Vol. 24, No. 1
INFECTION AND IMMUNITY, Apr. 1979, p. 1-6
0019-9567/79/04-0001/06$02.00/0
Suppression of In Vitro Antibody Response by Spleen Cells of Mice Infected with Friend-Associated Lymphatic Leukemia Virus MAURO BENDINELLI,'* DONATELLA MATTEUCCI,1 ANTONIO TONIOLO,'
AND
HERMAN
FRIEDMAN2
Institute of Microbiology, University of Pisa, 56100 Pisa, Italy,' and Department of Microbiology and Immunology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 191412 Received for publication 29 December 1978
The ability of spleen cells of mice infected with oncornaviruses to depress the in vitro antibody responsiveness of normal lymphoid cells was exploited in an attempt to clarify the role played by the lymphatic leukemia virus (LLV) component in the immunodepressive properties of the Friend leukemia complex. Spleen cells of mice infected with LLV or, for comparison, with the entire complex were added to cultures of sheep erythrocyte-primed uninfected spleen cells, and the antibody-forming cells produced by the latter, after antigen restimulation, were assayed. The addition within 2 days from culture initiation of low numbers of cells infected with either virus preparation suppressed all stages of the response affecting the production of both immunoglobulin M and immunoglobulin G antibody. The activity of infected cells resisted doses of ultraviolet radiation which inhibit cell multiplication but was abolished by disrupting the cells and was prevented by the presence of anti-LLV antibodies. The LLV-infected spleen cells responsible for suppression were not removed by treatments which selectively remove or kill macrophages and exhibited surface properties of B lymphocytes. These results were interpreted as indicating that the effect is due to virus (or viral products) released by B cells. The suppressing cells in the spleens of mice in the early days of Friend leukemia complex infection presented superimposable properties, supporting the concept that their activity is also due to the LLV they release in large quantities. However, in later stages of infection, the spleens of Friend leukemia complex-infected mice also contained non-B-suppressing cells possibly derived from the proliferation of nonlymphoid LLV-producing cells caused by the neoplastic process.
The ability of Friend leukemia complex (FLC) to depress the immune response of the host has
been intensively investigated, but the mechanisms of the phenomenon, as well as its relevance in the pathogenesis of leukemia, are still poorly understood (2, 14). The recent observation that FLC-infected spleen cells are capable of suppressing the antibody response of normal lymphoid cell cultures (4, 15, 21, 33) provides an in vitro system that, being amenable to various manipulations, can help in understanding some unresolved problems. We have used this experimental system in an attempt to clarify the role played by the lymphatic leukemia virus (LLV) component in the immunodepressive properties of the complex. The results are presented in this paper. MATERIALS AND METHODS Animals. Inbred female BALB/c mice were from our own colony. They were 8 to 12 weeks old at the
time spleens were taken for tissue culture. Viruses and infection. Details on the history, preparation, and titration of the viruses were given in a previous paper (5). Virus stocks used for inoculation were from the plasma of syngeneic mice and therefore should contain no immunizing nonviral antigens. The Rowson-Parr isolate (28) of LLV (RP-LLV) was obtained from adult animals 8 days after infection and had a titer of 104 mean infectious doses per 0.1 ml. FLC, anemia-inducing strain, was obtained from adult mice 3 weeks after infection and contained 103 mean infectious doses of erythroleukemia-inducing activity and 1045 mean infectious doses of LLV per 0.1 ml. Both RP-LLV and FLC were freed of lactate dehydrogenase virus. Unless otherwise stated, the donors of infected spleen cells were injected intravenously with 0.1 ml of virus preparation 7 days before removal of the spleens. Antigen. Sheep erythrocytes in Alsever solution were purchased from Sclavo (Siena, Italy) and washed several times in saline before use. For priming, the donors of responder cells were injected intravenously with 4 x 107 sheep erythrocytes in saline 4 days before
2
BENDINELLI ET AL.
