Experimental cutaneous leishmaniasis. I. Nonspecific immunodepression in BALB/c mice infected with Leishmania tropica

Leishmania tropica in BALB/c mice causes a fatal infection accompanied by the development of multiple metastatic lesions. Spleen cells from these mice were shown to have depressed proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS). Coinciding with this immunodepression was the development of a cell population capable of suppressing normal spleen cell responses to Con A. This suppressor cell activity was first observed at 6 wk and was present throughout the remainder of the infection. At 12 wk the suppressor cells could be removed by Sephadex G-10 passage or carbonyl iron treatment; however, Sephadex G-10 passage could not reverse the suppression at 18 wk. Indomethacin, a prostaglandin synthetase inhibitor, was found to abrogate the activity of the adherent suppressor cell, suggesting that prostaglandin production may be involved in the immunosuppression seen in these mice. In addition, Sephadex G-10 passage and indomethacin were found to markedly augment spleen cell responses to leishmanial antigen, indicating that the adherent suppressor cell is capable of regulating specific immunologic responses.     

Suppressor cell activity in mice bearing a progressively growing Simian virus 40-induced sarcoma

Summary General and cell-mediated immunity (CMI) were investigated in BALB/c mice bearing progressively growing Simian virus 40-induced (mKSA) sarcoma by means of the Winn tumor cell neutralization (WN), ¹²⁵I isotopic footpad (IFP), lymphoproliferative (LP) and plaque-forming cell (PFC) assays. Correlation between depressed antitumor immunity and the IFP responses was observed in tumor-bearing (TB) mice. Depressed LP responses to both T- and B-cell mitogens were observed in both early and late stages of tumor growth. Results obtained with the PFC assay similarly demonstrated depressed humoral immunity to sheep red blood cells (SRBC). Suppressor cell activity was demonstrated in cocultivation experiments in which spleen cells of TB mice were mixed with normal spleen cells. Treatment of TB spleen cells by passage through Sephadex G-10 columns or incubation on plastic surfaces to deplete the adherent cells restored LP responses. Cocultivation of Sephadex G-10- or plastic-adherent cells from TB mice with normal spleen cells significantly reduced mitogen-induced LP responses of normal cells. Examination of cell surface markers indicated an increase in the proportion of spleen cells bearing Fc receptors, which correlated with progressive mKSA tumor growth. There was also a correlation between Fc receptor-bearing spleen cells and macrophages, as shown by nonspecific esterase staining. These results indicate that depressed LP and PFC responses and the appearance of suppressor cells in mKSA tumor-bearing mice parallel an impaired ability to recognize (IFP responses) and neutralize (WN responses) tumor cells.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983     

Stimulation of prostaglandin-dependent macrophage suppressor cells by the subcutaneous injection of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs), in the form of pure linoleic, linolenic, or arachidonic acid, were injected subcutaneously into male C57Bl/6 mice daily for 10 days. Injection of 3.6 mg/day of PUFA resulted in up to a two- to threefold increase in spleen weight. Spleen cell response to mitogens was reduced by about 70%; mixed lymphocyte responses were reduced by about 90% when compared to normal values. In admixture experiments, spleen cells from PUFA treated mice suppressed the mitogen induced blastogenic response of control spleen cells by up to 90%. Fractionation of spleen cells from PUFA treated mice by G-10 adherence resulted in an enrichment of suppressive activity in the adherent cells. The suppressive effect of G-10 adherent cells was abolished by the addition of indomethacin as well as by depletion of macrophages by treatments with agents such as carbonyl iron and leucine methyl ester. These studies indicate that the administration of PUFA has marked immunosuppressive effects in mice. These effects may be related to increased prostaglandin production and appear to be mediated by a macrophage type cell.     

