by FCM analysis. Cell samples exposed to second-step reagent (FITClabeled anti-rat IgG antibody) were used as negative controls. 1
2
We would like to thank Beat Imhof for advice on staining oftissue sections, Ms. Birgit Kugelberg for the excellent preparation of embryo sections and slides, and Mr. Hans- Peter Stahlhiv-gerfor the illustrations. We are also grateful to Werner Haas. Damir Vidovic. Klaus Karjalainen and Harald von Boehmer for critical reading of the manuscript, and Ms. Janette Millar for skillful preparation of thc manuscript.
Rcceived July 12. 1090; in revised form September 4. 1990.
Pro-T lymphocytc development
Eur. J. Immunol. 1991. 21: 109-113
11 I
Day 10
Day 9
CONTROL
Figure 2. Formation of JORO 37-5+ cells in the developing liver. The presence of JORO 37-5+ cells in C57BL/6 embryo sections was assessed by immunofluorescence staining and fluorescence microscopy. Slides in which JORO 37-5 mAb was omitted in the staining procedure were used as negative controls.
112
(A)
Eur. J. Irnrnunol. 1991. 21: 109-113
R . Palacios and J. Samaridis Day 10
Pro-T lymphocyte development
Eur. J. Immunol. 1991. 21: 109-113
(B)
113
Day 14
Figure 3. Thymus colonization by 37-5+ cells.The presence of JORO 37-5+ thymocytes was studied by immunofluorescence staining and fluorescence microscopy of embryo sections at the days of gestation indicatcd. Slides in which JORO 37-5 mAb was omitted in the staining procedure were used as negative controls (see Sect. 2 for further details).
4 References 1 Metcalf, D. and Moore, M. A. S.. Hemopoietic cells. NorthHolland Publishing Company, AmsterdadLondon 1971. p. 70. 2 Moore. M. A . S. and Metcalf, D., Br. J. Imrnunol. 1070. 18: 274. 3 Tyan. M. J., J. Immunol. 1968. 100: 535. 4 Moore. M. A. S. and Owen, J. J.T., Lancet 1967. ii: 658. 5 Till, J. E. and McCulloch, E. A., Radiat. Res 1061. 14: 213. 6 Abrahamson. S., Miller, R. and Phillips, R..J. Exp. Med. 1977. 145: 1567. 7 Spangrude. G.. Heinfeld, S. and Weissmann, I. L., Science 1988. 241: 58.
8 Moller. G.. Editor. T. cell precursors. Imtnunol. Her! 1Y88. 104: 5 . 0 Adkins. B.. Mueller. C., Okada, C., Reichart. R.. Weissmann. 1. L. and Sprangrude, G.. Annu. Rev. Imtnuriol. 1Y87. 5: 325. 10 Palacios, R., Samaridis. J.. Thorpe, D. and Leu, T.. J. Exp. Med. 1990. 172: 219. 11 Palacios. R.. Sideras. P. and Von Boehmer. H.. EMBO J. 1987. 6: 91. 12 Palacios, R., Samaridis. J., Studer. S.. and Pelkonen. J.. E M B O J. 1989. 8: 4053. 13 Moore. M. A. S. and Owen, J. J. T.. J. Exp. M e d . lYh7. 126: 715.
Ronald Palacios and Jacqueline Samaridis Basel Institute for Immunologyv, Basel
Pro-T lymphocyte development
109
Thymus colonization in the developing mouse embryo We have directly followed the formation of and the thymus colonization by pro-T lymphocytes in the developing C57BL/6 mouse embryo by using the monoclonal antibody JORO 37-5 specific for pro-T lymphocytes, immunofluorescence staining and flow fluorocytometry or microscopy analysis. The results show that JORO 37-5+ cells begin to appear in the liver at day 9 of gestation. These JORO 37-5+ cells migrate to and colonize the thymus 1 day later, where they expand vigorously during the next 4-5 days and, subsequently, switch off expression of JORO 37-5 as they further differentiate into mature thymocytes.
