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Effect of thymosin on lymphoid cells from human fetal organs
Effect of thymosin on lymphoid from human fetal organs
cells
Shigeyoshi Suzuki, M.D., Osamu Kawano, M.D., Masao Oguri, M.D., Ryuzo Tomidokoro, M.D., and Takayoshi Kuroume, M.D. Mtrdxishi, Gutmu. Juptrn
It is now generally accepted that T-cell differentiation is affected by several thymic factors’, 3. ‘. “‘which are produced by thymic reticuloepithelial cells and are circulating in the blood. However, little is known of the process by which primitive lymphoid cells evolve into functional cells during embryogenesis. Until now, the response of lymphocytes to mitogens”’ or allogeneic cells,” lymphocytotoxicity,‘” or the development of T and B cell populations’. x. ‘-I has been used by various investigators to map the functional development of immunocytes in human fetuses. The present study was designed to elucidate the responsiveness of lymphoid cells from human fetal organs to thymosin. Our results suggest that the fetal spleen plays a crucial role in the development of primitive immunocytes which, by the influence of thymic hormone, develop into mature cells. MATERIALS AND METHODS Thymosin preparation Fraction-V of thymosin was prepared from neonatal calf thymus according to the modified method of Hooper et al.!’
Fresh calf thymus obtained from a local slaughterhousewas homogenized with five volumes of 0.15 M NaCl and centrifuged at 14,000 x g for 60 min. The clear supernatanc was heatedat 80” C for 15 min in a water bath. The heavy precipitate was removed by centrifugation at 14,000 x g. The clear supernate was poured. under stirring, into IO volumes of cold acetone and dried in a desiccator. The resulting powder was suspended in IO volumes of IO mM NaPO, (pH, 7.0) and adjusted to a protein concentration of 25 mgiml. To this solution, a one-third volume of saturated ammonium sulfate (pH, 7.0) was added and stirred for I hr. The supernatant.removedby centrifugation at 10,000 x g for 30 min, was then acidified to pH 4.0 with 10% acetic acid. Solid (NH,) SO, (14.6 gmidl) was added and the
mixture stirred for another hour. The precipitate was dissolved in 10mM tris-HCl buffer (pH, 8.0) at a concentration of 10 mgidl protein, subjected to negative pressure ultrafiltration in a Visking tube, and lyophilized (thymosin-V). Thymosin-V was fractionated on a 3 x 80 cm Sephadex G-25 column equilibrated with deionized water. The active fraction (thymosin-Vl) was adjustedto a protein concentration of 1 mgiml with 0.01 M tric-HCI buffer (PH. 8.0). Human fetal cells Twelve 16- to 24-wk-old therapeutic abortion from
From the Department of Pediatrics, Gunma University, School of Medicine. Supported by Grant No. 248222 from the Japanese Ministry of Education. Culture and Science, and the Ministry of Health. Received for publication Nov. 3, 1978. Accepted for publication June I I, 1979. Reprint requests to: Shigeyoshi Suzuki, M.D., Department of Pediatrics. Cunma University School of Medicine, Maebashi, Gunma. Japan. Vol. 64, No. 6, Part 1, pp. 522-525
human fetuses were obtained by patients giving prior informed
consent. The gestational age was determined from crownto-heel length and expressed as weeks, calculated from the last menstrual period. The fetal thymus. spleen, liver, and kidney, finely teased on stainless steel mesh, were passed through four layers of gauze to obtain a single-cell suspension of each organ.
Cell suspensionsand heparinized peripheral blood were purified
by Ficoll-Conray
density-gradient
0091-6749/79/120522+04$00.40/00
centrifugation
1979 The C.V. Mosby
Co.
VOLUME NUMBER
Thymosin
64 6, PART 1
TABLE I. Percentage
of E-RFC found
Crown-heel length km)
Fetus No.
12.0 14.0 16.0 I’).0 21 .o 22.0 22.5 23.0 24.0 26.5 27.0 28.5
I
2 3 4 5 6 I 8 9 I0 II I2 Mean
among
lymphoid
Thymus
523
cells from human fetal organs
Spleen
Blood
Liver
Kidney
2 9 1 2 I ND 21 5 ND 5 6 5
55 71 ND 73 76 63 83 66 85 75 61 59
ND ND ND 26 30 6 25 28 I9 IO 26 31
ND NC. 24 27 36 35 ND 32 ND 29 25 21
2 12 0 0 2 3 II 2 I 4 6 4
69.7 + 3.1
22.3 2 3.0
28.6 -+ I.9
4.1 t I.2
5.7 c I.9
ND: Not done. TABLE II. Percentage Fetus No.
