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Delayed type hypersensitivity responses to radiation leukemia virus variants
CELLULAR
IMMUNOLOGY
70, 196- 199 ( 1982)
Delayed Type Hypersensitivity Responses Virus Variants’
to Radiation
Leukemia
GIDEON STRASSMANNAND NECHAMA HARAN-GHERA Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel Received December 17, 1981; accepted April 6, 1982 DTH responseswere evaluated in different strains of mice shown to be resistant or sensitive to leukemogenesis by the radiation leukemia virus variants A-RadLV and D-RadLV. A significant responsewas observed only in the H-2 complex-linked resistant haplotypes to RadLV leukemogenesis. The DTH response could be transferred by immune cells of mice resistant to the appropriate RadLV variant. Thus, an inverserelationship between the leukemogenic activity of the virus and its immunization ability expressed by DTH responsewas demonstrated in different mouse strains.
INTRODUCTION Leukemogenicity to RadLV’ variants, D-RadLV and A-RadLV (1, 2), was shown to be associated with Z region linked genes of the H-2 complex (3). A survey of the H-2 haplotypes associated with resistance or sensitivity to leukemia development indicated that D-RadLV and A-RadLV induce opposite resistance patterns in H-2’ and H-2” hosts, H-2’ mice being sensitive to A-RadLV leukemogenesis and resistant to D-RadLV (the reverse in H-2” mice). The resistant haplotypes were shown to harbor preleukemic cells, their leukemogenic potential demonstrable only upon transfer into appropriate recipients (4). Thus, in the resistant hosts the proliferation of these preleukemic cells into overt leukemia is “arrested.” The resistant haplotype can be immunized by intrathymic virus infection and rejects syngeneic leukemic cell grafts whereas the sensitive haplotype does not (5). This immunity can be transferred by lymphoid cells (4, 6) and can be established by preleukemic bone marrow cells as the immunogen (7). It is still unresolved whether the preleukemic cells in resistant and sensitive haplotypes are similar. The Zr-controlled immune responsivenessmight involve the function of different H-2 defined presentations by similar target cells or by different target cells, having different immunogenicity. Previous findings have indicated that in BL/6 mice A-RadLV has an immunosuppressive effect on the T-cell lineage (helper cells) whereas D-RadLV abrogates the function of lymphoid precursors in the bone marrow (2,8). It seemed therefore of interest to further elucidate the T-cell-dependent component of the r This investigation was supported by PHS Grant lRO1 CA-29657-01 awarded by the National Cancer Institute, DHHS. 2 Abbreviations used: RadLV, radiation leukemia virus; A-RadLV, autonomous RadLV; D-RadLV, dependent RadLV; H-2, major histocompatibility complex of the mouse; DTH, delayed-type hypersensitivity; N-CFC, normal cell free centrifugate; R, right; L, left; ‘%JdR , s-[ ‘251]iodo-2’-deoxyuridine; PPD, purified protein derivative; CFA, complete Freund’s adjuvant. 196 OOO8-8749/82/090196-04$02.00/O Copyright Q 1982 by Acdemic Prm~. Inc. All rights of reproduction ia any form memd.
