CELLULAR
IbIiWJNOLOCY
22, 389-393
(1976)
Inhibition of Erythroid Allogeneic Lymphoid
Cell Growth in irradiated Mice by Cells: Specificity of the Response
G~RAN LUNDEGKRDH, Bo LILLIEH~~~K, Department
of Tumor Biology,
Karolinska
AND HENRIC BLOMGREN
Imtitutet,
Received December
S-104 01 Stockholm,
Sweden
15.1975
Allogeneic lymphocytes can inhibit proliferation of erythroid cells in the spleens of irradiated mice grafted with syngeneic bone marrow cells. Since there is a linear relationship between the number of injected lymphocytes and erythroid activity, the method is frequently used as a graft-vs-host assay. In this investigation we show that the reduction of erythroid activity is due to both a reaction of the lymphocytes against the tissues of the host and against the grafted bone marrow cells. Thus, reduced erythropoietic activity does not necessarily indicate that the bone marrow targets possess antigens against which the lymphocytes are reactive.
INTRODUCTION Previously it was observed that parental strain lymphocytes can inhibit the growth of allogeneic bone marrow cells in the spleens of irradiated mice genetically identical with the grafted bone marrow. This reaction can be strongly enhanced by prior sensitization of the lymphocytes against the relevant transplantation antigens. The strongest immunizing effect is obtained by sensitizing the lymphocytes in the spleens of heavily irradiated allogeneic recipients (educated cells). Such lymphocyte populations do not display any detectable reactivity against irrelevant bone marrow targets (1, 2). This finding shows that the response of the educated cells is immunologically specific. However, the mechanism by which the injected lymphocytes inhibit growth of the erythroid cells is unknown; for instance, do they react against the cells of the host, thereby indirectly preventing proliferation of the grafted cells, or do they act directly on the bone marrow cells ? In this investigation we have educated mouse lymphocytes against allogeneic cells and tested their capacity to inhibit growth of relevant or irrelevant bone marrow cells injected into syngeneic or allogeneic hosts. MATERIALS
AND METHODS
Mice. The following inbred mouse strains and their F1 hybrids were used : C57Bl (H-Zb), CBA (H-2k), A/Sri (H-28), A/Sri X C57B1, and CBA x C57Bl. Animals of both sexes, 2-4 months old, were used. Sensitization of Zymphocytes. Lymph node cells, 10’ cells, suspended in a balanced salt solution, were injected iv into 700-R-exposed semiallogeneic recipients. Five days later cell suspensions were prepared from the spleens. Such cells will hereafter be termed educated. Technical details concerning preparation of cell suspensions and X-ray exposure of mice have been described previously (1). 389 Copyright 1976 by Academic Press, Inc. All rights oP reproduction in any form reserved.
390
SHORT
COMMUNICATIONS
Inhibition of evythroid cell growth. This graft-vs-host (GvH) assay has been described before (2). Briefly, mice are exposed to 700 R and then injected iv with 2 X 10” syngeneic bone marrow cells either alone or together with allogeneic lymphocytes. Six days later the animals received 0.2 &i of 5aFe-citrate, and 16 hr later the radioactivity of the spleens is determined. Radioactivity, expressed as counts per minute (cpm), in the spleens of mice which had received bone marrow cells and lymphocytes is related to the values obtained in control mice infused with bone marrow cells only. The results are expressed as log10 percent. The following modifications of the method have been introduced: (i) In some tests