I 2 3 3 5 6 7 8 Y IO II I2 Mean
of EAC-RFC found among
Crown-heel length (cm)
12.0 14.0 16.0 19.0 21.0 22.0 22.5 23.0 24.0 26.5 27.0 28.5
Thymus
lymphoid
cells from human fetal organs
Spleen
Blood
Liver
32 56 ND 46 I2 56 55 33 5 37 61 42
ND ND 5 8 4 I5 24 3 II 6 31 34
ND ND I 7 I4 I2 ND 21 ND II I8 28
24 IO I 6 II 4 5 0 7 I 3 32
39.5 ? 5.5
14.1 23.7
II.3 t 2.9
8.7 i- 3.1
Kidney
5 5 ND 3 0 ND I4 I ND 8 9 x 6.6 t I.5
ND: Not done
according to the method of BGyum.” To deplete the adherent cells, cells were suspended in medium containing 50% fetal calf serum (FCS) and incubated for 30 min at 37” C on glass dishes. More than 95% of the separated cells did not take up trypan blue. All manipulations were performed under aseptic conditions.
incubation
of cells with thymosin-VI
Preliminary experiments revealed that 100 pg of thymosin-VI per 5 x IO” cells represented the optimal dose for increasing the erythrocyte rosette formation of peripheral blood lymphocytes from immunodeficient patients. Therefore, 5 x IO” cells/O. I ml medium were incubated with 100 pg of thymisin-VI for 30 min at 37” C, then washed twice and resuspended in 0.1 ml medium. For the control, cells were incubated in medium.
E-rosette
formation
The percentage of E-RFC in the lymphocyte preparation was determined by the method of spontaneous rosette formation, using sheep red blood cells (SRBC) treated with 50 pg neuraminidase (Behringwerke AC, Milano. Italy) per I x IOx SRBC. The neuraminidase-treated SRBC were suspended in FCS absorbed beforehand with I x IOx SRBUml. Lymphocyte (5 x IO”iml) and SRBC suspensions (100 ~1 each) were mixed at 37” C for I5 min, centrifuged at I50 x g for 5 min. and further incubated in ice water for I hr.
EAC-rosette
formation
EAC-RFC were detected by the following method. Equal volumes of thrice-washed SRBC suspended in Hanks’ balanced salt solution (HBSS) at 2 x IO’ cells/ml and IgM rabbit anti-SRBC antibody at subagglutination dilution were
524
Suzuki et
J ALLERGY
al.
CLIN. IMMUNOL DECEMBER 1979
100 .
0
Control
@$$$ Thymosin-treated Control &\$
Thymosin-treated
50
0 Spleen
Thymus
Blood
Liver
Kidney
FIG. 1. The change in E-RFC population of lymphoid cells from human fetal organs following incubation with 100 pg thymosin-VI. The increase of E-RFC in spleen cell preparations was statistically significant from the basal value (p < 0.05).
Thymus
following
of E-RFC
in fetal
with
thymosin-Vl
incubation
spleen
cells
Fetus No.
Control (%I
Treated with 100 pg thymosin-Vl(%)
‘I 5 6 x 9 IO II I2
26 30 6 28 I9 IO 26 31
3.5 40 22 39 61 I3 31 38
Mea”:’ SE
22 3.33
36 4.96
*p < 0.05 tar difi’erence between the means (Student’s t test).
incubated for 30 min at 30” C. After thorough washing. the erythrocytes were sensitized with human AB-positive or heat-denatured serum previously absorbed with SRBC at a final dilution of 1 : 50 as a source of complement. EAC were washed four times, and a suspension of 1 x IO’ cells/ml of HBSS was prepared. EAC and lymphoid cell suspensions t 100 ~1 each) were incubated with shaking at 37” C for I hr and allowed to settle for I hr at room temperature. To exclude the possibility that we were measuring EA,,,, rosettes rather than EAC rosettes. we confirmed that the SRBC (EA,,,,) sensitized with heated AB serum did not form rosettes with lymphocytes.
Blood
Liver
Kidney
FIG. 2. The change of EAC-RFC population of lymphoid cells from human fetal organs following incubation with 100 pg thymosin-Vl.