Characteristics of DTH suppressor cells in mice infected with Candida albicans

Inoculation of 10⁸ C. albicans intraperitoneally into Balb/c mice at given dosage was reported to induce suppression of antigen-specific delayed-type hypersensitivity. Adoptive transfer of spleen cells into normal syngeneic mice pre-treated with Cyclophosphamide confirmed the existence of suppressor cells in mice. Such cells were sensitive to treatment with anti- serum and complement, non-adherent to Sephadex G-10. A pretreatment of the mice with Cyclophosphamide eliminated DTH suppression. Treatment with antimacrophage agents via intraperitoneal abrogated suppression only if being effected before inoculation of alive 10⁸ Candida albicans.It is concluded that the spleen suppressor cell is a T-lymphocyte whose precursor is Cyclophosphamide-sensitive, requiring the macrophage to be induced.     

Immunoregulation in experimental schistosomiasis: III. Role of macrophages in soluble egg antigen-induced chronic spleen cell augmentation of baseline lymphocyte reactivity

Spleen cells from mice infected for 20 weeks with Schistosoma mansoni, exposed in vitro to soluble schistosomal egg antigens (SEA), treated with mitomycin C (Mc), and cocultured with syngeneic responder spleen cells increased the baseline proliferation of the otherwise unstimulated responder cells in cocultures. The role of macrophages in this “spontaneous” thymidine incorporation was studied directly by removal of macrophages on Sephadex G-10 columns. Removal of esterase-positive, Sephadex G-10-adherent cells (macrophages) greatly reduced the amount of SEA-induced, chronically infected spleen cell-mediated stimulation observed in cocultures. It also reduced an elevated background of spontaneous DNA synthesis seen with control cultures of spleen cells from infected animals. Depletion of T lymphocytes from chronic spleen cell populations by treatment with anti-Thy 1.2 serum and complement prior to exposure to SEA partially abrogated the augmentation effect. Comparison of these results with mitogen (concanavalin A)induced spleen cell-mediated stimulation (which is elevated, rather than reduced, by macrophage removal) and with known alterations in splenic T- and B-lymphocyte ratios in chronic murine Schistosomiasis suggests that antigen-stimulated, chronically infected splenic macrophage-de-pendent baseline augmentation may depend on specific T-lymphocyte-derived lymphokine induction. These results may reflect a general mechanism whereby animals harboring a persistent, chronic infection can respond quickly to a second or challenge infection or a flareup of the primary infection.     

Modulation of lymphoproliferation and oxidative burst by herpes-transformed tumors.

In this study, herpes simplex virus Type 2 (HSV-2)-transformed cells (H238) and conditioned medium (CM) from H238 cell cultures were studied with respect to their effects on lymphoproliferation and the chemiluminescent oxidative burst of phagocytic cells. The H238 cells expressed a nuclear antigen detectable by fluorescent antibody testing using pooled sera from tumor-bearing mice, but no HSV-1 or HSV-2 cell membrane antigens could be found using specific monoclonal antibodies. BALB/c mice subcutaneously injected with 1 X 10(6) H238 cells developed progressively growing fibrosarcomas and depressed T lymphocyte blastogenesis in response to phytohemagglutinin (PHA) by 6 weeks post-injection when compared to non-injected controls. In contrast, oxygen radical production was increased by nearly 28-fold in the tumor-bearing subjects at this time. Incubation of normal mouse spleen cells in 100 microliters to 500 microliters of CM/ml resulted in significant dose-dependent suppression of PHA-induced lymphoproliferation. This was seen when the total spleen cell population was used, as well as after removal of the adherent cells, thereby suggesting that the inhibitory effect was not due to activation of adherent suppressor cells by the CM. However, the oxidative burst of total and adherent spleen cells from normal mice was significantly enhanced by the presence of either the H238 cells or their CM. In contrast, oxygen radical production by J774A.1 cells (a BALB/c mouse macrophage cell line) was depressed by H238 cells. Our results show that H238 tumors can alter lymphocyte as well as phagocytic cell functions both in vivo and in vitro. These tumor-induced modulations may occur via secretion of soluble factors or direct cell-to-cell interactions and, thus, may influence the outcome of immunotherapy in the tumor-bearing host.     

