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Induction of immunodeficiency in mice by injection with syngeneic splenocytes immune to semi-allogeneic hybrid cells
CELLULARIMMUNOLOGY88,222-221(1984)
Induction of lmmunodeficiency in Mice by Injection with Syngeneic Splenocytes Immune to Semi-allogeneic Hybrid Cells BYUNG S. KIM Departments of Microbiology-Immunology and Pathology, Medical and Dental Schools, Northwestern University, Chicago, Illinois 6061I Received March 8. 1984; accepted June 19, I984 Intravenous transfer of BALB/c spleen cells (l-10 X 10’) immunized against semi-allogeneic hybrid cells bearing H-2’ antigens into BALB/c mice resulted in splenomegaly (2- to 6-fold). As few as 2 X 104spleen cells could transfer splenomegaly. A significant spleen enlargement was seen from 2 weeks after cell transfer and reached the peak level at 3 weeks. Those mice with splenomegaly displayed markedly reduced levels of proliferative responsesto concanavalin A and lipopolysaccharide. However, the levels of the major Ig classes(i.e., I&I, I&I, and 184) in the immunodeficient mice were significantly elevated. Such induction of immunodeficiency in mice may help further to understand the acquired immunodeficiency syndrome of humans. Q 1984Academic FTes,Inc.
INTRODUCTION The acquired immune deficiency syndrome (AIDS) has recently been described in homosexual men, drug abusers, hemophiliacs, and Haitian refugees (l-7). The syndrome exhibits progressive impairment of immune responses and diminished lymphocyte proliferation responses to various mitogens (4-6). These patients ultimately become defenselessagainst opportunistic infections. Unfortunately, a good animal model system for this diseasehas not yet been established. We report here that transferable splenomegaly and immunodeficiency are induced following serial transfers of spleen cells immune to semi-allogeneic hybrid cells (H-2d/H-2k). Striking similarities between these experimentally induced mouse immune deficiencies and AIDS are noted. This induction/transfer of immune deficiencies in mice may serve as a model system for investigations of AIDS. MATERIALS
AND METHODS
Mice. BALB/c mice were purchased from Cumberland View Farms (Clinton, Tenn.) and used throughout the study. Semi-allogeneic hybrids. Hybrids were derived by fusion of LM(TK-) and TEEC15 plasmacytoma cells as described previously (8, 9) and the LTC-1 clone (10) was used to induce splenomegaly in this study. The hybrid clone was maintained in tissue culture medium consisting of Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum and a 1% mixture of penicillin and streptomycine (Grand Island Biological Co., Grand Island, N.Y.). The hybrid cells exhibited H-2k and H-2d antigens of both parental haplotypes (8, 9). 222 0008-8749184$3.00 Copyright Q 1984byAcademic Press, Inc. All t’igbts of repdudon in anyform-4.
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COMMUNICATIONS
223
Mitogens. Concanavalin A (Con A) was purchased from Sigma Chemical Company (St. Louis, MO.). Lipopolysaccharide (LPS) from E. coli B55:05 (Difco, Detroit, Mich.) was used. Serial transfer. Spleens from mice immunized ip against semi-allogeneic LTC-1 hybrid clone (three times weekly with 1 X lo6 cells) were teased in cold Hanks’ balanced salt solution (BSS). Single-cell suspensions of the spleens were washed three times with BSS. Aliquots (0.2 ml) of the spleen cell suspension containing 1 X 106-2 X 10’ nucleated cells were injected iv into normal recipient mice. Twenty days after the cell transfer (unless otherwise indicated), spleens from the recipient mice were weighed and their proliferative responseswere measured. In addition, an aliquot of the spleen cells was similarly transferred into another set of mice for further passages.Three to five recipient mice were used per group. Thymidine uptake in vivo. t3H]Thymidine (TdR; 25 &i/mouse) was injected iv into individual mice. Two hours after the injection, single-cell suspensions were prepared by forcing the spleens through a wire mesh. The spleen cells were washed three times with BSS following brief treatment with Tris-buffered ammonium chloride solution to remove erythrocytes. The washed nucleated spleen cells were then solubilized in 5 ml of