Rosette TABLE III. Change
Spleen
determination
The reaction mixture was gently resuspended. Cells that did and did not form rosettes were counted in a hemocytometer using a microscope at ~400 magnification. The percentage of RFC was calculated by counting at least 200 nucleated ceils. Only those cells that attached more than three erythrocytes were considered as rosette-positive.
Statistical
evaluation
RFC populations in thymosin-treated and thymosinuntreated lymphocytes were submitted to Student’s t test to determine whether there was a statistical significance to the difference.
RESULTS Tables I and II summarize the percentage of E-RFC and EAC-RFC found among lymphoid ceils from various organs of 12 human fetuses. Thymus cells showed the highest percentage of E-RFC and EACRFC, and their means were 69.7 -C 3.1% and 39.5 + .55%, respectively. E-RFC and EAC-RFC populations varied from fetus to fetus, and there was no correlation with crown-to-heel length. In the thymus, spleen, and blood, the mean percentages of E-RFC were higher than those of EAC-RFC, while EAC-RFC were predominant in the liver and kidney. This finding represents indirect evidence that E-RFC and EAC-RFC in the liver and kidney were not due to peripheral blood contamination. Furthermore, we observed the change in E-RFC and EAC-RFC populations following incubation of
VOLUME NUMBER
6,J 6, PART 1
the fetal cells with 100 /.~g thymosin-Vl. Incubation with thymosin-VI did not increase E-RFC in preparations from any of the organs, however, with the exception of spleen cell preparations, where the E-RFC was raised from 22% basal level to 36% (p < 0.05) (Fig. 1 and Table III). On the other hand, EAC-RFC was increased 1% to 8% by incubation with thymosin-VI in all preparation, but the increase was not statistically significant (Fig. 2).
Thymosin
2. Bach J-F, Dardenne
3. 4.
5.
DISCUSSION It is now generally accepted that several thymic factors play a role in the final differentiation of cells with a propensity to enter the T-cell pathway. The in vitro transformation in the presence of thymic factors of immature human lymphoid cells from peripheral blood of patients with immune deficiencylx or from bone marrow’. !‘. ‘K Ifi and cord blood’. 4 into mature T lymphocytes has been reported. Our investigation demonstrated that E-RFC populations of fetal spleen cells significantly increased following the incubation with 100 wg thymosin-Vl. Aiuti et al.’ reported that incubation with thymic factor prepared from young pig thymus resulted in a significantly greater E-RFC increase in lymph node cells than in those from fetal spleen or thymus. Their investigation, however, was based on a single 16wk-old fetus and thus does not permit a final conclusion. The cultivation of nude mouse spleen cells on a thymic reticuloendothelial cell monolayer induced a marked increase in characteristic T cell reactivity .li and nude mouse spleen was suggested to contain T lineage lymphocytes.” If we can apply the same rule to the human fetus, it is conceivable that the fetal spleen plays a crucial role in the development of primitive immunocytes and that this development is affected by thymic hormone. Furthermore, there is a strong possibility that lymphocytes are transported from the spleen to the thymus. We thank Mr. Kinichi Kobayashi for his excellent technical assistance. We are also grateful to Mrs. Reiko Ikawa for typing the manuscript. REFERENCES I. Aiuti F. Schirrmacher V. Ammirati P, Fiorilli M: Effect of thymus factor on human precursor T lymphocytes. Clin Exp Immunol 30:499. 1975.
6. 7.
8.
9.
10.
I I. 12.
13.
14.
15. 16.
17.
18.
M. Pleau JM, Bach M-A:
Isolation.