removal of the spleen for culture. In vitro, the standard dose of sheep erythrocytes was 2 x 106/culture. Anti-RP-LLV serum. Mice were injected with RPLLV and 2 months later challenged with a high dose of FLC. Three weeks later all mice had spleen weights in the normal range. Their sera, which have been shown to contain antibodies neutralizing both components of FLC (5), were used as antisera. Normal serum was obtained from coeval mice. Before use, the sera were heated at 60'C for 1 h, absorbed with sheep erythrocytes, and sterilized by filtration. Preparation of spleen cell suspensions. Primed, normal, and infected cell suspensions were obtained by gently teasing the spleens of three or more mice in RPMI 1640 (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum. The cells were washed twice in the cold, their viability was tested by trypan blue exclusion, and they were resuspended at the required density. Assay of the suppressive activity of infected cells. The responder cells were primed spleen cells restimulated with sheep erythrocytes in 2.6 ml of RPMI 1640 supplemented with fetal calf serum, nutrient cocktail, and antibiotics, as described (20), and cultured in 16-mm wells of Costar-24 tissue culture plates (Costar, Cambridge, Mass.). To these responder cultures RP-LLV-infected or, for comparison, FLCinfected or normal cells were added, unless otherwise noted, at the initiation of cultivation, and direct and indirect plaque-forming cells (PFC) were assayed by the method of Jerne 4 days later. The addition of uninfected unstimulated spleen cells (hereafter called normal cells) can adversely affect the PFC response of primed cultures (7, 8; unpublished data). Preliminary experiments had shown that this type of suppression in our system (i) required high proportions of normal cells; (ii) was dependent on the total number of cells in each well (for instance, with ratios of normal to primed cells equal to one, suppression was absent or negligible if the total number of cells was lower than 8 x 106); and (iii) varied considerably with the batch of fetal calf serum used. Therefore, to study the suppressive effects of infected cells without the interference of those that can be exerted by normal cells, the following conditions were adopted: (i) a low number of primed cells, usually 3 x 106, were cultured; (ii) infected cells were added at ratios lower than one, usually 1:3; (iii) one single batch of fetal calf serum was used throughout. As a further precaution, in most experiments controls with the addition of normal cells were established. Moreover, to avoid the contribution of the added cells to the PFC response of the cultures, a batch of fetal calf serum (Rehatuin, lot P 30603, Reheis, Phoenix, Ariz.) was chosen which under our conditions did not support the generation of an efficient primary response (3 x 10r virgin cells constantly produced fewer than 250 PFC). Treatments of spleen cells. For sonication, cells suspended at 106/ml were treated at 100-W intensity with a Blackstone probe, model BP-10, CW-10 (Blackstone Ultrasonics, Sheffield, Pa.), for 30 s followed by 30 s of rest. This was repeated three times, keeping the cells on ice. After such treatment, no whole cells were microscopically detectable.
INFECT. IMMUN. Ultraviolet irradiation was carried out in open dishes placed on ice with a 30-W Philips low-pressure mercury lamp at an intensity of 12 ergs/mm2 per s (fluency rate determined by a Latarjet dosimeter). The cell concentration was 106/ml, and the thickness of the suspensions in the dish was about 1 mm. Macrophage depletion was performed: (i) by adherence to plastic surfaces (26); (ii) by treatment with silica (Dorentrop, lot DQ 12; particle size,
INFECTION AND IMMUNITY, Apr. 1979, p. 1-6
0019-9567/79/04-0001/06$02.00/0
Suppression of In Vitro Antibody Response by Spleen Cells of Mice Infected with Friend-Associated Lymphatic Leukemia Virus MAURO BENDINELLI,'* DONATELLA MATTEUCCI,1 ANTONIO TONIOLO,'
AND
HERMAN
FRIEDMAN2
Institute of Microbiology, University of Pisa, 56100 Pisa, Italy,' and Department of Microbiology and Immunology, Albert Einstein Medical Center, Philadelphia, Pennsylvania 191412 Received for publication 29 December 1978
The ability of spleen cells of mice infected with oncornaviruses to depress the in vitro antibody responsiveness of normal lymphoid cells was exploited in an attempt to clarify the role played by the lymphatic leukemia virus (LLV) component in the immunodepressive properties of the Friend leukemia complex. Spleen cells of mice infected with LLV or, for comparison, with the entire complex were added to cultures of sheep erythrocyte-primed uninfected spleen cells, and the antibody-forming cells produced by the latter, after antigen restimulation, were assayed. The addition within 2 days from culture initiation of low numbers of cells infected with either virus preparation suppressed all stages of the response affecting the production of both immunoglobulin M and immunoglobulin G antibody. The activity of infected cells resisted doses of ultraviolet radiation which inhibit cell multiplication but was abolished by disrupting the cells and was prevented by the presence of anti-LLV antibodies. The LLV-infected spleen cells responsible for suppression were not removed by treatments which selectively remove or kill macrophages and exhibited surface properties of B lymphocytes. These results were interpreted as indicating that the effect is due to virus (or viral products) released by B cells. The suppressing cells in the spleens of mice in the early days of Friend leukemia complex infection presented superimposable properties, supporting the concept that their activity is also due to the LLV they release in large quantities. However, in later stages of infection, the spleens of Friend leukemia complex-infected mice also contained non-B-suppressing cells possibly derived from the proliferation of nonlymphoid LLV-producing cells caused by the neoplastic process.
