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The regulation of the T-lymphocytes precursor pool by a humoral factor released by the thymus
CELLULAR
IMMUNOLOGY
34, 420-423
The Regulation Humoral
(1977)
of the T-Lymphocytes Factor
Pool by a
Released by the Thymus
GEORGES E. ROELANTS 1 AND KATHLEEN Basel In&de
Precursor
S. MAYOR-WITHEY
for I~~~m~~ology, Base& Switzerland Received July 28,1977
Studies in congenitally athymic nude mice grafted with syngeneic thymus enclosed or not in a cell-impermeable Millipore chamber show that the thymus secretes one or several humoral factors controlling the size of the pool of early T-lineage cells produced by the bone marrow.
INTRODUCTION Congenitally athymic nude mice are essentially devoid of mature T lymphocytes but possess T-lineage cells (l-3). These cells are characterized by the absence of surface immunoglobulin and the presence of a low density of 6 (Thy-l determined) antigen and of the Tla antigen in Tla+ strains of mice. They have a slow electrophoretic mobility and do not recirculate through the thoracic duct. They are produced in the bone marrow of adult animals, accumulate essentially in the spleen, and have a life span of 1 to 2 days. They are found in early fetal thymuses prior to the appearance of thymocytes, and the maturation of thymocytes in those thymuses can be obtained independently of further cell entry (4). Thus we suggested that they represent precursor cells for thymocytes, i.e., “prethymocytes,” which are already committed to the T pathway but need the thymus microenvironment to differentiate further. This was demonstrated by Sato et al. (S), who separated these cells with the use of a fluorescence-activated cell sorter and obtained further differentiation by culture with thymic reticuloepithelial cells. The differentiation of prethymocytees from multipotential stem cells appears to be completely independent of the thymus for it also occurs in nude mice born from homozygous nu/nu parents and examined at birth (6). On the contrary, preliminary results showed that the production of these cells in the bone marrow is submitted to the homeostatic regulation of the thymus, for they appear rapidly in normal mice after surgical deprivation of the thymus and disappear rapidly when athymic mice are grafted with a thymus (3). In the present report we confirm this regulation and show that it is mediated through a humoral pathway and is a unique property of the thymus for it cannot be achieved by other lymphoid organs or cells. lAddress correspondence to Dr. Roelants at the International Animal Diseases, P.O. Box 30709, Nairobi, Kenya.
Laboratory
for Research on
420 Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0008-8749
(1) (2) (3) (1) (2) (3) (1) (2) (3) (4) (I) (2) (3) (4) (1) (2) (3) (4) (1) (2)
No treatment Sham subcutaneous graft Empty Millipore chamber Thymus Lymph node Nu/nu lymph node Thymus Lymph node Nu/nu lymph node Spleen Thymocytes Lymph node cells Nu/nu lymph node cells Spleen cells Thymus Lymph node Nu/nu lymph node Spleen Lymph node cells Nu/nu lymph node cells
TABLE
1
36 5 25 40 5 5 31 22 15 16 IO 10 5 IO 12 10 5 10 1.5 5
8 1 5 8 1 1 8 4 3 4 2 2 1 2 2 2 1 2 3 1
Number of miceb
Number of experiments
in the Spleen of Nude Mice after Various
9.8 8 12.6 2.6 9 6 1.9 6.0 7.3 10.5 15.0 8.0 10 10.5 12.0 13.5 7 13.5 9.3 9
Arithmetic mean6 (%I
Treatments’
1.5 4.1 4.0 6.4 1.4 4.5 4.9 4.2 2.1 9.2 4.7
3.5 3.0 1.5 6.5 3.8
5.2 2.7
2.9
6.5 2.7
1.5
3.5 3.0
3 ;f 5 5 z m
E 1.0 3.2 3.2
0
2.0 2.3
2.3 1.0
1.0
Standard error of the mean’ (%I
chambers or intraafter 7 days.
Standard deviation” (%)
1.4 3.5 3.2 5.6 1.0 3.2
4.1 2.5
2.7
Standard errord (o/o)
a Recipient mice received half a thymus lobe, the lymph node equivalent of one donor, half a spleen, or 50 X lo6 viable cells in Miliipore venously. “Epithelium” was prepared by implanting organs in Millipore chambers into nude mice and transferring them to a new recipient b Some spleens were examined individually, others were pooled. ’ X = Xi/n* d SE = [(xi - g)z/,]‘. e.SD = [(xi - g)z/(, - l)]+. f SERI = [(Xi - S)z/n(n - I)]“.