525
bio-
chemical characteristics, and biological activity of a circulating thymic hormone in the mouse and in the human. Ann NY Acad Sci 249:186, 1975. Boyum A: Separation of leukocytes from blood and bone matrow. Stand J Clin Lab Invest 21(suppl.):97. 1968. Fiorilli M. Aiuti F. Ammirdti P. Luzi G. Schirrmacher V: Effect of thymic factor on human lymphoid cells of umbilical cord blood and of children with T cell deticiency. Int Atrh Allergy Appl lmmunol 53:242. 1977. Goldstein AL. Slater FD, White A: Preparation. assay. and partial puritication of a thymic Iymphocytopoietic factot (thymosin). Proc Nat1 Acad Sci USA 56:lOlO. 1966. Goldstein G: Isolation of bovine thymourn: A polypeptide hormone of the thymus. Nature 247: I 1, 1974. Hayward AR. Ever G: Development of lymphocyte populations in the human foetal thymus and spleen. Clin Exp Immunol 17:169. 1976. Hayward AR. Graham L: Increased E-rosette formation by foetal liver and spleen cells incubated with theophylline. Clin Exp Immunol 23:279. 1976. Hooper JA. McDaniel MC, Thurman GB. Cohen GH. Schulof RS. Goldstein AL: Purification and properties ot hovinc thymoain. Ann NY Acad Sci 249:125. 1975. Kook Al, Yakir Y, Trainin N: Isolation and partial chemical characterization of THF, a thymus hormone involved in immune maturation of lymphoid cells. Cell Immunol 19:lSl. 1975. Loor F. Rtrlants GE: High frequency of T lineage lymphocyte in nude mouse spleen. Nature 251:229. 1974. Pirofsky B, Davies GH, Ramirez-Mateoa JC, Newton BW: Cellular immune competence in the human fetus. Cell Immunol 6~257. 1973. Stites DP. Carr MC, Fudenberg HH: Ontogeny of cellular immunity in the human fetus. Development of responses to phytohemagglutinin and to allogenic cells. Cell Immunol 11:257. 1974. Stites DP, Wybran 1. Carr MC, Fudenberg HH: Development of cellular immune competence in man, in Ontogeny of acquired immunity. Ciba Foundation Symposium, Amsterdam, 1972, Elsevier, p. 113. Timonen T, Saksela E: Cell-mediated anti-embryo cytotoxicity in human pregnancy. Clin Exp lmmunol 23:462, 1976. Touraine JL, Lncefy GS, Touraine F. Rho YM, Good RA: Differentiation of human bone marrow cell\ into T Iymphocytes by in vitro incubation with thymic extracts, Clin Exp Immunol 17:151, 1974. Wakaal SD, Gohen IR. Waksal HW, St. Pierre RL: In vitro differentiation of T cells. Interaction of T cell precursors and thymus epithelium. Fed Proc 33:736, 1974. Wara DW, Goldstein AL, Dovle NE. Ammann AJ: Thymosin activity in patients with cellular immunodeticiency. New Engl J Med 292:70, 197.5.
cells
Shigeyoshi Suzuki, M.D., Osamu Kawano, M.D., Masao Oguri, M.D., Ryuzo Tomidokoro, M.D., and Takayoshi Kuroume, M.D. Mtrdxishi, Gutmu. Juptrn
It is now generally accepted that T-cell differentiation is affected by several thymic factors’, 3. ‘. “‘which are produced by thymic reticuloepithelial cells and are circulating in the blood. However, little is known of the process by which primitive lymphoid cells evolve into functional cells during embryogenesis. Until now, the response of lymphocytes to mitogens”’ or allogeneic cells,” lymphocytotoxicity,‘” or the development of T and B cell populations’. x. ‘-I has been used by various investigators to map the functional development of immunocytes in human fetuses. The present study was designed to elucidate the responsiveness of lymphoid cells from human fetal organs to thymosin. Our results suggest that the fetal spleen plays a crucial role in the development of primitive immunocytes which, by the influence of thymic hormone, develop into mature cells. MATERIALS AND METHODS Thymosin preparation Fraction-V of thymosin was prepared from neonatal calf thymus according to the modified method of Hooper et al.!’