the infused bone marrow cells were not syngeneic to the irradiated recipient, When testing the proliferation of C57Bl-derived bone marrow cells in CBA X C57Bl recipients, we injected 8 X lo6 cells; C57Bl bone marrow exhibits an impaired proliferation in CBA X C57Bl hosts, most likely due to histocompatibility resistance (3, 4). In all other tests we used 2 X lo6 cells. (ii) In some tests the infused lymphocytes were syngeneic with the recipient. RESULTS Table 1 shows the capacity of CBA lymphocytes, educated against C57B1, to inhibit growth of various bone marrow cells injected into irradiated CBA x C57Bl or CBA hosts. Growth of CBA x C57B1, C57B1, and CBA bone marrow cells was inhibited on transplantation to irradiated CBA X C57Bl recipients. Such educated cells were also able to inhibit growth of CBA x C57Bl but not CBA bone marrow cells in irradiated CBA hosts. These results were confirmed in analogous tests using A/Sri lymphocytes educated in irradiated A/Sri X C57Bl mice (Table 2). Thus, erythroid cell growth was inhibited when the host animal and/or the bone marrow cells possessed transplantation antigens against which the lymphocytes were sensitized. DISCUSSION The present results indicate that the inhibition of erythroid cell proliferation in the spleens of irradiated allogeneic mice is due to a response against both the host animal and the specific bone marrow target cells. Possibly the lymphocytes destroy specific radioresistant cells in the splenic stroma which creates the inductive microenvironment necessary for the differentiation of the multipotent stem cells into an erythroid pathway (5, 6) or, alternatively, lymphocytes may release toxic factors during reaction with the host antigens (7,s). This investigation shows that the results using this G-v-H assay must be interpreted with caution. For instance, CBA lymphocytes, which have been sensitized in the H-2-compatible but M-antigen-incompatible hybrid C3H x CBA, exhibit strong, specific capacity to inhibit growth of C3H X CBA bone marrow cells injected into C3H X CBA hosts (9), suggesting that not only B cells and macrophages (10-12) but also bone marrow cells may posses M antigens (9). The present study, however, shows that such an interpretation may be wrong. ACKNOWLEDGMENTS The work on which this publication is based was performed pursuant to Contract No. l-CB33868 within the Division of Cancer Biology and Diagnosis, National Cancer Institute, Department of Health, Education and Welfare, Washington, D.C. Grants were also received from the Swedish Cancer Society.
2.000 2.011 1.923 1.674 1.158
f f f f f
0.022 0.037 0.064 0.164 0.146
CBA X C57Bl
of CBA Lymphocytes, C57Bl
(13)c (8) (9) (11) (11)
BMa
X C57Bl
(9) (8) (9) (8)
0.125 0.175 0.061 0.062
f f f f
1.773 1.300 0.989 0.904
BM
recipients
0.070 (10)
C57Bl
TABLE
1
1.809 1.903 1.629 1.089 0.964
f f f f f
2.000 f 0.038 0.068 0.071 0.069 0.060
(5) (13) (14) (13) (8)
0.019 (12)
CBA BM
0.113 (9) 0.140 (7) 0.062 (7) 0.056 (7) 0.069 (7)
0.891 f 0.931 f
BM
1.235 f 1.099 f
X C57Bl
2.000 f
CBA
1.938 zt 0.110 (8) 1.974 f 0.104 (7) 1.988 f 0.081 (7)
0.067 (9)
CBA BM
CBA and
2.000 f
CBA recipients
X C57Bl Mice, to Inhibit Growth of Bone Marrow Cells from Mice of Strains in the Spleens of Irradiated CBA X C57Bl or CBA Recipientsa
2.000 f
CBA
Educated in CBA or their Fr Hybrid
a Mean log percent of 59Fe uptake &SE is shown. BM, bone marrow. * Control mice injected with bone marrow cells only. c Number of mice used.
0* 0.03 0.06 0.12 0.25 0.50 1 .oo
Number of lymphocytes injected (millions)
Capacity
; ?I 3 z z [A
z
8
Ts
a, b and c, Table 1.