SHORT
COMMUNICATIONS
197
immune reaction elicited by the RadLV variants. The delayed-type hypersensitivity (DTH) response in the mouse appears to be mediated by T lymphocytes and is transferable to normal mice by antigen-activated T lymphocytes. DTH responses to a wide variety of antigens can be enhanced by pretreatment with cyclophosphamide which affects suppressor cells (9). Since the sensitivity to RadLV leukemogenesis seems to be related with thymocyte-virus interaction (2), it seemed of interest to compare DTH responsesto RadLV variants in strains of mice resistant or sensitive to leukemogenesis following intrathymic virus inoculation in adults. Thus, mice were sensitized with A-RadLV or D-RadLV and the DTH response was measured following virus challenge. MATERIALS AND METHODS Mice. C57BL/6, BlO.S, and BIO.A mice, 2-3 months old, were obtained from the Breeding Center of our Institute. Virus. Both virus variants were prepared from thymic lymphomas induced in C57BL/6 mice by D-RadLV or A-RadLV as described previously (1). The same virus batches were used throughout the experiments. Immunization procedure. Mice were injected subcutaneously with 200 mg/kg cyclophosphamide (Sigma, St. Louis, MO.). Two days later each mouse was immunized with 200 ~1 of 1:1 dilution of virus preparation in complete Freund’s adjuvant (Difco, Detroit, Mich.) in four footpads. DTH measurement. This technique was originally described by Vadas et al. (10). Five days following immunization, mice were challenged with 10 ~1 of either virus preparation or cell free centrifugate from normal thymus (N-CFC), or purified protein derivative (PPD 1 mg/ml, Statens Seruminstitute Co., Denmark) in the right (R) ear. The left (L) ear was injected with phosphate-buffered saline. Ten hours following challenge mice received 5-fluorodeoxyuridine and 20 min later 2 PCi of 12’1-5-iodo-2’-deoxyuridine ( 1251UdR,Radiochemical Center, Amersham). Ears were cut 24-30 hr after challenge and counted in a gamma counter. The results are expressed as the ratio of radioactivity in the right ear to that of the left ear (R/L ‘251UdR index). The results are expressed as the arithmetic mean of all mice in the group + standard error. P values were calculated by the Student’s t test. Transfer of DTH. Five days following the immunization protocol, 50 X lo6 spleen cells of C57BL/6 mice sensitized to either D- or A-RadLV were transferred intravenously into syngeneic recipients. RESULTS AND DISCUSSION In order to find out whether there is any relationship between the leukemogenic activity of RadLV variants and the ability to develop DTH reactions, different mice sensitive or resistant to RadLV variants were immunized to either A-RadLV or D-RadLV and 5 days later assayed for DTH manifestations. As demonstrated in Table 1, C57BL/6 (H-2b) mice were able to develop DTH responsesto D-RadLV (to which they are resistant) but not to A-RadLV (to which they are sensitive). The same experiment was repeated three times, using different batches of A- and D-RadLV, in order to exclude the possibility of batch dependency of the reactions. Hence the results of ‘these experiments were found to be similar to those presented in Table 1.
N-CFC A-RadLV
N-CFC
D-RadLV A-RadLV
A-RadLV
5 5
5
5 11
11
mice tested
No. of
-
-
+
Resistance’
1.18 f 0.07 1.10 + 0.09
1.45 + 0.14
1.50 2 0.09 1.51 f 0.08
2.56 e.O.32’ i P < 0.01
Index
4
5 11
lib
No. of mice tested
-
_ +
-
Resistance”
Index
1.03 + 0.05
BIOS
’ Resistance to leukemia development following virus inoculation into the thymus of adult mice. b Significant difference between the first and the third groups.
D-RadLV A-RadLV
D-RadLV
D-RadLV
-
Antigen challenge
Sensitizing virus
C57BL/6
DTH response (R/L ‘*‘IUdR index + SE)
DTH Responses in Resistant and Sensitive Strains to Variants of RadLV
TABLE 1
5
4 6
6
No. of mice tested
-
-
-
Resistance”
Bl0.A
1.04 + 0.07
1.00 -+ 0.08 1.16 + 0.08
1.02 + 0.04
Index
z VI
G 2
K 2
8
m _-
199
SHORT COMMUNICATIONS TABLE 2 Transfer of DTH Responses by Immune Splenocytes in C57BL/6 Recipients” Sensitizng virus
Challenging virus
D-RadLV D-RadLV A-RadLV A-RadLV
D-RadLV A-RadLV A-RadLV D-RadLV
R/L ‘*%IdR index f SE 1.90 f 1.98 f 1.24 f 1.26 f
O.lb 0.096 0.08 0.1
’ Each mouse received 50 X lo6 RadLV immunized splenocytes. Five mice were tested per group. b Significant difference from the last group: P < 0.01.