1 Introduction In the developing mouse embryo, hematopoietic pluripotent stem cells are first formed in the 7-8-day yolk sac, probably from cells that migrate out of particular regions of the primitive streak [l-31. Although stem cells in the yolk sac differentiate into first-generation erythrocytes, some of them divide and form further stem cells which, at the time of initiation of circulation (day 9), reach the bloodstream [2] and seed the liver, BM and spleen, where they differentiate into various hematopoietic cell types [2, 41. In postnatal life, the stem cell pool, probably derived from the stem cells in these embryonic sites, reside in the BM and are responsible for the maintenance of hemopoiesis during the life span of the mouse [5-71. Over the last 8 years, a great deal of knowledge has been accumulated on the sequence and timing of expression of theTcR genes, phenotypic characteristics and production of growth factors and expression of their corresponding receptors by the developing thymocytes (for reviews see [8, 91. However,very little is known about the formation of the very early T cell progenitors (pro-T lymphocytes), their migration to and colonization of the thymus because of the lack of specific cell surface markers for these cells. Elsewhere [lo], we describe the generation and characterization of rat mAb specific for mouse pro-T lymphocytes. In the present study we have used one of these antibodies, JORO 37-5, to directly follow the formation of and the thymus colonization by these cells in the developing embryo.
2 Materials and methods All embryos were obtained from C57BL/6 females, mated with C57BL/6 males, which were raised in the animal facility of our Institute. The age of the embryos was determined by scoring the day of the appearance of vaginal plugs, which is taken as day 0. Further assessment of the age
of gestation was based on the following criteria; 8 days, a well-developed ectoplacental cone and early membrane formation; 9 days, a well-developed yolk sac, a conspicuous heart and no liver pigment; 10 days, early pigmentation in the liver; 11 to 12 days, a well-pigmented liver and the absence of a differentiated thymus; 13 to 18 days, the relative size and development of the fetus. As there was at times variation in the individual development of the embryos within a single pregnancy, the estimates in some instances may be in error by 1/2 to 1 day. Cell suspensions were prepared from liver and thymus, free of erythrocytes, as previously described [ 11, 121. Immunofluorescence staining and FCM analysis was carried out as described [12], using a FACScan instrument (Becton Dickinson, Mountain View, CA) with purified JORO 37-5 mAb [lo] and FITC-anti-rat IgG-specific antibody (Jackson Immunoresearch), as second-step reagent. Dead cells were excluded from the analysis by using propidium iodide. Fluorescence emitted by single viable cells was detected with logarithmic amplification. The data collected from 2.5 x lo4-5 x lo4 cells were analyzed with Consort 30 Software. Embryo sections (4-6 pm) were prepared and acetone-fixed on glass slides and stored at - 20°C until use. Immunofluorescence staining of the slides (four previously selected slides per embryo) was carried out using a previously determined optimal concentration of purified JORO 37-5 mAb in staining buffer (PBS 2% BSA 0.1% sodium azide), slides were then incubated at 20°C for 1 h. Following two washes with buffer, FITC-anti-rat IgG-specific antibody (final dilution 1 : 200) was added and the slides were incubated at 4°C for 1 h. Following three washes with buffer (each for 10 min), the slides were mounted under glass cover slips using 50 mM Tris (pH 8.6) containing gelvatol and analyzed, both under phase contrast and green fluorescence with an Axiophot Zeiss microscope. In all cases the entire embryo was scanned for the presence of cells that specifically bound the JORO 37-5 mAb. JORO 37-5+ cells usually displayed a ring-dotted pattern of fluorescence, and sometimes a patchy pattern on one pole of the cells was also observed.
+
+
[I 87351 The Basel Institute was founded and is supported by E Hoffmann-La Roche Ltd., Basel, Switzerland.