0.088 0.076 0.151 0.114 0.083 0.095
a See footnotes
f f i f f zk
2.000 2.285 2.162 1.100 0.596 0.419
X C57Bl
Lymphocytes, C57Bl
A/Sri
of A/%
O= 0.06 0.12 0.25 0.50 1.00 2.00
Number of lymphocytes injected (millions)
Capacity
(12)” (6) (8) (8) (8) (8)
BM5
A/Sri
1.143 0.976 0.941 0.799
2
0.109 (8)
BM
recipients
1.787 1.892 1.504 1.540
zk f zt f
2.000 f
A/Sri
BM
0.117 0.041 0.101 0.087
(8) (7) (8) (8)
2.000 1.888 1.490 1.289 0.965 0.632
A/Sri
BM
f 0.082 (16) f 0.118 (12) f 0.148 (13) f 0.125 (11) f 0.116 (7) f 0.122 (5)
X C57Bl
A/Sri
A/Sri
BM
A/Sri
and
1.973 f 0.118 (6) 1.675 f 0.245 (6) 1.976 f 0.088 (5)
2.000 ). 0.062 (13)
recipients
Growth of Bone Marrow Cells from Mice of Strains AjSn X C57Bl or A/Sri Recipientsa
0.040 (8)
X C57Bl Mice, to Inhibit in the Spleens of Irradiated
f 0.098 (7) f 0.094 (6) f 0.075 (7) f 0.065 (7)
2.000 f
C57Bl
X C57Bl
Educated in A/.% or Their Fr Hybrid
TABLE
SHORT
COMMUNICATIOKS
303
REFERENCES 1. Blomgren, H., and Andersson, B., Cell. I~lz~mtnol. 3, 318, 1972. 2. Blomgren, H., and Andersson, B., Cell. Intmunol. 7, 453, 1973. 3. Cudkowics, G. In “The Proliferation and Spread of Neoplastic Cells,” pp. 661-691. Williams and Wilkins, Baltimore, 1968. 4. Cudkowics, G., and Lotzov’a, E., Transplant. Proc. 5, 1399, 1973. 5. Seno, S., Acta Pathol. Jap. 16, 457, 1966. 6. Curry, J. L., Trentin, J. J., and Wolf, N., J. Exp. Med. 125. 703, 1967. 7. Granger, G. A., and Williams, T. W., Natwe (London) 218, 1253, 1968. 8. Kolb, W. P., and Granger, G. A., Proc. Nat. Acad. Sci. U.S.A. 61, 1250, 1968. 9. LilliehGijk, B., Jacobsson, H., and Blomgren, H., Stand. I. I+mrznnol. 4, 463, 1975. 10. Click, R. E., J. Exp. Med. 139, 1628, 1974. 11. von Boehmer, H., and Sprent, J., Nature (Lowdon) 249, 363, 1974. 12. Schirrmacher, V., Peiia-Martinez, J., and Festenstein, H., Nature (Lorzdon) 255, 155, 1975.
IbIiWJNOLOCY
22, 389-393
(1976)
Inhibition of Erythroid Allogeneic Lymphoid
Cell Growth in irradiated Mice by Cells: Specificity of the Response
G~RAN LUNDEGKRDH, Bo LILLIEH~~~K, Department
of Tumor Biology,
Karolinska
AND HENRIC BLOMGREN
Imtitutet,
Received December
S-104 01 Stockholm,
Sweden
15.1975
Allogeneic lymphocytes can inhibit proliferation of erythroid cells in the spleens of irradiated mice grafted with syngeneic bone marrow cells. Since there is a linear relationship between the number of injected lymphocytes and erythroid activity, the method is frequently used as a graft-vs-host assay. In this investigation we show that the reduction of erythroid activity is due to both a reaction of the lymphocytes against the tissues of the host and against the grafted bone marrow cells. Thus, reduced erythropoietic activity does not necessarily indicate that the bone marrow targets possess antigens against which the lymphocytes are reactive.
INTRODUCTION Previously it was observed that parental strain lymphocytes can inhibit the growth of allogeneic bone marrow cells in the spleens of irradiated mice genetically identical with the grafted bone marrow. This reaction can be strongly enhanced by prior sensitization of the lymphocytes against the relevant transplantation antigens. The strongest immunizing effect is obtained by sensitizing the lymphocytes in the spleens of heavily irradiated allogeneic recipients (educated cells). Such lymphocyte populations do not display any detectable reactivity against irrelevant bone marrow targets (1, 2). This finding shows that the response of the educated cells is immunologically specific. However, the mechanism by which the injected lymphocytes inhibit growth of the erythroid cells is unknown; for instance, do they react against the cells of the host, thereby indirectly preventing proliferation of the grafted cells, or do they act directly on the bone marrow cells ? In this investigation we have educated mouse lymphocytes against allogeneic cells and tested their capacity to inhibit growth of relevant or irrelevant bone marrow cells injected into syngeneic or allogeneic hosts. MATERIALS
AND METHODS
Mice. The following inbred mouse strains and their F1 hybrids were used : C57Bl (H-Zb), CBA (H-2k), A/Sri (H-28), A/Sri X C57B1, and CBA x C57Bl. Animals of both sexes, 2-4 months old, were used. Sensitization of Zymphocytes. Lymph node cells, 10’ cells, suspended in a balanced salt solution, were injected iv into 700-R-exposed semiallogeneic recipients. Five days later cell suspensions were prepared from the spleens. Such cells will hereafter be termed educated. Technical details concerning preparation of cell suspensions and X-ray exposure of mice have been described previously (1). 389 Copyright 1976 by Academic Press, Inc. All rights oP reproduction in any form reserved.