On the other hand, BIO.S mice that were shown to be resistant to A-RadLV and sensitive to D-RadLV leukemogenesis responded only to A-RadLV sensitization and challenge, but not to D-RadLV. In contrast to these two mouse strains, BIO.A mice (H-2”) were found to be sensitive to both virus variants (3). As shown in Table 1 no DTH response was observed in BIO.A mice to both A-RadLV and D-RadLV. Under the same experimental conditions BIO.A were tested for their ability to respond to PPD which is the major antigenic determinant of CFA (data not shown in the table). Thus, when BIO.A mice were immunized with either Aor D-RadLV emulsified in CFA, significant DTH responsesto PPD were monitored (R/L ‘*‘IUdR indexes 1.93 f 0.21 and 2.0 + 0.3, respectively). The transfer of DTH reactions was tested by pretreating C57BL/6 mice with cyclophosphamide and then immunizing them to either D or A-RadLV. Five days following immunization 50 X lo6 splenocytes of these immunized mice were transferred into syngeneic recipients and their ability to mediate DTH reaction was further tested. DTH responsescould be transferred by immune cells from C57BL/ 6-D-RadLV-treated mice only (not from A-RadLV-sensitive treated donors) (Table 2). The immunological cross reactivity between leukemic cells induced by both variants, demonstrated already in previous studies (5), was confirmed in this experiment using the DTH response (Table 2). Thus, the DTH response to RadLV variants seemsto be correlated with resistance to RadLV leukemogenesis afforded by the proliferation arrest of preleukemic cells (4). Sensitivity to RadLV leukemogenesis related to thymocyte-virus interaction (8) on the other hand seems to prevent DTH responses. REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9. 10.
Haran-Ghera, N., Inr. J. Cancer 1, 81, 1966. Haran-Ghera, Ben-Yaakov, M., and Peled, A., J. Immunol. 118, 600, 1977. Lonai, P., and Haran-Ghera, N., Immunogenerics 11, 21, 1980. Lonai, P., Katz, E., Peled, A., and Haran-Ghera, N., Immunogenetics 12, 423, 1981. Haran-Ghera, N., and Rubio, N., J. Immunol. 118, 607, 1977. Peled, A., Leukemia Res. 1, 333, 1977. Haran-Ghera, N., Rubio, N., Leef, F., and Goldstein, G., Cell. Immunol. 37, 308, 1978. Haran-Ghera, N., and Peled, A., Advan. Cancer Rex 30, 45, 1979. Gill, H. L., and Liew, F. W., Eur. J. Immunol. 8, 172, 1978. Vadas, M. A., Miller, J. F. A. P., Gamble, J., and Whitelaw, A., Znt. Arch. Allerg. Appl. Immunol. 49, 670, 1975.
IMMUNOLOGY
70, 196- 199 ( 1982)
Delayed Type Hypersensitivity Responses Virus Variants’
to Radiation
Leukemia
GIDEON STRASSMANNAND NECHAMA HARAN-GHERA Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel Received December 17, 1981; accepted April 6, 1982 DTH responseswere evaluated in different strains of mice shown to be resistant or sensitive to leukemogenesis by the radiation leukemia virus variants A-RadLV and D-RadLV. A significant responsewas observed only in the H-2 complex-linked resistant haplotypes to RadLV leukemogenesis. The DTH response could be transferred by immune cells of mice resistant to the appropriate RadLV variant. Thus, an inverserelationship between the leukemogenic activity of the virus and its immunization ability expressed by DTH responsewas demonstrated in different mouse strains.
INTRODUCTION Leukemogenicity to RadLV’ variants, D-RadLV and A-RadLV (1, 2), was shown to be associated with Z region linked genes of the H-2 complex (3). A survey of the H-2 haplotypes associated with resistance or sensitivity to leukemia development indicated that D-RadLV and A-RadLV induce opposite resistance patterns in H-2’ and H-2” hosts, H-2’ mice being sensitive to A-RadLV leukemogenesis and resistant to D-RadLV (the reverse in H-2” mice). The resistant haplotypes were shown to harbor preleukemic cells, their leukemogenic potential demonstrable only upon transfer into appropriate recipients (4). Thus, in the resistant hosts the proliferation of these preleukemic cells into overt leukemia is “arrested.” The resistant haplotype can be immunized by intrathymic virus infection and rejects syngeneic leukemic cell grafts whereas the sensitive haplotype does not (5). This immunity can be transferred by lymphoid cells (4, 6) and can be established by preleukemic bone marrow cells as the immunogen (7). It is still unresolved whether the preleukemic cells in resistant and sensitive haplotypes are similar. The Zr-controlled immune responsivenessmight involve the function of different H-2 defined presentations by similar target cells or by different target cells, having different immunogenicity. Previous findings have indicated that in BL/6 mice A-RadLV has an immunosuppressive effect on the T-cell lineage (helper cells) whereas D-RadLV abrogates the function of lymphoid precursors in the bone marrow (2,8). It seemed therefore of interest to further elucidate the T-cell-dependent component of the r This investigation was supported by PHS Grant lRO1 CA-29657-01 awarded by the National Cancer Institute, DHHS. 2 Abbreviations used: RadLV, radiation leukemia virus; A-RadLV, autonomous RadLV; D-RadLV, dependent RadLV; H-2, major histocompatibility complex of the mouse; DTH, delayed-type hypersensitivity; N-CFC, normal cell free centrifugate; R, right; L, left; ‘%JdR , s-[ ‘251]iodo-2’-deoxyuridine; PPD, purified protein derivative; CFA, complete Freund’s adjuvant. 196 OOO8-8749/82/090196-04$02.00/O Copyright Q 1982 by Acdemic Prm~. Inc. All rights of reproduction ia any form memd.