Correspondence: Ronald PaIacios, Basel Institute for Immunology Grenzacherstrasse 487. CH-4058 Basel, Switzerland 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
3 Results and discussion The JORO 37-5 mAb was obtained from a fusion of LN cells of a rat immunized with the mouse BM pro-T 0014-2980/91/0101-0109$3.50+ .25/0
110
R. Palacios and J. Samaridis
Eur. J. Immunol. 1991. 21: lo!-113
lymphocyte clone C4-77 [ 101. This mAb binds to all pro-T lymphocyte clones from BM or fetal thymus tested, but it does not react with cell lines representing later stages of T cell development, B lymphocyte lineage cells (from pro-B to plasma cells), myeloid cells or fibroblasts. The two embryonic stem cell lines tested were also negative. JORO 37-5+ cells, purified by cell sorter from the BM of 3-week-old normal mice or from 14-15-day fetal liver, gave rise to mature T lymphocytes, but not to B lymphocytes, following their transfer into sublethally irradiated scid mice [lo] and unpublished results). In young C57BL/6 mice, which were exposed to 600 rad of y-rays, JORO 37-5+ cells enter the thymus 2 days post-irradiation, where they expand during the next 4 days. and subsequently differentiate into JORO 37-5- thymocytes. At day 8 post-irradiation, JORO 37-5+ cells constitute < 1% of thymocytes [ 101. We have now studied the formation of and the thymus colonization by JORO 37-5+ cells in the developing C57BL/6 mouse embryo. By immunofluorescence staining and FCM analysis of cell suspensions from C57BL/6 embryos we could determine the frequency of JORO 37-5+ cells in the liver and thymus from day 14 of gestation onwards. Fig. 1 shows that in the liver about 3% to 5% of mononuclear cells are JORO 37-5+ at day 14 and 15.Their number then declines to < 1% at birth. In the thymus, most cells are JORO 37-5+ at day 14. Subsequently, the frequency progressively declines to < 1% of the thymocytes at birth (Fig. 1). As we could not obtain sufficient numbers of cells from embryos before day 14, we searched for JORO 37-5+ cells by immunofluorescence staining and fluorescence microscopy of tissue sections from day 9 to day 16 C57BL/6 embryos. Scanning of the entire mouse embryos at all ages studied showed that cells specifically staining with JORO 37-5 were only present in the liver and thymus. The results of these studies are illustrated with some examples in Fig. 2 (liver) and Fig. 3 (thymus). Very few JORO 37-5+ cells were found in the liver primordium at day 9 of gestation; these cells increased both in numbers and brightness at day 10. They were often found in clusters (Fig. 2). Embryo liver sections in which
the JORO 37-5 mAb was omitted in the staining procedure showed no positive cells (control, Fig. 2). Very few JORO 37-5+ cells were first detected in the day-10 thymus anlage; these JORO 37-5+ thymocytes underwent an impressive expansion at days 13-15 of gestation, but their frequency declined from day 16 of gestation (Fig. 3). Staining without inclusion of the JORO 37-5 mAb showed n o positive cells (control, Fig. 3). The same type of results were obtained in three independent experiments. The timing of thymus colonization by JORO 37-5+ that we find here is in good agreement with that reported by Moore and Owen [ 131. As JORO 37-5+ cells were found in the d a y 4 liver primordium (Fig. 2 ) it was important to test for the presence of these cells in day-8-9 yolk sac. No detectable JORO 37-5+ cells were found in this tissue. Taken together, our results suggest the following sequence of events. Circulating yolk sac-derived hemopoietic stem cells seed the liver, where some differentiate into JORO 37-5+ cells from day 9 of gestation. The JORO 37-5+ cells migrate to and colonize the thymus 1 day later, where they vigorously expand during the next 4-5 days and subsequently switch off expression of JORO 37-5 as they differentiate. Interestingly, there seems to be a correlation, both between the time at which JORO 37-5+ thymocytes vigorously expand and the initiation of rearrangement and expression of TcR genes and between the time when the developing thymocytes switch off expression of JORO 37-5 and the beginning of expression of TcR complexes, and CD8 and CD4 molecules, on the cell membrane. Thus, previous studies have shown that at day 14 of gestation, the time of massive expansion of JORO 37-5+ thymocytes, the developing thymocytes start of rearrangeTcR genes (6, y. f3). At day 16-17 of gestation, when most thymocytes have become JORO 37-5-, thymocytes bearing y/6 and a/f3TcR heterodimers, CD8 and CD4 surface markers begin to appear [8, 91. Our previous in vivo studies with purified JORO 37-5+ cells from either BM of young mice or 14-15-day fetal liver show their restricted functional potential to develop along the T lymphocyte pathway ([lo] and unpublished results). We have thus far not been able to obtain sufficient numbers of purified JORO 37-5+ cells from day-9 liver to directly assess their functional potential. Hence, we cannot exclude the possibility that the JORO 37-5+ cells in the day4 embryonic liver have a less restricted functional potential than those in the BM of young mice. In any case, it will be most interesting to try now to understand the events which lead to the differentiation of stem cells into the first JORO 37-5+ cells.