Fresh calf thymus obtained from a local slaughterhousewas homogenized with five volumes of 0.15 M NaCl and centrifuged at 14,000 x g for 60 min. The clear supernatanc was heatedat 80” C for 15 min in a water bath. The heavy precipitate was removed by centrifugation at 14,000 x g. The clear supernate was poured. under stirring, into IO volumes of cold acetone and dried in a desiccator. The resulting powder was suspended in IO volumes of IO mM NaPO, (pH, 7.0) and adjusted to a protein concentration of 25 mgiml. To this solution, a one-third volume of saturated ammonium sulfate (pH, 7.0) was added and stirred for I hr. The supernatant.removedby centrifugation at 10,000 x g for 30 min, was then acidified to pH 4.0 with 10% acetic acid. Solid (NH,) SO, (14.6 gmidl) was added and the
mixture stirred for another hour. The precipitate was dissolved in 10mM tris-HCl buffer (pH, 8.0) at a concentration of 10 mgidl protein, subjected to negative pressure ultrafiltration in a Visking tube, and lyophilized (thymosin-V). Thymosin-V was fractionated on a 3 x 80 cm Sephadex G-25 column equilibrated with deionized water. The active fraction (thymosin-Vl) was adjustedto a protein concentration of 1 mgiml with 0.01 M tric-HCI buffer (PH. 8.0). Human fetal cells Twelve 16- to 24-wk-old therapeutic abortion from
From the Department of Pediatrics, Gunma University, School of Medicine. Supported by Grant No. 248222 from the Japanese Ministry of Education. Culture and Science, and the Ministry of Health. Received for publication Nov. 3, 1978. Accepted for publication June I I, 1979. Reprint requests to: Shigeyoshi Suzuki, M.D., Department of Pediatrics. Cunma University School of Medicine, Maebashi, Gunma. Japan. Vol. 64, No. 6, Part 1, pp. 522-525
human fetuses were obtained by patients giving prior informed
consent. The gestational age was determined from crownto-heel length and expressed as weeks, calculated from the last menstrual period. The fetal thymus. spleen, liver, and kidney, finely teased on stainless steel mesh, were passed through four layers of gauze to obtain a single-cell suspension of each organ.
Cell suspensionsand heparinized peripheral blood were purified
by Ficoll-Conray
density-gradient
0091-6749/79/120522+04$00.40/00
centrifugation
1979 The C.V. Mosby
Co.
VOLUME NUMBER
Thymosin
64 6, PART 1
TABLE I. Percentage
of E-RFC found
Crown-heel length km)
Fetus No.
12.0 14.0 16.0 I’).0 21 .o 22.0 22.5 23.0 24.0 26.5 27.0 28.5
I
2 3 4 5 6 I 8 9 I0 II I2 Mean
among
lymphoid
Thymus
523
cells from human fetal organs
Spleen
Blood
Liver
Kidney
2 9 1 2 I ND 21 5 ND 5 6 5
55 71 ND 73 76 63 83 66 85 75 61 59
ND ND ND 26 30 6 25 28 I9 IO 26 31
ND NC. 24 27 36 35 ND 32 ND 29 25 21
2 12 0 0 2 3 II 2 I 4 6 4
69.7 + 3.1
22.3 2 3.0
28.6 -+ I.9
4.1 t I.2
5.7 c I.9
ND: Not done. TABLE II. Percentage Fetus No.
I 2 3 3 5 6 7 8 Y IO II I2 Mean
of EAC-RFC found among
Crown-heel length (cm)
12.0 14.0 16.0 19.0 21.0 22.0 22.5 23.0 24.0 26.5 27.0 28.5
Thymus
lymphoid
cells from human fetal organs
Spleen
Blood
Liver
32 56 ND 46 I2 56 55 33 5 37 61 42
ND ND 5 8 4 I5 24 3 II 6 31 34
ND ND I 7 I4 I2 ND 21 ND II I8 28
24 IO I 6 II 4 5 0 7 I 3 32
39.5 ? 5.5
14.1 23.7
II.3 t 2.9
8.7 i- 3.1
Kidney
5 5 ND 3 0 ND I4 I ND 8 9 x 6.6 t I.5
ND: Not done
according to the method of BGyum.” To deplete the adherent cells, cells were suspended in medium containing 50% fetal calf serum (FCS) and incubated for 30 min at 37” C on glass dishes. More than 95% of the separated cells did not take up trypan blue. All manipulations were performed under aseptic conditions.
incubation
of cells with thymosin-VI
Preliminary experiments revealed that 100 pg of thymosin-VI per 5 x IO” cells represented the optimal dose for increasing the erythrocyte rosette formation of peripheral blood lymphocytes from immunodeficient patients. Therefore, 5 x IO” cells/O. I ml medium were incubated with 100 pg of thymisin-VI for 30 min at 37” C, then washed twice and resuspended in 0.1 ml medium. For the control, cells were incubated in medium.
E-rosette
formation
The percentage of E-RFC in the lymphocyte preparation was determined by the method of spontaneous rosette formation, using sheep red blood cells (SRBC) treated with 50 pg neuraminidase (Behringwerke AC, Milano. Italy) per I x IOx SRBC. The neuraminidase-treated SRBC were suspended in FCS absorbed beforehand with I x IOx SRBUml. Lymphocyte (5 x IO”iml) and SRBC suspensions (100 ~1 each) were mixed at 37” C for I5 min, centrifuged at I50 x g for 5 min. and further incubated in ice water for I hr.