Recruitment of null cells during graft versus host reactions in the chicken

Untreated SC (B2/B2) chicken spleen or thymus cells (2 × 10⁷) caused significantly increased [³H]thymidine incorporation in spleens of heavily irradiated FP (B15/B21) recipient chicks on Day 4 after iv injection. Mitomycin-treated SC spleen cells or spleen cells treated with rabbit anti-T-cell serum and complement failed to raise the [³H]thymidine incorporation over that in uninjected, bursa cell-injected or FP spleen cell-injected controls. However, the combination of mitomycin-treated spleen or thymus cells and anti-T-treated spleen cells caused an increased [³H]thymidine uptake, suggesting the recruitment of non-T cells into proliferation by alloreactive mitomycin-treated T cells. Bursa cells did not proliferate during GVH reactions even though they could be shown to undergo proliferation in vivo upon mitogen (lipopolysaccharide and dextran sulfate) stimulation. In contrast, anti-T-treated spleen cells from agammaglobulinemic chickens were recruited into proliferation, suggesting that the recruited cell was not only not a T cell, but also no pre-B or B cell and most likely represented a cell of the monocyte-macrophage series.     

Effect of tumor immunity on the distribution of labeled lymphoid cells in tumor-challenged mice

The distribution of ⁵¹Cr-labeled lymphoid cells from normal mice and mice immunized against a tumor were compared after intravenous inoculation of the labeled cells into normal syngeneic recipients. Spleen cell preparations from immune donors contained increased percentages of spleen and bone marrow-seeking cells, thus suggesting expansion of these cell populations when immunity to a tumor exists. Homing of labeled normal cells in tumor cell-injected normal animals was somewhat different from that seen in tumor cell-inoculated mice that were immunized against the tumor. In the latter case, accumulations of lymph node and spleen cells in recipient lymph nodes and bone marrow were consistently lower. In contrast, lymphoid cells from animals immunized against the tumor were found to accumulate in virtually the same percentages in lymphoid organs of normal and immune recipients. The behavior of lymphoid cell populations from thymus or bone marrow that consist mainly of precursor cells was unaffected by presence of malignancy and/or tumor immunity.     

The induction in vitro of immunological tolerance in T lymphocytes: an inhibitory role of macrophages.

Hapten-reactive helper T cells were generated in the spleen of C57BL/6 mice primed with sulfanilated syngeneic IgG (S-MGG). Specific immunological tolerance was induced in vitro in these helper T cells, when spleen cell suspension was passed through Sephadex G-10 column to remove adherent cells and cultured in the presence of soluble S-MGG for 21 to 24 hours. On the other hand, tolerance was not inducible in unfractionated, primed spleen cells. When G-10-passed spleen cells were added to the culture dishes containing phagocytic, adherent cells of the spleen, tolerance was no more inducible in these reconstituted cell population. From these experimental results, it was concluded that macrophages played an interfering role in tolerance induction. The experimental data were also discussed in terms of macrophage function in the recognition of antigen by T lymphocytes.     

Production of a factor inhibiting tumor cell migration by spleen cells from tumor-bearing mice

A factor inhibiting tumor cell migration was found in the culture supernatants of spleen cells of BALB/c mice transplanted with sarcoma cells 20 days before or injected with killed sarcoma cells 14 days before, when the spleen cells were stimulated in vitro for 2 or 24 hr with a 3 M KCl extract of the sarcoma. This factor did not inhibit the migration of guinea-pig macrophages or of theophylline-pretreated sarcoma cells. When the supernatants were fractioned on Sephadex G-100 columns, active fractions with molecular weights lower than 14,000 were distributed broadly.     

Suppression of Con A mitogen-induced proliferation of normal spleen cells by macrophages from chickens with hereditary muscular dystrophy

Spleen cells from chickens with hereditary muscular dystrophy (MD) give low blastogenic responses to the T cell mitogen concanavalin A (Con A) while exhibiting normal mitogen stimulated blastogenic responses to the T cell mitogen phytohemagglutinin (PHA). The addition of MD spleen cells to normal spleen cells caused a marked suppression of the Con A response of the normal cells while not affecting the PHA response of the normal cells. The suppressive activity by the MD spleen cells requires viable cells and is contact mediated. The suppressive activity is attributed to the presence in MD spleens of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by complement-mediated lysis using anti-T or anti-B sera, but it was reversible by treatment with carrageenan or carbonyl iron magnet, by passage through a Sephadex G-10 column, and by adherence to plastic petri dishes or glass beads. MD spleen cells depleted of the suppressor cell population remained unable to respond to Con A.     

