Suppression of BCG cell wall-induced delayed-type hypersensitivity by BCG pre-treatment. II. Induction of suppressor T cells by heat-killed BCG injection.

Previous intravenous (i.v.) injection of heat-killed Bacillus Calmette-Guérin (BCG) in mice produced a suppression of delayed-type hypersensitivity (DTH) induced with oil-treated BCG cell walls. This phenomenon was analysed by the macrophage migration inhibition (MI) test in which non-adherent spleen cells from mice which had been injected with heat-killed BCG (K-Non-ad. cells) were mixed with peritoneal exudate cells from BCG cell wall-immunized mice (effector-PEC). The test showed that the K-Non-ad. cells suppressed the MI activity in the effector-PEC, which indicated that the suppressor cells were induced in the spleen by the heat-killed BCG injections. Moreover, the suppressive effect of the K-Non-ad. cells disappeared after treatment with anti-brain associated theta serum (BA theta) and guinea-pig complement, and operated across the H-2 barrier. The suppressor cells inhibited the production or release of the migration inhibition factor from the BCG cell wall-sensitized cells in the presence of the specific antigen, purified protein derivatives (PPD). It was concluded that the injections of heat-killed BCG produced antigen non-specific suppressor T cells in DTH suppression.     

Killed Listeria-induced suppressor T cells involved in suppression of delayed-type hypersensitivity and protection against Listeria infection.

Pretreatment of mice by intravenous injection with killed Listeria provided neither delayed-type hypersensitivity to Listeria protoplasm nor protection against Listeria infection. Assuming that this suppression is due to suppressor cells, we attempted to clarify their induction and characterization. Pretreatment with killed BCG instead of killed Listeria suppressed the induction of DTH and protection in subsequent Listeria-immunized mice. Conversely, pretreatment with killed Listeria suppressed subsequent induction of DTH to PPD or protection from tuberculosis. Thus, these suppressions were induced antigen nonspecifically. Transfer of splenic non-adherent cells from killed Listeria-injected mice which had been treated with anti-BA theta serum plus complement, or had been passed through Sephadex G-10 columns, resulted in both afferent and efferent DTH suppression, suggesting that the DTH suppression is closely associated with suppressor T cells. Moreover, the splenic nonadherent cells from killed Listeria-injected mice showed suppression in vitro of listericidal activity of PEC from Listeria-immune mice in the presence of Listeria protoplasm.     

An essential role for endogenous interferon-gamma in the generation of protective T cells against Mycobacterium bovis BCG in mice.

Protective CD4+ T cells against Mycobacterium bovis bacillus Calmette-Guérin (BCG), which are characterized by the ability to produce interferon-gamma (IFN-gamma), could be induced by immunization of mice with viable BCG but not with killed BCG. A high level of IFN-gamma mRNA was observed when normal spleen cells were stimulated with viable but not killed BCG. By comparing mice immunized with either viable or killed M. bovis BCG, it was found that a high level of IFN-gamma mRNA was expressed only after immunization with viable BCG. This finding prompted us to investigate the effect of neutralizing the IFN-gamma on the final generation of protective T cells against M. bovis BCG. When endogenous IFN-gamma was neutralized by administration of anti-IFN-gamma monoclonal antibody in mice immunized with viable BCG, the generation of protective T cells was significantly impaired, as revealed by the adoptive transfer of spleen T cells. The generation of BCG-specific, IFN-gamma-producing T cells was also abolished. These results clearly demonstrate that endogenous IFN-gamma actually plays a critical role in the generation of protective T cells against M. bovis BCG in vivo. Moreover, this study suggests that the lack of IFN-gamma-inducing ability is responsible for the inability of killed M. bovis BCG to induce protective T cells in mice.     