(D) Cells injected intravenously
(E) “Epithelium” in Millipore chamber
(D) Cells in Millipore chamber
(C) Organs in Millipore chamber
(B) Subcutaneous graft
(A) Controls
Ig-@+ Lymphocytes
422
SHORT
MATERIALS
COMMUNICATIONS
AND
METHODS
The experimental principle was to introduce into BALB/c nude mice (8-12 weeks old ; G. Bomholtgaard, Ltd., Ry, Denmark) various organs or cells (from 4- to S-week-old syngeneic mice) included or not in a cell-impermeable Millipore chamber, GSWPO 1300 with an average pore size of 0.22 pm (instead of the HAWPO 1300 with an average pore size of 0.45 pm used previously) (3) and to look at the spleen 3 to 26 days later for the presence of Ig-B+ cells using immunofluorescence techniques. The life span of Ig-$+weak cells being 1 to 2 days and the reconstitution of peripheral T cells in thymus-grafted nude mice starting only after weeks (7), depression of Ig-8+weak cell production could be easily detected. The characterization and use of rabbit antibody raised against mouse brain-associated 0 antigen made specific by absorption in viva and of AKR anti-BCSH reagents have been reported in detail (l-3, 8). The only slight difference from reported techniques was that the cells were passed through Ficoll-Urovison before staining. RESULTS
AND
DISCUSSION
Results presented in Table 1 show that an intact thymus introduced subcutaneously (group Bl) depresses the production of “prethymocytes.” This effect remains when the thymus is enclosed within a cell-impermeable Millipore chamber (group Cl) showing that it is mediated through one or several humoral factors. The remaining 2.61.9s 8’ cells may represent 8+strongcells known to be present in small quantities in nude mice (1, 2, 9). Other lymphoid organs such as spleen or lymph nodes do not have the same effect. The regulation does not appear to be mediated by the thymocytes’ physiological or degradation products since it did not appear in animals receiving Millipore chambers containing only thymocytes (group Dl). Chambers containing thymuses which were recovered after l-week in viva implantation, a technique used to obtain mainly epithelial tissue (10, ll), were not functional in our assay. This is surprising since left in situ they continue to exert their activity for at least 26 days. Controls of bone marrow and peripheral blood and lymph nodes showed that the reduction of Ig-9+ cells in the spleen of thymus-deprived mice was not due to a mere redistribution of these cells in the organism. The absolute amount of other cell types, i.e., Ig+B and Ig-&, in the spleens of those mice also remained unchanged. In conclusion it appears that the thymus, while not inducing the early commitment of multipotential stem cells to the T lineage (6), exerts a humoral negative homeostatic control on the size of the T-lineage precursor pool. The relationship of this factor with other described thymic hormones (12) remains to be elucidated. REFERENCES 1. Loor, F., and Roelants, G. E., Nature (Lo&on) 251,229, 1974. 2. Roelants, G. E., Loor, F., von Boehmer, H., Sprent, J., Hagg, L. B., Mayor, K. S., and 3. 4. 5. 6.
Ryden, A., Em-. J. Immunol. 5, 127, 1975. Roelants, G. E., Mayor, K. S., Hagg, L. B., and Loor, F., Eur. J. Zmmzlnol. 6, 75, 1976. Owen, J. J. T., and Raff, M. C.,.J. Exp. Med. 132, 1216, 1970. Sato, V. L., Waksal, S. D., and Herzenberg, L. A., Cell. Immunol. 24, 173, 1976. Loor, F., Amstutz, H., Hagg, L. B., Mayor, K. S., and Roelants, G. E., Eur. J. Zmmulzol. 6, 663, 1976.
SHORT
COMMUNICATIONS
423
7. Loor, F., Roelants, G. E., Kindred, B., Mayor, K. S., and Hagg, L. B., In. “Progress in Differentiation Research” (N. Muller-Berat, Ed.), pp. 559-566. North-Holland, Amsterdam, 1976. 8. Loot-, F., and Roelants, G. E., Ann. N. Y. Acad. Sci. 254,226, 1975. 9. Raff, M. C., Nature (London), 246, 350, 1973. 10. Levey, R. H., Trainin, N., and Low, L. W., I. Nat. Cancer. Inst. 31, 199, 1963. 11. Osoba, D., and Miller, J. F. A. P., J. Exp. Med. 119, 177, 1954. 12. Van Bekkum, D. W. (Ed.), “The Biological Activity of Thymic Hormones.” Kooyker Scientific Publication, Rotterdam, 1975.