The ability of Friend leukemia complex (FLC) to depress the immune response of the host has
been intensively investigated, but the mechanisms of the phenomenon, as well as its relevance in the pathogenesis of leukemia, are still poorly understood (2, 14). The recent observation that FLC-infected spleen cells are capable of suppressing the antibody response of normal lymphoid cell cultures (4, 15, 21, 33) provides an in vitro system that, being amenable to various manipulations, can help in understanding some unresolved problems. We have used this experimental system in an attempt to clarify the role played by the lymphatic leukemia virus (LLV) component in the immunodepressive properties of the complex. The results are presented in this paper. MATERIALS AND METHODS Animals. Inbred female BALB/c mice were from our own colony. They were 8 to 12 weeks old at the
time spleens were taken for tissue culture. Viruses and infection. Details on the history, preparation, and titration of the viruses were given in a previous paper (5). Virus stocks used for inoculation were from the plasma of syngeneic mice and therefore should contain no immunizing nonviral antigens. The Rowson-Parr isolate (28) of LLV (RP-LLV) was obtained from adult animals 8 days after infection and had a titer of 104 mean infectious doses per 0.1 ml. FLC, anemia-inducing strain, was obtained from adult mice 3 weeks after infection and contained 103 mean infectious doses of erythroleukemia-inducing activity and 1045 mean infectious doses of LLV per 0.1 ml. Both RP-LLV and FLC were freed of lactate dehydrogenase virus. Unless otherwise stated, the donors of infected spleen cells were injected intravenously with 0.1 ml of virus preparation 7 days before removal of the spleens. Antigen. Sheep erythrocytes in Alsever solution were purchased from Sclavo (Siena, Italy) and washed several times in saline before use. For priming, the donors of responder cells were injected intravenously with 4 x 107 sheep erythrocytes in saline 4 days before
2
BENDINELLI ET AL.
removal of the spleen for culture. In vitro, the standard dose of sheep erythrocytes was 2 x 106/culture. Anti-RP-LLV serum. Mice were injected with RPLLV and 2 months later challenged with a high dose of FLC. Three weeks later all mice had spleen weights in the normal range. Their sera, which have been shown to contain antibodies neutralizing both components of FLC (5), were used as antisera. Normal serum was obtained from coeval mice. Before use, the sera were heated at 60'C for 1 h, absorbed with sheep erythrocytes, and sterilized by filtration. Preparation of spleen cell suspensions. Primed, normal, and infected cell suspensions were obtained by gently teasing the spleens of three or more mice in RPMI 1640 (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum. The cells were washed twice in the cold, their viability was tested by trypan blue exclusion, and they were resuspended at the required density. Assay of the suppressive activity of infected cells. The responder cells were primed spleen cells restimulated with sheep erythrocytes in 2.6 ml of RPMI 1640 supplemented with fetal calf serum, nutrient cocktail, and antibiotics, as described (20), and cultured in 16-mm wells of Costar-24 tissue culture plates (Costar, Cambridge, Mass.). To these responder cultures RP-LLV-infected or, for comparison, FLCinfected or normal cells were added, unless otherwise noted, at the initiation of cultivation, and direct and indirect plaque-forming cells (PFC) were assayed by the method of Jerne 4 days later. The addition of uninfected unstimulated spleen cells (hereafter called normal cells) can adversely affect the PFC response of primed cultures (7, 8; unpublished data). Preliminary experiments had shown that this type of suppression in our system (i) required high proportions of normal cells; (ii) was dependent on the total number of cells in each well (for instance, with ratios of normal to primed cells equal to one, suppression was absent or negligible if the total number of cells was lower than 8 x 106); and (iii) varied considerably with the batch of fetal calf serum used. Therefore, to study the suppressive effects of infected cells without the interference of those that can be exerted by normal cells, the following conditions were adopted: (i) a low number of primed cells, usually 3 x 106, were cultured; (ii) infected cells were added at ratios lower than one, usually 1:3; (iii) one single batch of fetal calf serum was used throughout. As a further precaution, in most experiments controls with the addition of normal cells were established. Moreover, to avoid the contribution of the added cells to the PFC response of the cultures, a batch of fetal calf serum (Rehatuin, lot P 30603, Reheis, Phoenix, Ariz.) was chosen which under our conditions did not support the generation of an efficient primary response (3 x 10r virgin cells constantly produced fewer than 250 PFC). Treatments of spleen cells. For sonication, cells suspended at 106/ml were treated at 100-W intensity with a Blackstone probe, model BP-10, CW-10 (Blackstone Ultrasonics, Sheffield, Pa.), for 30 s followed by 30 s of rest. This was repeated three times, keeping the cells on ice. After such treatment, no whole cells were microscopically detectable.
INFECT. IMMUN. Ultraviolet irradiation was carried out in open dishes placed on ice with a 30-W Philips low-pressure mercury lamp at an intensity of 12 ergs/mm2 per s (fluency rate determined by a Latarjet dosimeter). The cell concentration was 106/ml, and the thickness of the suspensions in the dish was about 1 mm. Macrophage depletion was performed: (i) by adherence to plastic surfaces (26); (ii) by treatment with silica (Dorentrop, lot DQ 12; particle size,