Soluen-100 overnight at 40°C. The aliquots of solubilized spleen cells were mixed (1: 10) with counting cocktail (3a70B; Research Products International Co.), and counted in a scintillation counter. The thymidine uptake by the spleen cells was calculated as the cpm/mg of spleen weight. In vitro proliferation assay. Cell proliferation was determined as a function of the [3H]TdR uptake by the spleen cells. Briefly, 3 X lo5 nucleated cells per well of a 96-well plate (Falcon Plastics, Oxnard, Calif.) were incubated for 3 days in 200 ~1 of medium containing mitogen (Con A, 0.3 pg; and LPS, 0.2 pg). Twenty-four hours before cell harvest, 35~1 of [3H]TdR in culture medium (sp act, 0.2 mCi/ml) was added to cultures. Cells were washed extensively with saline and harvested using a Mash II cell harvester (Flow Laboratories, Inglewood, Calif.). Radial d#ibon. The method described by Mancini et al. (11) was applied on cellulose-acetate plates as modified by Nerenberg (12). The cellulose-acetate plate (Titan III X 100, Helena Laboratory, Beaumont, Tex.) was immersed in goat antimouse IgG, IgM, or IgA and then 0.5 ~1 serum was applied onto the plate. After 18-48 hr of incubation at room temperature, the plate was washed with saline and stained with 0.5% Ponceau S in 7.5% trichloroacetate. RESULTS Induction of Splenomegaly BALB/c mice were injected intraperitoneally three times weekly with 1 X lo6 semi-allogeneic hyrid cells (LTC-1; H-2d/H-2k) derived from a fusion of TEPC-15 plasmacytoma and LM(TK-) (8- 10). Either untreated or irradiated (10,000 R) LTC-1 cells grown in tissue culture were used for immunization. The spleens of the immune mice did not exhibit significant enlargement when examined 2 to 3 weeks after the last injection. In addition, the spleens did not contain viable LTC-1 cells based on determinations of morphology and colony formation in the soft agar (8). This is probably due to the rejection by the host recognizing the allogeneic H-2 antigens on the hybrids. The immune spleen cells (1 X 10’ nucleated cells/mouse)
224
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were serially transferred into normal BALB/c mice every 2-3 weeks by intravenous injections. During the cell passages,the progeny of the original immune spleen cells might have been transferred. After two to three passagesof the immune cells, the recipients displayed a significant splenomegaly when compared to either normal mice or mice receiving normal splenocyte passages.The splenomega.ly could be continuously transferred (>9 passages)into naive recipients by serial transfer of immune spleen cells and/or their progeny cells in normal BALB/c mice. Time Course of Splenomegaly In an attempt to determine the time course for the maximal splenomegaly induction, either normal splenocytes or immune spleen cells (2 X 10’ cells) at seventh passagewere injected iv into normal BALB/c mice and the spleen size in the recipients were determined alter 5, 10, 17, 23, 33, and 60 days (Fig. 1A). The results indicated that the size of spleen increased only after 10 days and reached a maximum around 20-30 days. Once induced, the splenomegaly persisted for a prolonged period: Their spleen size remained significantly larger than that of control mice even 60 days alter the cell transfer. Proliferation of the splenocytes during the course of splenomegaly induction was measured to correlate with the increase in spleen size (Fig. 1B). More active cell proliferation occurred in the immune cell recipients than in the normal cell recipients several days prior to the weight increase. These results suggestthat increase in spleen size is due to increase in the cell number following cell proliferation.
A
0
5
u-i 0
IO
20 TIME (days)
M
60
TIME (days)
FIG. 1. Time course of the development of splenomegaly. Either normal spleen cells (0) or spleen cells from mice with splenomegaly (0) were injected iv into normal BALB/c mice. The spleen weights and [3H]thymidine uptake by the spleen cells were measured after the indicated time period. Four mice were used for each determination. Cl Indicates the weights of the spleens of uninjected normal BALB/c mice. The splenomegaly was expressed by the arithmetic means of we.t wt f standard error of the individual spleens.