SHORT
COMMUNICATIONS
197
immune reaction elicited by the RadLV variants. The delayed-type hypersensitivity (DTH) response in the mouse appears to be mediated by T lymphocytes and is transferable to normal mice by antigen-activated T lymphocytes. DTH responses to a wide variety of antigens can be enhanced by pretreatment with cyclophosphamide which affects suppressor cells (9). Since the sensitivity to RadLV leukemogenesis seems to be related with thymocyte-virus interaction (2), it seemed of interest to compare DTH responsesto RadLV variants in strains of mice resistant or sensitive to leukemogenesis following intrathymic virus inoculation in adults. Thus, mice were sensitized with A-RadLV or D-RadLV and the DTH response was measured following virus challenge. MATERIALS AND METHODS Mice. C57BL/6, BlO.S, and BIO.A mice, 2-3 months old, were obtained from the Breeding Center of our Institute. Virus. Both virus variants were prepared from thymic lymphomas induced in C57BL/6 mice by D-RadLV or A-RadLV as described previously (1). The same virus batches were used throughout the experiments. Immunization procedure. Mice were injected subcutaneously with 200 mg/kg cyclophosphamide (Sigma, St. Louis, MO.). Two days later each mouse was immunized with 200 ~1 of 1:1 dilution of virus preparation in complete Freund’s adjuvant (Difco, Detroit, Mich.) in four footpads. DTH measurement. This technique was originally described by Vadas et al. (10). Five days following immunization, mice were challenged with 10 ~1 of either virus preparation or cell free centrifugate from normal thymus (N-CFC), or purified protein derivative (PPD 1 mg/ml, Statens Seruminstitute Co., Denmark) in the right (R) ear. The left (L) ear was injected with phosphate-buffered saline. Ten hours following challenge mice received 5-fluorodeoxyuridine and 20 min later 2 PCi of 12’1-5-iodo-2’-deoxyuridine ( 1251UdR,Radiochemical Center, Amersham). Ears were cut 24-30 hr after challenge and counted in a gamma counter. The results are expressed as the ratio of radioactivity in the right ear to that of the left ear (R/L ‘251UdR index). The results are expressed as the arithmetic mean of all mice in the group + standard error. P values were calculated by the Student’s t test. Transfer of DTH. Five days following the immunization protocol, 50 X lo6 spleen cells of C57BL/6 mice sensitized to either D- or A-RadLV were transferred intravenously into syngeneic recipients. RESULTS AND DISCUSSION In order to find out whether there is any relationship between the leukemogenic activity of RadLV variants and the ability to develop DTH reactions, different mice sensitive or resistant to RadLV variants were immunized to either A-RadLV or D-RadLV and 5 days later assayed for DTH manifestations. As demonstrated in Table 1, C57BL/6 (H-2b) mice were able to develop DTH responsesto D-RadLV (to which they are resistant) but not to A-RadLV (to which they are sensitive). The same experiment was repeated three times, using different batches of A- and D-RadLV, in order to exclude the possibility of batch dependency of the reactions. Hence the results of ‘these experiments were found to be similar to those presented in Table 1.