IMMUNOLOGY
34, 420-423
The Regulation Humoral
(1977)
of the T-Lymphocytes Factor
Pool by a
Released by the Thymus
GEORGES E. ROELANTS 1 AND KATHLEEN Basel In&de
Precursor
S. MAYOR-WITHEY
for I~~~m~~ology, Base& Switzerland Received July 28,1977
Studies in congenitally athymic nude mice grafted with syngeneic thymus enclosed or not in a cell-impermeable Millipore chamber show that the thymus secretes one or several humoral factors controlling the size of the pool of early T-lineage cells produced by the bone marrow.
INTRODUCTION Congenitally athymic nude mice are essentially devoid of mature T lymphocytes but possess T-lineage cells (l-3). These cells are characterized by the absence of surface immunoglobulin and the presence of a low density of 6 (Thy-l determined) antigen and of the Tla antigen in Tla+ strains of mice. They have a slow electrophoretic mobility and do not recirculate through the thoracic duct. They are produced in the bone marrow of adult animals, accumulate essentially in the spleen, and have a life span of 1 to 2 days. They are found in early fetal thymuses prior to the appearance of thymocytes, and the maturation of thymocytes in those thymuses can be obtained independently of further cell entry (4). Thus we suggested that they represent precursor cells for thymocytes, i.e., “prethymocytes,” which are already committed to the T pathway but need the thymus microenvironment to differentiate further. This was demonstrated by Sato et al. (S), who separated these cells with the use of a fluorescence-activated cell sorter and obtained further differentiation by culture with thymic reticuloepithelial cells. The differentiation of prethymocytees from multipotential stem cells appears to be completely independent of the thymus for it also occurs in nude mice born from homozygous nu/nu parents and examined at birth (6). On the contrary, preliminary results showed that the production of these cells in the bone marrow is submitted to the homeostatic regulation of the thymus, for they appear rapidly in normal mice after surgical deprivation of the thymus and disappear rapidly when athymic mice are grafted with a thymus (3). In the present report we confirm this regulation and show that it is mediated through a humoral pathway and is a unique property of the thymus for it cannot be achieved by other lymphoid organs or cells. lAddress correspondence to Dr. Roelants at the International Animal Diseases, P.O. Box 30709, Nairobi, Kenya.
Laboratory
for Research on
420 Copyright Q 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0008-8749
(1) (2) (3) (1) (2) (3) (1) (2) (3) (4) (I) (2) (3) (4) (1) (2) (3) (4) (1) (2)
No treatment Sham subcutaneous graft Empty Millipore chamber Thymus Lymph node Nu/nu lymph node Thymus Lymph node Nu/nu lymph node Spleen Thymocytes Lymph node cells Nu/nu lymph node cells Spleen cells Thymus Lymph node Nu/nu lymph node Spleen Lymph node cells Nu/nu lymph node cells
TABLE
1
36 5 25 40 5 5 31 22 15 16 IO 10 5 IO 12 10 5 10 1.5 5
8 1 5 8 1 1 8 4 3 4 2 2 1 2 2 2 1 2 3 1
Number of miceb
Number of experiments
in the Spleen of Nude Mice after Various
9.8 8 12.6 2.6 9 6 1.9 6.0 7.3 10.5 15.0 8.0 10 10.5 12.0 13.5 7 13.5 9.3 9
Arithmetic mean6 (%I
Treatments’
1.5 4.1 4.0 6.4 1.4 4.5 4.9 4.2 2.1 9.2 4.7
3.5 3.0 1.5 6.5 3.8
5.2 2.7
2.9
6.5 2.7
1.5
3.5 3.0
3 ;f 5 5 z m
E 1.0 3.2 3.2
0
2.0 2.3
2.3 1.0
1.0
Standard error of the mean’ (%I
chambers or intraafter 7 days.
Standard deviation” (%)
1.4 3.5 3.2 5.6 1.0 3.2
4.1 2.5
2.7
Standard errord (o/o)
a Recipient mice received half a thymus lobe, the lymph node equivalent of one donor, half a spleen, or 50 X lo6 viable cells in Miliipore venously. “Epithelium” was prepared by implanting organs in Millipore chambers into nude mice and transferring them to a new recipient b Some spleens were examined individually, others were pooled. ’ X = Xi/n* d SE = [(xi - g)z/,]‘. e.SD = [(xi - g)z/(, - l)]+. f SERI = [(Xi - S)z/n(n - I)]“.