loo]
U I
1'4 1'5 1's 117
Days of gestation
1-
116 Newborn
Days of gestation
Fipure 1 . The freouencv o f JORO 37-5+ cells in mononuclear cell " suspensions from liver and thymus of C57BL/6 mouse embryos (from day 14 of gestation until birth) was determined by FCM analysis. Cell samples exposed to second-step reagent (FITClabeled anti-rat IgG antibody) were used as negative controls. 1
2
We would like to thank Beat Imhof for advice on staining oftissue sections, Ms. Birgit Kugelberg for the excellent preparation of embryo sections and slides, and Mr. Hans- Peter Stahlhiv-gerfor the illustrations. We are also grateful to Werner Haas. Damir Vidovic. Klaus Karjalainen and Harald von Boehmer for critical reading of the manuscript, and Ms. Janette Millar for skillful preparation of thc manuscript.
Rcceived July 12. 1090; in revised form September 4. 1990.
Pro-T lymphocytc development
Eur. J. Immunol. 1991. 21: 109-113
11 I
Day 10
Day 9
CONTROL
Figure 2. Formation of JORO 37-5+ cells in the developing liver. The presence of JORO 37-5+ cells in C57BL/6 embryo sections was assessed by immunofluorescence staining and fluorescence microscopy. Slides in which JORO 37-5 mAb was omitted in the staining procedure were used as negative controls.
112
(A)
Eur. J. Irnrnunol. 1991. 21: 109-113
R . Palacios and J. Samaridis Day 10
Pro-T lymphocyte development
Eur. J. Immunol. 1991. 21: 109-113
(B)
113
Day 14
Figure 3. Thymus colonization by 37-5+ cells.The presence of JORO 37-5+ thymocytes was studied by immunofluorescence staining and fluorescence microscopy of embryo sections at the days of gestation indicatcd. Slides in which JORO 37-5 mAb was omitted in the staining procedure were used as negative controls (see Sect. 2 for further details).
4 References 1 Metcalf, D. and Moore, M. A. S.. Hemopoietic cells. NorthHolland Publishing Company, AmsterdadLondon 1971. p. 70. 2 Moore. M. A . S. and Metcalf, D., Br. J. Imrnunol. 1070. 18: 274. 3 Tyan. M. J., J. Immunol. 1968. 100: 535. 4 Moore. M. A. S. and Owen, J. J.T., Lancet 1967. ii: 658. 5 Till, J. E. and McCulloch, E. A., Radiat. Res 1061. 14: 213. 6 Abrahamson. S., Miller, R. and Phillips, R..J. Exp. Med. 1977. 145: 1567. 7 Spangrude. G.. Heinfeld, S. and Weissmann, I. L., Science 1988. 241: 58.
8 Moller. G.. Editor. T. cell precursors. Imtnunol. Her! 1Y88. 104: 5 . 0 Adkins. B.. Mueller. C., Okada, C., Reichart. R.. Weissmann. 1. L. and Sprangrude, G.. Annu. Rev. Imtnuriol. 1Y87. 5: 325. 10 Palacios, R., Samaridis. J.. Thorpe, D. and Leu, T.. J. Exp. Med. 1990. 172: 219. 11 Palacios. R.. Sideras. P. and Von Boehmer. H.. EMBO J. 1987. 6: 91. 12 Palacios, R., Samaridis. J., Studer. S.. and Pelkonen. J.. E M B O J. 1989. 8: 4053. 13 Moore. M. A. S. and Owen, J. J. T.. J. Exp. M e d . lYh7. 126: 715.