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225
Cell Numbers Required for Transfer of Splenomegaly In order to determine the number of immune cells required for transfer of splenomegaly, varying numbers of spleen cells from mice receiving immune splenocytes were injected iv into normal BALB/c mice and spleen weights of the recipients were measured after 20 days (Fig. 2). Transfer of as few as 2 X lo4 cells induced significant splenomegaly (>Zfold) and 2 X lo5 cells transferred a maximal level of splenomegaly (>3.5-fold). Injection of 2 X lo6 normal spleen cells did not result in a significant enlargement of the recipient spleens. Mitogen Responsesand Ig Levels of Mice with Splenomegaly In order to correlate the splenomegaly with the immune response, responses to Con A and LPS by spleen cells from mice with splenomegaly were compared with those of normal spleen cells. The typical responses to the mitogens are shown in the Table 1. Mice bearing splenomegaly exhibited drastic decreases(>90%) in their proliferative responsesto both mitogens. Patients with AIDS having severeimpairment in lymphocyte proliferation responses to mitogens often display elevated levels of Ig (4-7). To examine the possible parellelism with our mouse system, levels of Ig in mice with splenomegaly were examined and correlated with the deficiencies in proliferative responses to Con A and LPS (Table 1). Mice exhibiting splenomegaly and deficiencies in their mitogenic responsesdisplayed markedly elevated levels of the major Ig classestested, i.e., IgM, IgG, and IgA.
oL
zrtd ~~02 2x10 iwo' ~9 mea zx~d NUMBER DFCELLS TRANSFERRED
FIG. 2. Dose relationship between the number of donor ceils injected and the increases in the weight of spleens. Spleen cells (2 X IO*-2 X 10’) from mice with splenomegaly (0) were transferred into normal BALB/c mice and the weights of the spleenswere measured after 20 days. As a control for the background level, 2 X IO6 normal spleen cells (0) were similarly transferred.
226
SHORT COMMUNICATIONS TABLE 1 Serum Ig Levels and Proliferative Responsesto Mitogens in Mice with Splenomegaly Ig levels (mg/ml)
[‘H]TdR uptake @pm)” Treatment Splenomegaly Normal Splenomegaly/ Normal
Spleen wt (mt3)
Con A
LPS
I&f
W
4 12 + 50 7,522 + 8,368 11,372 zk 9,177 9.2 2 1.3 28.0 f 1.5 159 f 40 302,515 f 27,976 147,295 f 17,146 2.6 f 0.8 14.2 + 4.8 2.6
0.025
0.077
3.5
2.0
IgA 7.6 + 0.4 3.3 + 0.1 2.3
’ The cell proliferation is expressed as the geometric means and standard error of radioactivity taken up by 4 identical cultures of 3 spleens per group.
DISCUSSION We present here evidence that splenomegaly and acquisition of deficiency for proliferative responses to T- or B-cell mitogens can be induced in BALB/c mice following consecutive transfer of immune spleen cells against semi-allogeneic hybrid cells (Table 1). Despite the lack of these responses,serum Ig levels in these immunedeficient mice were significantly elevated (Table 1). Ig levels (IgG and IgA) were also frequently elevated in patients with AIDS (4-7). These parameters of immune deficiency are strikingly similar to those in patients with AIDS (l-7). One can speculate that AIDS in homosexual men may be due to the introduction of allogeneic leukocytes into the recipients via regions of rectum (13). Although the mechanisms involved in the initiation of the syndrome are not yet clear, Shearer and coworkers (13) have proposed that the host may recognize donor Ia antigens and the repetitive transfer of Ia-positive cells from an active to a passive partner could initiate a nontransferable suppressivesignal in the recipient. In our experimental scheme, semi-allogeneic hybrid cells lacking surface Ia antigens were used to immunize the host