EAC-rosette
formation
EAC-RFC were detected by the following method. Equal volumes of thrice-washed SRBC suspended in Hanks’ balanced salt solution (HBSS) at 2 x IO’ cells/ml and IgM rabbit anti-SRBC antibody at subagglutination dilution were
524
Suzuki et
J ALLERGY
al.
CLIN. IMMUNOL DECEMBER 1979
100 .
0
Control
@$$$ Thymosin-treated Control &\$
Thymosin-treated
50
0 Spleen
Thymus
Blood
Liver
Kidney
FIG. 1. The change in E-RFC population of lymphoid cells from human fetal organs following incubation with 100 pg thymosin-VI. The increase of E-RFC in spleen cell preparations was statistically significant from the basal value (p < 0.05).
Thymus
following
of E-RFC
in fetal
with
thymosin-Vl
incubation
spleen
cells
Fetus No.
Control (%I
Treated with 100 pg thymosin-Vl(%)
‘I 5 6 x 9 IO II I2
26 30 6 28 I9 IO 26 31
3.5 40 22 39 61 I3 31 38
Mea”:’ SE
22 3.33
36 4.96
*p < 0.05 tar difi’erence between the means (Student’s t test).
incubated for 30 min at 30” C. After thorough washing. the erythrocytes were sensitized with human AB-positive or heat-denatured serum previously absorbed with SRBC at a final dilution of 1 : 50 as a source of complement. EAC were washed four times, and a suspension of 1 x IO’ cells/ml of HBSS was prepared. EAC and lymphoid cell suspensions t 100 ~1 each) were incubated with shaking at 37” C for I hr and allowed to settle for I hr at room temperature. To exclude the possibility that we were measuring EA,,,, rosettes rather than EAC rosettes. we confirmed that the SRBC (EA,,,,) sensitized with heated AB serum did not form rosettes with lymphocytes.
Blood
Liver
Kidney
FIG. 2. The change of EAC-RFC population of lymphoid cells from human fetal organs following incubation with 100 pg thymosin-Vl.
Rosette TABLE III. Change
Spleen
determination
The reaction mixture was gently resuspended. Cells that did and did not form rosettes were counted in a hemocytometer using a microscope at ~400 magnification. The percentage of RFC was calculated by counting at least 200 nucleated ceils. Only those cells that attached more than three erythrocytes were considered as rosette-positive.
Statistical
evaluation
RFC populations in thymosin-treated and thymosinuntreated lymphocytes were submitted to Student’s t test to determine whether there was a statistical significance to the difference.
RESULTS Tables I and II summarize the percentage of E-RFC and EAC-RFC found among lymphoid ceils from various organs of 12 human fetuses. Thymus cells showed the highest percentage of E-RFC and EACRFC, and their means were 69.7 -C 3.1% and 39.5 + .55%, respectively. E-RFC and EAC-RFC populations varied from fetus to fetus, and there was no correlation with crown-to-heel length. In the thymus, spleen, and blood, the mean percentages of E-RFC were higher than those of EAC-RFC, while EAC-RFC were predominant in the liver and kidney. This finding represents indirect evidence that E-RFC and EAC-RFC in the liver and kidney were not due to peripheral blood contamination. Furthermore, we observed the change in E-RFC and EAC-RFC populations following incubation of
VOLUME NUMBER
6,J 6, PART 1
the fetal cells with 100 /.~g thymosin-Vl. Incubation with thymosin-VI did not increase E-RFC in preparations from any of the organs, however, with the exception of spleen cell preparations, where the E-RFC was raised from 22% basal level to 36% (p < 0.05) (Fig. 1 and Table III). On the other hand, EAC-RFC was increased 1% to 8% by incubation with thymosin-VI in all preparation, but the increase was not statistically significant (Fig. 2).
Thymosin
2. Bach J-F, Dardenne
3. 4.
5.