Suppression of BCG cell wall induced delayed-type hypersensitivity by BCG pre-treatment. I. Induction of adherent suppressor cells by live BCG injection and their characterization

Previous injections of live Bacillus Calmette-Guérin (BCG) in mice produced a suppression of delayed-type hypersensitivity (DTH) induced by oil-treated BCG cell walls (CW). This phenomenon was analysed by the macrophage migration inhibition (MI) test in which peritoneal exudate cells (PEC) from live BCG-injected mice were mixed with PEC from BCG CW-immunized mice, with the result that the former cells suppressed the MI activity in the latter. We considered the Mi test to be a reliable method for demonstrating the existence of suppressor cells induced by the injection of live BCG. Moreover, we found that the adherent cells of PEC possessed a suppressive effect which was retained even after treatment with either anti-mouse Ig or anti-brain associated theta (BA theta) antigen; that the PEC from mice injected with live BCG on at least the 12th day before cell harvesting showed the suppression; and that the suppression operated across the H-2 barrier.     

Production of migration inhibitory factor by Listeria-immune mouse T lymphocytes, but not B lymphocytes

Antigens and B cell mitogens have been reported to induce migration inhibition factor (MIF) production by mouse B cells. Immune resistance to the intracellular bacterium, Listeria monocytogenes is thought to involve T cells, but not B cells. Since Listeria-derived components are B cell, but not T cell mitogens, it was important to determine whether these materials could stimulate secretion of the lymphokine, MIF by T cells, B cells, or both. Thus populations of whole, unfractionated spleen cells, obtained from normal and Listeria-immune BDF1 mice, were cultured with or without 100 micrograms/ml of Listeria intracellular product (LIP). The culture supernatants obtained 24 h later were assayed for MIF activity using the in vitro macrophage migration inhibition assay. Data obtained show that immune T lymphocytes release MIF in response to specific Listeria antigens, but that spleen B cells from immune and normal mice, obtained as immune, nylon-wool-adherent cells treated with anti-T-cell serum plus complement, are not capable of releasing MIF. This suggests that release of lymphokines by Listeria-immune or normal B cells stimulated with Listeria-derived antigens and mitogens is unlikely to contribute to resistance against Listeria in vivo.     

Induction of suppressor T cells in delayed-type hypersensitivity to Mycobacterium bovis BCG in low-responder mice.

The induction of delayed-type hypersensitivity to Mycobacterium bovis BCG was specifically inhibited by suppressor T cells in C3H/He, a strain of mice which is a low responder to BCG. The existence of these suppressor cells was confirmed by an adoptive transfer of spleen cells of BCG-injected mice into cyclophosphamide-treated recipients. The suppressor cells appeared in the spleens of the mice 2 to 7 days after intravenous BCG injection. They were sensitive to anti-theta serum and complement and did not adhere to Sephadex G-10. A pretreatment of the mice with cyclophosphamide eliminated the suppression of delayed-type hypersensitivity. These suppressor cells effectively inhibited the induction of delayed-type hypersensitivity to BCG, but showed only weak effect on the expression of it.     

Intrathymic injection of antigen: a potent procedure for the induction of suppressor T cells.

Immunological tolerance is induced in mice by intrathymic injection of HSA. The tolerance thus induced is mediated by suppressor T cells. Strong tolerance persists more than 56 days after the induction, and the high efficiency of the tolerance thus induced is accounted for in terms of the number or the potentiality of suppressor cells. Possible mechanisms of suppressor T cell induction by iT injection of the antigen are discussed briefly.     

Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.     

Effects of intrathymic injection of organ-specific autoantigens,parietal cells,at the neonatal stage on autoreactive effector and suppressor T cell precursors