390
SHORT
COMMUNICATIONS
Inhibition of evythroid cell growth. This graft-vs-host (GvH) assay has been described before (2). Briefly, mice are exposed to 700 R and then injected iv with 2 X 10” syngeneic bone marrow cells either alone or together with allogeneic lymphocytes. Six days later the animals received 0.2 &i of 5aFe-citrate, and 16 hr later the radioactivity of the spleens is determined. Radioactivity, expressed as counts per minute (cpm), in the spleens of mice which had received bone marrow cells and lymphocytes is related to the values obtained in control mice infused with bone marrow cells only. The results are expressed as log10 percent. The following modifications of the method have been introduced: (i) In some tests the infused bone marrow cells were not syngeneic to the irradiated recipient, When testing the proliferation of C57Bl-derived bone marrow cells in CBA X C57Bl recipients, we injected 8 X lo6 cells; C57Bl bone marrow exhibits an impaired proliferation in CBA X C57Bl hosts, most likely due to histocompatibility resistance (3, 4). In all other tests we used 2 X lo6 cells. (ii) In some tests the infused lymphocytes were syngeneic with the recipient. RESULTS Table 1 shows the capacity of CBA lymphocytes, educated against C57B1, to inhibit growth of various bone marrow cells injected into irradiated CBA x C57Bl or CBA hosts. Growth of CBA x C57B1, C57B1, and CBA bone marrow cells was inhibited on transplantation to irradiated CBA X C57Bl recipients. Such educated cells were also able to inhibit growth of CBA x C57Bl but not CBA bone marrow cells in irradiated CBA hosts. These results were confirmed in analogous tests using A/Sri lymphocytes educated in irradiated A/Sri X C57Bl mice (Table 2). Thus, erythroid cell growth was inhibited when the host animal and/or the bone marrow cells possessed transplantation antigens against which the lymphocytes were sensitized. DISCUSSION The present results indicate that the inhibition of erythroid cell proliferation in the spleens of irradiated allogeneic mice is due to a response against both the host animal and the specific bone marrow target cells. Possibly the lymphocytes destroy specific radioresistant cells in the splenic stroma which creates the inductive microenvironment necessary for the differentiation of the multipotent stem cells into an erythroid pathway (5, 6) or, alternatively, lymphocytes may release toxic factors during reaction with the host antigens (7,s). This investigation shows that the results using this G-v-H assay must be interpreted with caution. For instance, CBA lymphocytes, which have been sensitized in the H-2-compatible but M-antigen-incompatible hybrid C3H x CBA, exhibit strong, specific capacity to inhibit growth of C3H X CBA bone marrow cells injected into C3H X CBA hosts (9), suggesting that not only B cells and macrophages (10-12) but also bone marrow cells may posses M antigens (9). The present study, however, shows that such an interpretation may be wrong. ACKNOWLEDGMENTS The work on which this publication is based was performed pursuant to Contract No. l-CB33868 within the Division of Cancer Biology and Diagnosis, National Cancer Institute, Department of Health, Education and Welfare, Washington, D.C. Grants were also received from the Swedish Cancer Society.