DISCUSSION It is now generally accepted that several thymic factors play a role in the final differentiation of cells with a propensity to enter the T-cell pathway. The in vitro transformation in the presence of thymic factors of immature human lymphoid cells from peripheral blood of patients with immune deficiencylx or from bone marrow’. !‘. ‘K Ifi and cord blood’. 4 into mature T lymphocytes has been reported. Our investigation demonstrated that E-RFC populations of fetal spleen cells significantly increased following the incubation with 100 wg thymosin-Vl. Aiuti et al.’ reported that incubation with thymic factor prepared from young pig thymus resulted in a significantly greater E-RFC increase in lymph node cells than in those from fetal spleen or thymus. Their investigation, however, was based on a single 16wk-old fetus and thus does not permit a final conclusion. The cultivation of nude mouse spleen cells on a thymic reticuloendothelial cell monolayer induced a marked increase in characteristic T cell reactivity .li and nude mouse spleen was suggested to contain T lineage lymphocytes.” If we can apply the same rule to the human fetus, it is conceivable that the fetal spleen plays a crucial role in the development of primitive immunocytes and that this development is affected by thymic hormone. Furthermore, there is a strong possibility that lymphocytes are transported from the spleen to the thymus. We thank Mr. Kinichi Kobayashi for his excellent technical assistance. We are also grateful to Mrs. Reiko Ikawa for typing the manuscript. REFERENCES I. Aiuti F. Schirrmacher V. Ammirati P, Fiorilli M: Effect of thymus factor on human precursor T lymphocytes. Clin Exp Immunol 30:499. 1975.
6. 7.
8.
9.
10.
I I. 12.
13.
14.
15. 16.
17.
18.
M. Pleau JM, Bach M-A:
Isolation.
525
bio-
chemical characteristics, and biological activity of a circulating thymic hormone in the mouse and in the human. Ann NY Acad Sci 249:186, 1975. Boyum A: Separation of leukocytes from blood and bone matrow. Stand J Clin Lab Invest 21(suppl.):97. 1968. Fiorilli M. Aiuti F. Ammirdti P. Luzi G. Schirrmacher V: Effect of thymic factor on human lymphoid cells of umbilical cord blood and of children with T cell deticiency. Int Atrh Allergy Appl lmmunol 53:242. 1977. Goldstein AL. Slater FD, White A: Preparation. assay. and partial puritication of a thymic Iymphocytopoietic factot (thymosin). Proc Nat1 Acad Sci USA 56:lOlO. 1966. Goldstein G: Isolation of bovine thymourn: A polypeptide hormone of the thymus. Nature 247: I 1, 1974. Hayward AR. Ever G: Development of lymphocyte populations in the human foetal thymus and spleen. Clin Exp Immunol 17:169. 1976. Hayward AR. Graham L: Increased E-rosette formation by foetal liver and spleen cells incubated with theophylline. Clin Exp Immunol 23:279. 1976. Hooper JA. McDaniel MC, Thurman GB. Cohen GH. Schulof RS. Goldstein AL: Purification and properties ot hovinc thymoain. Ann NY Acad Sci 249:125. 1975. Kook Al, Yakir Y, Trainin N: Isolation and partial chemical characterization of THF, a thymus hormone involved in immune maturation of lymphoid cells. Cell Immunol 19:lSl. 1975. Loor F. Rtrlants GE: High frequency of T lineage lymphocyte in nude mouse spleen. Nature 251:229. 1974. Pirofsky B, Davies GH, Ramirez-Mateoa JC, Newton BW: Cellular immune competence in the human fetus. Cell Immunol 6~257. 1973. Stites DP. Carr MC, Fudenberg HH: Ontogeny of cellular immunity in the human fetus. Development of responses to phytohemagglutinin and to allogenic cells. Cell Immunol 11:257. 1974. Stites DP, Wybran 1. Carr MC, Fudenberg HH: Development of cellular immune competence in man, in Ontogeny of acquired immunity. Ciba Foundation Symposium, Amsterdam, 1972, Elsevier, p. 113. Timonen T, Saksela E: Cell-mediated anti-embryo cytotoxicity in human pregnancy. Clin Exp lmmunol 23:462, 1976. Touraine JL, Lncefy GS, Touraine F. Rho YM, Good RA: Differentiation of human bone marrow cell\ into T Iymphocytes by in vitro incubation with thymic extracts, Clin Exp Immunol 17:151, 1974. Wakaal SD, Gohen IR. Waksal HW, St. Pierre RL: In vitro differentiation of T cells. Interaction of T cell precursors and thymus epithelium. Fed Proc 33:736, 1974. Wara DW, Goldstein AL, Dovle NE. Ammann AJ: Thymosin activity in patients with cellular immunodeticiency. New Engl J Med 292:70, 197.5.