mice. Therefore, immunosuppression due to the graft-vs-host reaction may not be involved in the induction of transferable splenomegaly and immunosuppression. It is widely speculated that AIDS may be transmitted by a virus-like infectious agent because the disease appears to be spread through blood transfusion, through transfer of cell-free blood products (hemophiliacs), or by close family association (4, 6, 14). This is consistent with our results that the immune deficiency and splenomegaly can be transferred continuously in the syngeneic recipients (Fig. 1 and Table 1). Moreover, our preliminary result (data not shown) indicating that the immune deficiency can be transferred through cell-free extracts and culture supernatants of spleen cells supports this possibility. An ubiquitous latent virus in mice may be activated during an intense cell proliferation and/or immunologically compromised state such as allogeneic disease or autoimmune reaction during vigorous immune responses to allogeneic cells ( 15, 16). Once activated, such a virus may be infectious and may have been transmitted during subsequent serial transfer of the immune cells. Alternatively, a suppressor cell population (or factors) may be involved in this serial transfer of immune deficiencies following the initial induction of suppressor cells during alloimmune reactions. Specific and nonspecific immunosuppressions in many
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227
experimental systems can be transferred into normal naive animals by introducing either suppressor cells or their cellular products (suppressor factors) (17, 18). Nevertheless,further investigations clarifying the causative agent(s) for the induction/ transfer of immune deficiencies in this system may lead to a better understanding of the life-threatening immune deficiency syndrome in humans. REFERENCES 1. Masur, H., Michaelis, M. A., Greene, J. B., Onorato, I., Vandestouwe, R. A., Holzman, R. S., Wormser, G., Brettman, L., Lange, M., and Cunningham-Rundles, S., N. Engl. J. Med. 305, 1431, 1981. 2. Siegal, F. P., Lopez, F., Hammer, G. S., Brown, A. E., Komfeld, S. J., Gold, J., Hassett, J.,
Hirschman, S. Z., Cunningham-Rundles, C., Adelsburg, B. R., Parham, D. M., Siegal, M., Cunningham-Rundles, S., and Armstrong, D. A., N. Engl. J. Med. 305, 1439, 1981. 3. Gottlieb, M. S., Schroff, R., Schanker, H. M., Weisman, J. D., Fan, P. T., Wolf, R. A., and Saxon, A., N. Engl. J. Med. 305, 1425, 1981. 4. Poon, M.-C., Landay, A., Prasthofer, E. F., and Stagno, S., Ann. Intern. Med. 98, 287, 1983. 5. Pitchenik, A. E., Fischl, M. A., Dickinson, G. M., Becker, D. M., Foumier, A. M., O’Connell, M. T., Colton, R. M., and Spira, T. J., Ann. Intern. Med. 98, 277, 1983. 6. Talal, N., Zmmunol. Today 4, 180, 1983. 7. Friedman-Klein, A. E., Laubenstein, L. J., Rubinstein, P., Buimovici-Klein, E., Marmor, M., Stahl, R., Spigland, I., Kim, K. S., and Zolla-Pazner, S., Ann. Intern. Med. 96, 693, 1982. 8. Kim, B. S., Liang, W., and Cohen, E. P., J. Immunol. 123, 733, 1979. 9. Kim, B. S., J. Immunol. 123, 739, 1979. 10. Kim, B. S., Cell. Immunol. 65, 373, 1981. 1I. Mancini, G., Carbonara, A. O., and Heremans, J. F., Immunochemistry 2, 235, 1965. 12. Nerenberg, S. T., J. Lab. Clin. Med. 79, 673, 1972. 13. Shearer, G. M., Zmmunol. Toduy 4, 181, 1983. 14. Francis, D. P., Curran, J. W., and Essex, M., J. Natl. Cancer Inst. 71, 1, 1983. 15. Gershon, R., and Kondo, K., Immunology 23, 335, 1972. 16. Greene, M. I., Sy, M.-S., Nisonoff, A., and Benacerraf, B., Mol. Immunol. 17, 857, 1980. 17. Hirsch, M. S., Black, P. H., Tracy, G. S., Leibowitz, S., and Schwartz, R. S., Proc. Natl. Acud. Sci. USA 76, 1914, 1970. 18. Miller, G., and Enders, J. F., J. Viral. 2, 787, 1968.