2.000 2.011 1.923 1.674 1.158
f f f f f
0.022 0.037 0.064 0.164 0.146
CBA X C57Bl
of CBA Lymphocytes, C57Bl
(13)c (8) (9) (11) (11)
BMa
X C57Bl
(9) (8) (9) (8)
0.125 0.175 0.061 0.062
f f f f
1.773 1.300 0.989 0.904
BM
recipients
0.070 (10)
C57Bl
TABLE
1
1.809 1.903 1.629 1.089 0.964
f f f f f
2.000 f 0.038 0.068 0.071 0.069 0.060
(5) (13) (14) (13) (8)
0.019 (12)
CBA BM
0.113 (9) 0.140 (7) 0.062 (7) 0.056 (7) 0.069 (7)
0.891 f 0.931 f
BM
1.235 f 1.099 f
X C57Bl
2.000 f
CBA
1.938 zt 0.110 (8) 1.974 f 0.104 (7) 1.988 f 0.081 (7)
0.067 (9)
CBA BM
CBA and
2.000 f
CBA recipients
X C57Bl Mice, to Inhibit Growth of Bone Marrow Cells from Mice of Strains in the Spleens of Irradiated CBA X C57Bl or CBA Recipientsa
2.000 f
CBA
Educated in CBA or their Fr Hybrid
a Mean log percent of 59Fe uptake &SE is shown. BM, bone marrow. * Control mice injected with bone marrow cells only. c Number of mice used.
0* 0.03 0.06 0.12 0.25 0.50 1 .oo
Number of lymphocytes injected (millions)
Capacity
; ?I 3 z z [A
z
8
Ts
a, b and c, Table 1.
0.088 0.076 0.151 0.114 0.083 0.095
a See footnotes
f f i f f zk
2.000 2.285 2.162 1.100 0.596 0.419
X C57Bl
Lymphocytes, C57Bl
A/Sri
of A/%
O= 0.06 0.12 0.25 0.50 1.00 2.00
Number of lymphocytes injected (millions)
Capacity
(12)” (6) (8) (8) (8) (8)
BM5
A/Sri
1.143 0.976 0.941 0.799
2
0.109 (8)
BM
recipients
1.787 1.892 1.504 1.540
zk f zt f
2.000 f
A/Sri
BM
0.117 0.041 0.101 0.087
(8) (7) (8) (8)
2.000 1.888 1.490 1.289 0.965 0.632
A/Sri
BM
f 0.082 (16) f 0.118 (12) f 0.148 (13) f 0.125 (11) f 0.116 (7) f 0.122 (5)
X C57Bl
A/Sri
A/Sri
BM
A/Sri
and
1.973 f 0.118 (6) 1.675 f 0.245 (6) 1.976 f 0.088 (5)
2.000 ). 0.062 (13)
recipients
Growth of Bone Marrow Cells from Mice of Strains AjSn X C57Bl or A/Sri Recipientsa
0.040 (8)
X C57Bl Mice, to Inhibit in the Spleens of Irradiated
f 0.098 (7) f 0.094 (6) f 0.075 (7) f 0.065 (7)
2.000 f
C57Bl
X C57Bl
Educated in A/.% or Their Fr Hybrid
TABLE
SHORT
COMMUNICATIOKS
303
REFERENCES 1. Blomgren, H., and Andersson, B., Cell. I~lz~mtnol. 3, 318, 1972. 2. Blomgren, H., and Andersson, B., Cell. Intmunol. 7, 453, 1973. 3. Cudkowics, G. In “The Proliferation and Spread of Neoplastic Cells,” pp. 661-691. Williams and Wilkins, Baltimore, 1968. 4. Cudkowics, G., and Lotzov’a, E., Transplant. Proc. 5, 1399, 1973. 5. Seno, S., Acta Pathol. Jap. 16, 457, 1966. 6. Curry, J. L., Trentin, J. J., and Wolf, N., J. Exp. Med. 125. 703, 1967. 7. Granger, G. A., and Williams, T. W., Natwe (London) 218, 1253, 1968. 8. Kolb, W. P., and Granger, G. A., Proc. Nat. Acad. Sci. U.S.A. 61, 1250, 1968. 9. LilliehGijk, B., Jacobsson, H., and Blomgren, H., Stand. I. I+mrznnol. 4, 463, 1975. 10. Click, R. E., J. Exp. Med. 139, 1628, 1974. 11. von Boehmer, H., and Sprent, J., Nature (Lowdon) 249, 363, 1974. 12. Schirrmacher, V., Peiia-Martinez, J., and Festenstein, H., Nature (Lorzdon) 255, 155, 1975.