Mixed lymphocyte reactivity in spleen cells from F-9 tumor-bearing ice. I. Suppressor cell induction and characterization

The immune capacity of spleen cells from F-9 tumor-bearing mice was assessed in a one-way mixed leukocyte culture (MLR) and in MLR mixing experiments. Alloreactivity was found to be significantly depressed in mice bearing palpable tumors. Mixing experiments demonstrated that the reduced alloreactivity was due to suppressor cell activity in the spleens of the tumorous mice. Suppressor cells were characterized as adherent, Ly2⁺ T cells. This study provides the basis for an analysis of the cell surface antigens on F-9 embryonal carcinoma cells which lead to the induction of suppressor T cells in these mice.     

Effector cells for antibody-dependent cell mediated cytotoxicity. I. Increased cytotoxicity after priming with BCG-SS.

Intraperitoneal injection of an aqueous extract of Bacillus Calmette-Guérin (BCG-SS) is shown to increase the cytotoxicity of murine spleen cells which mediate antibody-dependent cellular Cytotoxicity (ADCC). Three to forty-four days after in vivo stimulation with BCG-SS, spleen cells were tested for their ability to lyse antibody-coated chicken red blood cells in a ⁵¹Cr release assay. Significantly increased lysis compared to non-BCG-SS primed litter mates was observed from 3 days through 3 weeks after priming. Aqueous extracts of other bacteria including Listcria, Brucella, Salmonella, Staphlococcus and Eschcrichia did not elicit the same cytotoxic response. Separation of BCG-SS stimulated spleen cells on columns of G-10 Sephadex showed increased cytotoxicity in both the adherent (presumptive macrophage and polymorph) and nonadherent (presumptive K lymphocyte) populations. The possible relationship of these results to BCG-mediated anti-tumor effects is discussed.     

Ly49h-deficient C57BL/6 mice: a new mouse cytomegalovirus-susceptible model remains resistant to unrelated pathogens controlled by the NK gene complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8(+) T cells in B6.Ly49h(-/-) mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H(+) NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h(-/-) mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H(+) NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H(+) NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.     

The autologous mixed lymphocyte reaction in strains of mice with autoimmune disease

We have studied the autologous mixed lymphocyte reaction (AMLR) in 3 strains of mice with autoimmune disease. T cell proliferation to autologous non-T cells occurs in young mice of these 3 strains (as it does in normal mice) but is absent or greatly reduced in older mice of strains with autoimmune disease. Reciprocal mixing experiments revealed that the defect in the AMLR of the older mice resides in the responder T cell population. Further analysis of the cells participating in the AMLR of young mice of the B/W F1 strain revealed that: 1) a Thy 1- and Ly1-positive responder cell was necessary at the start of the culture to initiate the AMLR; 2) the cells present after 5 days of culture contained very few, if any, Ly123 cells in the B/W F1 strain compared with the normal C57BL/6 strain; and 3) the stimulating cell appear to be a macrophage, and an Ia-bearing cell must be present for the reaction to occur.     

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.     

Mouse lymphotoxin.

The addition of PHA to C3H mouse spleen cells in tissue culture led to the production of lymphotoxin (LT). Cytotoxicity was assayed by addition of the culture fluids to syngeneic target cells labeled with tritiated thymidine; after an incubation period of 72 hr the amount of radioactivity released into the supernatant was measured. The LT activity in unfractionated culture fluids survived lyophilization, remained unchanged for many weeks at 4 degrees C, and progressively decreased on heating at 56 degrees C for periods from 1 to 9 hr. Based on the G-200 Sephadex distribution coefficients for several preparations, the m.w. of mouse lymphotoxin was about 41,000 daltons. Lymphotoxin from three different spleen cell production runs was recovered from isoelectric focusing columns in sharply focused peaks, the pH of which ranged from 4.4 to 4.8.