Induction of lmmunodeficiency in Mice by Injection with Syngeneic Splenocytes Immune to Semi-allogeneic Hybrid Cells BYUNG S. KIM Departments of Microbiology-Immunology and Pathology, Medical and Dental Schools, Northwestern University, Chicago, Illinois 6061I Received March 8. 1984; accepted June 19, I984 Intravenous transfer of BALB/c spleen cells (l-10 X 10’) immunized against semi-allogeneic hybrid cells bearing H-2’ antigens into BALB/c mice resulted in splenomegaly (2- to 6-fold). As few as 2 X 104spleen cells could transfer splenomegaly. A significant spleen enlargement was seen from 2 weeks after cell transfer and reached the peak level at 3 weeks. Those mice with splenomegaly displayed markedly reduced levels of proliferative responsesto concanavalin A and lipopolysaccharide. However, the levels of the major Ig classes(i.e., I&I, I&I, and 184) in the immunodeficient mice were significantly elevated. Such induction of immunodeficiency in mice may help further to understand the acquired immunodeficiency syndrome of humans. Q 1984Academic FTes,Inc.
INTRODUCTION The acquired immune deficiency syndrome (AIDS) has recently been described in homosexual men, drug abusers, hemophiliacs, and Haitian refugees (l-7). The syndrome exhibits progressive impairment of immune responses and diminished lymphocyte proliferation responses to various mitogens (4-6). These patients ultimately become defenselessagainst opportunistic infections. Unfortunately, a good animal model system for this diseasehas not yet been established. We report here that transferable splenomegaly and immunodeficiency are induced following serial transfers of spleen cells immune to semi-allogeneic hybrid cells (H-2d/H-2k). Striking similarities between these experimentally induced mouse immune deficiencies and AIDS are noted. This induction/transfer of immune deficiencies in mice may serve as a model system for investigations of AIDS. MATERIALS
AND METHODS
Mice. BALB/c mice were purchased from Cumberland View Farms (Clinton, Tenn.) and used throughout the study. Semi-allogeneic hybrids. Hybrids were derived by fusion of LM(TK-) and TEEC15 plasmacytoma cells as described previously (8, 9) and the LTC-1 clone (10) was used to induce splenomegaly in this study. The hybrid clone was maintained in tissue culture medium consisting of Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum and a 1% mixture of penicillin and streptomycine (Grand Island Biological Co., Grand Island, N.Y.). The hybrid cells exhibited H-2k and H-2d antigens of both parental haplotypes (8, 9). 222 0008-8749184$3.00 Copyright Q 1984byAcademic Press, Inc. All t’igbts of repdudon in anyform-4.
SHORT
COMMUNICATIONS
223
Mitogens. Concanavalin A (Con A) was purchased from Sigma Chemical Company (St. Louis, MO.). Lipopolysaccharide (LPS) from E. coli B55:05 (Difco, Detroit, Mich.) was used. Serial transfer. Spleens from mice immunized ip against semi-allogeneic LTC-1 hybrid clone (three times weekly with 1 X lo6 cells) were teased in cold Hanks’ balanced salt solution (BSS). Single-cell suspensions of the spleens were washed three times with BSS. Aliquots (0.2 ml) of the spleen cell suspension containing 1 X 106-2 X 10’ nucleated cells were injected iv into normal recipient mice. Twenty days after the cell transfer (unless otherwise indicated), spleens from the recipient mice were weighed and their proliferative responseswere measured. In addition, an aliquot of the spleen cells was similarly transferred into another set of mice for further passages.Three to five recipient mice were used per group. Thymidine uptake in vivo. t3H]Thymidine (TdR; 25 &i/mouse) was injected iv into individual mice. Two hours after the injection, single-cell suspensions were prepared by forcing the spleens through a wire mesh. The spleen cells were washed three times with BSS following brief treatment with Tris-buffered ammonium chloride solution to remove erythrocytes. The washed nucleated spleen cells were then solubilized in 5 ml of Soluen-100 overnight at 40°C. The aliquots of solubilized spleen cells were mixed (1: 10) with counting cocktail (3a70B; Research Products International Co.), and counted in a scintillation counter. The thymidine uptake by the spleen cells was calculated as the cpm/mg of spleen weight. In vitro proliferation assay. Cell proliferation was determined as a function of the [3H]TdR uptake by the spleen cells. Briefly, 3 X lo5 nucleated cells per well of a 96-well plate (Falcon Plastics, Oxnard, Calif.) were incubated for 3 days in 200 ~1 of medium containing mitogen (Con A, 0.3 pg; and LPS, 0.2 pg). Twenty-four hours before cell harvest, 35~1 of [3H]TdR in culture medium (sp act, 0.2 mCi/ml) was added to cultures. Cells were washed extensively with saline and harvested using a Mash II cell harvester (Flow Laboratories, Inglewood, Calif.). Radial d#ibon. The method described by Mancini et al. (11) was applied on cellulose-acetate plates as modified by Nerenberg (12). The cellulose-acetate plate (Titan III X 100, Helena Laboratory, Beaumont, Tex.) was immersed in goat antimouse IgG, IgM, or IgA and then 0.5 ~1 serum was applied onto the plate. After 18-48 hr of incubation at room temperature, the plate was washed with saline and stained with 0.5% Ponceau S in 7.5% trichloroacetate. RESULTS Induction of Splenomegaly BALB/c mice were injected intraperitoneally three times weekly with 1 X lo6 semi-allogeneic hyrid cells (LTC-1; H-2d/H-2k) derived from a fusion of TEPC-15 plasmacytoma and LM(TK-) (8- 10). Either untreated or irradiated (10,000 R) LTC-1 cells grown in tissue culture were used for immunization. The spleens of the immune mice did not exhibit significant enlargement when examined 2 to 3 weeks after the last injection. In addition, the spleens did not contain viable LTC-1 cells based on determinations of morphology and colony formation in the soft agar (8). This is probably due to the rejection by the host recognizing the allogeneic H-2 antigens on the hybrids. The immune spleen cells (1 X 10’ nucleated cells/mouse)
224
SHORT COMMUNICATIONS
were serially transferred into normal BALB/c mice every 2-3 weeks by intravenous injections. During the cell passages,the progeny of the original immune spleen cells might have been transferred. After two to three passagesof the immune cells, the recipients displayed a significant splenomegaly when compared to either normal mice or mice receiving normal splenocyte passages.The splenomega.ly could be continuously transferred (>9 passages)into naive recipients by serial transfer of immune spleen cells and/or their progeny cells in normal BALB/c mice. Time Course of Splenomegaly In an attempt to determine the time course for the maximal splenomegaly induction, either normal splenocytes or immune spleen cells (2 X 10’ cells) at seventh passagewere injected iv into normal BALB/c mice and the spleen size in the recipients were determined alter 5, 10, 17, 23, 33, and 60 days (Fig. 1A). The results indicated that the size of spleen increased only after 10 days and reached a maximum around 20-30 days. Once induced, the splenomegaly persisted for a prolonged period: Their spleen size remained significantly larger than that of control mice even 60 days alter the cell transfer. Proliferation of the splenocytes during the course of splenomegaly induction was measured to correlate with the increase in spleen size (Fig. 1B). More active cell proliferation occurred in the immune cell recipients than in the normal cell recipients several days prior to the weight increase. These results suggestthat increase in spleen size is due to increase in the cell number following cell proliferation.
A
0
5
u-i 0
IO
20 TIME (days)
M
60
TIME (days)
FIG. 1. Time course of the development of splenomegaly. Either normal spleen cells (0) or spleen cells from mice with splenomegaly (0) were injected iv into normal BALB/c mice. The spleen weights and [3H]thymidine uptake by the spleen cells were measured after the indicated time period. Four mice were used for each determination. Cl Indicates the weights of the spleens of uninjected normal BALB/c mice. The splenomegaly was expressed by the arithmetic means of we.t wt f standard error of the individual spleens.