(D) Cells injected intravenously
(E) “Epithelium” in Millipore chamber
(D) Cells in Millipore chamber
(C) Organs in Millipore chamber
(B) Subcutaneous graft
(A) Controls
Ig-@+ Lymphocytes
422
SHORT
MATERIALS
COMMUNICATIONS
AND
METHODS
The experimental principle was to introduce into BALB/c nude mice (8-12 weeks old ; G. Bomholtgaard, Ltd., Ry, Denmark) various organs or cells (from 4- to S-week-old syngeneic mice) included or not in a cell-impermeable Millipore chamber, GSWPO 1300 with an average pore size of 0.22 pm (instead of the HAWPO 1300 with an average pore size of 0.45 pm used previously) (3) and to look at the spleen 3 to 26 days later for the presence of Ig-B+ cells using immunofluorescence techniques. The life span of Ig-$+weak cells being 1 to 2 days and the reconstitution of peripheral T cells in thymus-grafted nude mice starting only after weeks (7), depression of Ig-8+weak cell production could be easily detected. The characterization and use of rabbit antibody raised against mouse brain-associated 0 antigen made specific by absorption in viva and of AKR anti-BCSH reagents have been reported in detail (l-3, 8). The only slight difference from reported techniques was that the cells were passed through Ficoll-Urovison before staining. RESULTS
AND
DISCUSSION
Results presented in Table 1 show that an intact thymus introduced subcutaneously (group Bl) depresses the production of “prethymocytes.” This effect remains when the thymus is enclosed within a cell-impermeable Millipore chamber (group Cl) showing that it is mediated through one or several humoral factors. The remaining 2.61.9s 8’ cells may represent 8+strongcells known to be present in small quantities in nude mice (1, 2, 9). Other lymphoid organs such as spleen or lymph nodes do not have the same effect. The regulation does not appear to be mediated by the thymocytes’ physiological or degradation products since it did not appear in animals receiving Millipore chambers containing only thymocytes (group Dl). Chambers containing thymuses which were recovered after l-week in viva implantation, a technique used to obtain mainly epithelial tissue (10, ll), were not functional in our assay. This is surprising since left in situ they continue to exert their activity for at least 26 days. Controls of bone marrow and peripheral blood and lymph nodes showed that the reduction of Ig-9+ cells in the spleen of thymus-deprived mice was not due to a mere redistribution of these cells in the organism. The absolute amount of other cell types, i.e., Ig+B and Ig-&, in the spleens of those mice also remained unchanged. In conclusion it appears that the thymus, while not inducing the early commitment of multipotential stem cells to the T lineage (6), exerts a humoral negative homeostatic control on the size of the T-lineage precursor pool. The relationship of this factor with other described thymic hormones (12) remains to be elucidated. REFERENCES 1. Loor, F., and Roelants, G. E., Nature (Lo&on) 251,229, 1974. 2. Roelants, G. E., Loor, F., von Boehmer, H., Sprent, J., Hagg, L. B., Mayor, K. S., and 3. 4. 5. 6.
Ryden, A., Em-. J. Immunol. 5, 127, 1975. Roelants, G. E., Mayor, K. S., Hagg, L. B., and Loor, F., Eur. J. Zmmzlnol. 6, 75, 1976. Owen, J. J. T., and Raff, M. C.,.J. Exp. Med. 132, 1216, 1970. Sato, V. L., Waksal, S. D., and Herzenberg, L. A., Cell. Immunol. 24, 173, 1976. Loor, F., Amstutz, H., Hagg, L. B., Mayor, K. S., and Roelants, G. E., Eur. J. Zmmulzol. 6, 663, 1976.
SHORT
COMMUNICATIONS
423
7. Loor, F., Roelants, G. E., Kindred, B., Mayor, K. S., and Hagg, L. B., In. “Progress in Differentiation Research” (N. Muller-Berat, Ed.), pp. 559-566. North-Holland, Amsterdam, 1976. 8. Loot-, F., and Roelants, G. E., Ann. N. Y. Acad. Sci. 254,226, 1975. 9. Raff, M. C., Nature (London), 246, 350, 1973. 10. Levey, R. H., Trainin, N., and Low, L. W., I. Nat. Cancer. Inst. 31, 199, 1963. 11. Osoba, D., and Miller, J. F. A. P., J. Exp. Med. 119, 177, 1954. 12. Van Bekkum, D. W. (Ed.), “The Biological Activity of Thymic Hormones.” Kooyker Scientific Publication, Rotterdam, 1975.