Thymectomy on day 3 after birth (3d-Tx) induces autoimmune gastritis (AIG) in 81%, and oophoritis (AIO) in 25% of BALB/c mice at the age of 2 to 3 months. Intrathymic, but not intraperitoneal injection of syngeneic parietal cells into sex-matched BALB/c mice within 24 h of birth resulted in almost complete prevention of the development of AIG in these mice in which 3d-Tx was performed. The prevention induced was parietal cell specific, since the development of AIO was not inhibited in female mice. Moreover, the injection of BALB/c liver cells, Mls-matched (BALB/c) and -disparate (DBA/2) B blasts which resulted in Vβ6 T cell deletion, as well as the injection of staphylococcal enterotoxin B failed to prevent the diseases. These findings suggested that recognition of an autoantigen in the thymus is necessary for the induction of tolerance, and that involvement of Mls-1 antigens in the pathogenesis of AIG, as has been suggested previously (Schwartz, R. H., Cell 1989. 57: 1073), was unlikely. T cells that suppress the development of organ-specific autoimmune diseases in 3d-Tx mice seem to maintain the unresponsiveness of autoreactive T cells at the periphery in normal mice. In agreement with our previous observations, we found that intraperitoneal (i.p.) injection of spleen cells from 3-month-old normal mice into 3d-Tx mice on day 10 after birth prevented the development of AIG, whereas spleen cells from age-matched AIG⁺ (mice with AIG) or AIG^? (mice without AIG) 3d-Tx mice failed to do this. This implies that the suppressor cells probably affect the differentiation of effector-precursor to effector. In fact, these suppressor cells did not inhibit the adoptive transfer of AIG to nu/nu BALB/c mice by spleen cells from 3d-Tx mice manifesting AIG. By negative selection using monoclonal antibody and complement, it was confirmed that the phenotype of the suppressor cell was CD4. In contrast to 3d-Tx, 10d-Tx did not induce AIG, indicating the peripheralization of the suppressor cell by that time. On the other hand, intrathymic injection of parietal cells immediately after birth did not affect suppressor cell generation, implying that some T cells, including suppressor cells, escape thymus selection. We postulate that these cells correspond to the precursors of the autoreactive effector T cells and suppressor T cells that are present in normal mice.     

Bacillus Calmette-Guérin (BCG) decreases resistance to Listeria monocytogenes infection in mice.

Bacillus Calmette-Guérin (BCG) inoculation has been shown to inhibit certain immune functions. To determine whether this inhibition adversely affects host defences against infection, the effect of BCG on Listeria infection in mice was investigated. Mice were injected intravenously (i.v.) with Listeria monocytogenes and 24-96 hr later were inoculated with 8 x 10(6) BCG. Mice given BCG and Listeria had a greater mortality and higher spleen Listeria counts than mice given Listeria alone. An increased number of bacteria in spleens was noted as early as 24 hr after BCG inoculation. Peritoneal macrophages from mice receiving both organisms had a decreased capacity to kill Listeria in vitro. In addition, BCG inoculation suppressed delayed hypersensitivity responses and in vitro spleen cell proliferative responses to Listeria antigen. Suppression of spleen cell proliferative responses was associated with an adherent, non-T lymphocyte subpopulation. The data indicate that BCG administration decreases resistance to intracellular pathogens by abrogating normal cellular defences.     

Selectivity in the Effects of Indomethacin on Bcg-Activated Suppressor Cell Populations

Intravenous inoculation of BCG into C57 B1/6 mice activated natural suppressor cells in the bone marrow and induced suppressor cells in the spleen. The suppressor activity of these cell populations was determined by co-cultivating them with normal lymphocytes being immunized against allogeneic P815 cells in vitro. Six million cells from the spleen or bone marrow of BCG treated mice inhibited by more than 50% the alloimmunization of twenty million normal syngeneic lymphocytes. The suppressor cells were found in the nylon wool adherent population in spleen and in both the nylon wool adherent and non-adherent POulations in bone marrow. Indomethacin, at a concentration of 10^--⁶M, completely blocked the suppression generated by unfractionated spleen or adherent spleen from BCG treated mice. However, 10^--⁶M and higher concentratlons of indomethacin only partially blocked the suppression generated by unfractionated marrow or adherent marrow from BCG treated mice. The suppression generated by non-adherent marrow from BCG treated mice was completely insensitive to indomethacin.     

Effect of Bacillus Calmette-Guérin on the in vitro generation of cytotoxic T lymphocytes. II. Role of interleukin-1-like factors and of soluble suppressor factors.