SHORT COMMUNICATIONS
225
Cell Numbers Required for Transfer of Splenomegaly In order to determine the number of immune cells required for transfer of splenomegaly, varying numbers of spleen cells from mice receiving immune splenocytes were injected iv into normal BALB/c mice and spleen weights of the recipients were measured after 20 days (Fig. 2). Transfer of as few as 2 X lo4 cells induced significant splenomegaly (>Zfold) and 2 X lo5 cells transferred a maximal level of splenomegaly (>3.5-fold). Injection of 2 X lo6 normal spleen cells did not result in a significant enlargement of the recipient spleens. Mitogen Responsesand Ig Levels of Mice with Splenomegaly In order to correlate the splenomegaly with the immune response, responses to Con A and LPS by spleen cells from mice with splenomegaly were compared with those of normal spleen cells. The typical responses to the mitogens are shown in the Table 1. Mice bearing splenomegaly exhibited drastic decreases(>90%) in their proliferative responsesto both mitogens. Patients with AIDS having severeimpairment in lymphocyte proliferation responses to mitogens often display elevated levels of Ig (4-7). To examine the possible parellelism with our mouse system, levels of Ig in mice with splenomegaly were examined and correlated with the deficiencies in proliferative responses to Con A and LPS (Table 1). Mice exhibiting splenomegaly and deficiencies in their mitogenic responsesdisplayed markedly elevated levels of the major Ig classestested, i.e., IgM, IgG, and IgA.
oL
zrtd ~~02 2x10 iwo' ~9 mea zx~d NUMBER DFCELLS TRANSFERRED
FIG. 2. Dose relationship between the number of donor ceils injected and the increases in the weight of spleens. Spleen cells (2 X IO*-2 X 10’) from mice with splenomegaly (0) were transferred into normal BALB/c mice and the weights of the spleenswere measured after 20 days. As a control for the background level, 2 X IO6 normal spleen cells (0) were similarly transferred.
226
SHORT COMMUNICATIONS TABLE 1 Serum Ig Levels and Proliferative Responsesto Mitogens in Mice with Splenomegaly Ig levels (mg/ml)
[‘H]TdR uptake @pm)” Treatment Splenomegaly Normal Splenomegaly/ Normal
Spleen wt (mt3)
Con A
LPS
I&f
W
4 12 + 50 7,522 + 8,368 11,372 zk 9,177 9.2 2 1.3 28.0 f 1.5 159 f 40 302,515 f 27,976 147,295 f 17,146 2.6 f 0.8 14.2 + 4.8 2.6
0.025
0.077
3.5
2.0
IgA 7.6 + 0.4 3.3 + 0.1 2.3
’ The cell proliferation is expressed as the geometric means and standard error of radioactivity taken up by 4 identical cultures of 3 spleens per group.
DISCUSSION We present here evidence that splenomegaly and acquisition of deficiency for proliferative responses to T- or B-cell mitogens can be induced in BALB/c mice following consecutive transfer of immune spleen cells against semi-allogeneic hybrid cells (Table 1). Despite the lack of these responses,serum Ig levels in these immunedeficient mice were significantly elevated (Table 1). Ig levels (IgG and IgA) were also frequently elevated in patients with AIDS (4-7). These parameters of immune deficiency are strikingly similar to those in patients with AIDS (l-7). One can speculate that AIDS in homosexual men may be due to the introduction of allogeneic leukocytes into the recipients via regions of rectum (13). Although the mechanisms involved in the initiation of the syndrome are not yet clear, Shearer and coworkers (13) have proposed that the host may recognize donor Ia antigens and the repetitive transfer of Ia-positive cells from an active to a passive partner could initiate a nontransferable suppressivesignal in the recipient. In our experimental scheme, semi-allogeneic hybrid cells lacking surface Ia antigens were used to immunize the host