N-CFC A-RadLV
N-CFC
D-RadLV A-RadLV
A-RadLV
5 5
5
5 11
11
mice tested
No. of
-
-
+
Resistance’
1.18 f 0.07 1.10 + 0.09
1.45 + 0.14
1.50 2 0.09 1.51 f 0.08
2.56 e.O.32’ i P < 0.01
Index
4
5 11
lib
No. of mice tested
-
_ +
-
Resistance”
Index
1.03 + 0.05
BIOS
’ Resistance to leukemia development following virus inoculation into the thymus of adult mice. b Significant difference between the first and the third groups.
D-RadLV A-RadLV
D-RadLV
D-RadLV
-
Antigen challenge
Sensitizing virus
C57BL/6
DTH response (R/L ‘*‘IUdR index + SE)
DTH Responses in Resistant and Sensitive Strains to Variants of RadLV
TABLE 1
5
4 6
6
No. of mice tested
-
-
-
Resistance”
Bl0.A
1.04 + 0.07
1.00 -+ 0.08 1.16 + 0.08
1.02 + 0.04
Index
z VI
G 2
K 2
8
m _-
199
SHORT COMMUNICATIONS TABLE 2 Transfer of DTH Responses by Immune Splenocytes in C57BL/6 Recipients” Sensitizng virus
Challenging virus
D-RadLV D-RadLV A-RadLV A-RadLV
D-RadLV A-RadLV A-RadLV D-RadLV
R/L ‘*%IdR index f SE 1.90 f 1.98 f 1.24 f 1.26 f
O.lb 0.096 0.08 0.1
’ Each mouse received 50 X lo6 RadLV immunized splenocytes. Five mice were tested per group. b Significant difference from the last group: P < 0.01.
On the other hand, BIO.S mice that were shown to be resistant to A-RadLV and sensitive to D-RadLV leukemogenesis responded only to A-RadLV sensitization and challenge, but not to D-RadLV. In contrast to these two mouse strains, BIO.A mice (H-2”) were found to be sensitive to both virus variants (3). As shown in Table 1 no DTH response was observed in BIO.A mice to both A-RadLV and D-RadLV. Under the same experimental conditions BIO.A were tested for their ability to respond to PPD which is the major antigenic determinant of CFA (data not shown in the table). Thus, when BIO.A mice were immunized with either Aor D-RadLV emulsified in CFA, significant DTH responsesto PPD were monitored (R/L ‘*‘IUdR indexes 1.93 f 0.21 and 2.0 + 0.3, respectively). The transfer of DTH reactions was tested by pretreating C57BL/6 mice with cyclophosphamide and then immunizing them to either D or A-RadLV. Five days following immunization 50 X lo6 splenocytes of these immunized mice were transferred into syngeneic recipients and their ability to mediate DTH reaction was further tested. DTH responsescould be transferred by immune cells from C57BL/ 6-D-RadLV-treated mice only (not from A-RadLV-sensitive treated donors) (Table 2). The immunological cross reactivity between leukemic cells induced by both variants, demonstrated already in previous studies (5), was confirmed in this experiment using the DTH response (Table 2). Thus, the DTH response to RadLV variants seemsto be correlated with resistance to RadLV leukemogenesis afforded by the proliferation arrest of preleukemic cells (4). Sensitivity to RadLV leukemogenesis related to thymocyte-virus interaction (8) on the other hand seems to prevent DTH responses. REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9. 10.
Haran-Ghera, N., Inr. J. Cancer 1, 81, 1966. Haran-Ghera, Ben-Yaakov, M., and Peled, A., J. Immunol. 118, 600, 1977. Lonai, P., and Haran-Ghera, N., Immunogenerics 11, 21, 1980. Lonai, P., Katz, E., Peled, A., and Haran-Ghera, N., Immunogenetics 12, 423, 1981. Haran-Ghera, N., and Rubio, N., J. Immunol. 118, 607, 1977. Peled, A., Leukemia Res. 1, 333, 1977. Haran-Ghera, N., Rubio, N., Leef, F., and Goldstein, G., Cell. Immunol. 37, 308, 1978. Haran-Ghera, N., and Peled, A., Advan. Cancer Rex 30, 45, 1979. Gill, H. L., and Liew, F. W., Eur. J. Immunol. 8, 172, 1978. Vadas, M. A., Miller, J. F. A. P., Gamble, J., and Whitelaw, A., Znt. Arch. Allerg. Appl. Immunol. 49, 670, 1975.