mice. Therefore, immunosuppression due to the graft-vs-host reaction may not be involved in the induction of transferable splenomegaly and immunosuppression. It is widely speculated that AIDS may be transmitted by a virus-like infectious agent because the disease appears to be spread through blood transfusion, through transfer of cell-free blood products (hemophiliacs), or by close family association (4, 6, 14). This is consistent with our results that the immune deficiency and splenomegaly can be transferred continuously in the syngeneic recipients (Fig. 1 and Table 1). Moreover, our preliminary result (data not shown) indicating that the immune deficiency can be transferred through cell-free extracts and culture supernatants of spleen cells supports this possibility. An ubiquitous latent virus in mice may be activated during an intense cell proliferation and/or immunologically compromised state such as allogeneic disease or autoimmune reaction during vigorous immune responses to allogeneic cells ( 15, 16). Once activated, such a virus may be infectious and may have been transmitted during subsequent serial transfer of the immune cells. Alternatively, a suppressor cell population (or factors) may be involved in this serial transfer of immune deficiencies following the initial induction of suppressor cells during alloimmune reactions. Specific and nonspecific immunosuppressions in many
SHORT COMMUNICATIONS
227
experimental systems can be transferred into normal naive animals by introducing either suppressor cells or their cellular products (suppressor factors) (17, 18). Nevertheless,further investigations clarifying the causative agent(s) for the induction/ transfer of immune deficiencies in this system may lead to a better understanding of the life-threatening immune deficiency syndrome in humans. REFERENCES 1. Masur, H., Michaelis, M. A., Greene, J. B., Onorato, I., Vandestouwe, R. A., Holzman, R. S., Wormser, G., Brettman, L., Lange, M., and Cunningham-Rundles, S., N. Engl. J. Med. 305, 1431, 1981. 2. Siegal, F. P., Lopez, F., Hammer, G. S., Brown, A. E., Komfeld, S. J., Gold, J., Hassett, J.,
Hirschman, S. Z., Cunningham-Rundles, C., Adelsburg, B. R., Parham, D. M., Siegal, M., Cunningham-Rundles, S., and Armstrong, D. A., N. Engl. J. Med. 305, 1439, 1981. 3. Gottlieb, M. S., Schroff, R., Schanker, H. M., Weisman, J. D., Fan, P. T., Wolf, R. A., and Saxon, A., N. Engl. J. Med. 305, 1425, 1981. 4. Poon, M.-C., Landay, A., Prasthofer, E. F., and Stagno, S., Ann. Intern. Med. 98, 287, 1983. 5. Pitchenik, A. E., Fischl, M. A., Dickinson, G. M., Becker, D. M., Foumier, A. M., O’Connell, M. T., Colton, R. M., and Spira, T. J., Ann. Intern. Med. 98, 277, 1983. 6. Talal, N., Zmmunol. Today 4, 180, 1983. 7. Friedman-Klein, A. E., Laubenstein, L. J., Rubinstein, P., Buimovici-Klein, E., Marmor, M., Stahl, R., Spigland, I., Kim, K. S., and Zolla-Pazner, S., Ann. Intern. Med. 96, 693, 1982. 8. Kim, B. S., Liang, W., and Cohen, E. P., J. Immunol. 123, 733, 1979. 9. Kim, B. S., J. Immunol. 123, 739, 1979. 10. Kim, B. S., Cell. Immunol. 65, 373, 1981. 1I. Mancini, G., Carbonara, A. O., and Heremans, J. F., Immunochemistry 2, 235, 1965. 12. Nerenberg, S. T., J. Lab. Clin. Med. 79, 673, 1972. 13. Shearer, G. M., Zmmunol. Toduy 4, 181, 1983. 14. Francis, D. P., Curran, J. W., and Essex, M., J. Natl. Cancer Inst. 71, 1, 1983. 15. Gershon, R., and Kondo, K., Immunology 23, 335, 1972. 16. Greene, M. I., Sy, M.-S., Nisonoff, A., and Benacerraf, B., Mol. Immunol. 17, 857, 1980. 17. Hirsch, M. S., Black, P. H., Tracy, G. S., Leibowitz, S., and Schwartz, R. S., Proc. Natl. Acud. Sci. USA 76, 1914, 1970. 18. Miller, G., and Enders, J. F., J. Viral. 2, 787, 1968.