The injection of BCG vaccine in C57BL/6J mice results in the suppression of the generation of cytotoxic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC) and of mitogenic reactions to concanavalin A (Con A). Suppression is mediated by macrophage-like suppressor cells. Since previous work had indicated that suppression involved the inhibition of the production of interleukin-2 (IL-2), the effects of BCG on interleukin-1 (IL-1), a monokine required for IL-2 production, were investigated. It was found that the release of IL-1-like activity in spleen cell cultures stimulated with LPS or Con A was increased by previous BCG treatment of the cell donors. In MLC, the release of IL-1-like activity was also increased by BCG. However, the detection of IL-1-like activity in MLC supernatants was prevented by the presence of a suppressor factor. In this case, the IL-1-like activity could be separated with gel filtration from the suppressor factor which had higher molecular weight. The production of IL-1-like activity by CBA/J spleen cells, which are not suppressed by BCG, was not significantly different from that of C57BL/6J cells, which are markedly suppressed. Moreover, the addition of IL-1 to the BCG-suppressed cultures not only did not restore normal reactivity, but actually further suppressed CTL formation. It was concluded that BCG-induced suppression cannot be attributed to decreased IL-1 activity. The suppressor factor discovered during these investigations may have a role in this type of suppression.     

Effect of pretreatment with Bacillus Calmette-Guerin on the course of a Listeria monocytogenes infection in normal and congenitally athymic (nude) mice.

The effect of pretreatment with BCG on the course of a Listeria monomcytogenes infection was studied in nu/nu mice and in their heterozygous littermates (+/nu). First, evidence was presented that the nu/nu mice used lacked functional T cells, since BCG treatment resulted only in skin reactivity to tuberculin in +/nu mice and not in nu/nu mice. Acquired resistance to Listeria (based on lower spleen counts) was only obtained in BCG pretreated +/nu mice. Evidence was presented that BCG pretreatment in nu/nu mice failed to increase non-specific resistance, both in terms of spleen counts and survival rate. These results seem to imply that functional T cells are required for this type of non-specific resistance to heterologous antigens. In this connection attention has been drawn to the possible implication of BCG treatment in man.     

The mechanism of reduction of cell-mediated cytotoxicity in neonatally thymectomized mice.

The effect of neonatal thymectomy at various times after birth (Tx-1, Tx-7) on effector and suppressor T cells responsible for cell-mediated cytotoxicity (CMC) for allogenic antigens was determined. Following in-vitro primary mixed lymphocyte cultures, in the absence of T-cell growth factor (TCGF), alloreactive CMC was not detected in spleen cells of Tx-1 mice, but was detected in spleen cells of Tx-7 mice at as high levels as in those of sham-operated mice. However, in the presence of TCGF, as much alloreactive CMC was detected in spleen cells of Tx-1 mice as in those of Tx-7 mice. Furthermore, TCGF production was not detected in spleen cells of Tx-1 mice but was detected in those of Tx-7 mice. In in-vivo experiments, inhibition of allogeneic tumour growth and CMC in spleen cells showed the same pattern as in in-vitro experiments. These results support the concept that the reduction of CMC in Tx-1 mice might be due to a defect in helper function (TCGF-producing capacity) rather than to a defect in cytotoxic T lymphocytes and/or cytotoxic T lymphocyte precursors. Alloreactive suppressor T cells could not be induced in spleen cells of Tx-1 mice but were induced in spleen cells of Tx-7 mice. Therefore, it was suggested that alloreactive suppressor T cells require the presence of the thymus for 7 days after birth in their development.     

Involvement of inflammatory cytokines and nitric oxide in the expression of non-specific resistance toListeria monocytogenesin mice induced by viable but not killedMycobacterium bovisBCG

The non-specific defense againstListeria monocytogenescould be induced by viable BCG but not by killed BCG in mice. In order to understand the mechanism of antilisterial activity, viable and killed BCG were compared for their ability of inducing cytokine gene expression in spleen cells. Both viable and killed BCG induced the same level of mRNA expression of interleukin 10 (IL-10), transforming growth factor beta (TGF-β), IL-12 and tumor necrosis factor alpha (TNF-α). Gene expression and production of IL-1αand gamma interferon (IFN-γ) could be induced by stimulation only with viable BCG. Viable BCG but not killed BCG induced the mRNA expression of inducible nitric oxide synthase (iNOS). Treatment of mice withN^G-monomethyl-L-arginine acetate (NMMA) significantly impaired the non-specific antilisterial action induced by viable BCG. These results demonstrated that NO is an important mediator for the non-specific antilisterial activity induced by viable BCG, and IFN-γ, IL-1αand TNF-αmay play a critical role in the non-specific antilisterial activity.     

The effect of peripheral immunization with Mls-1a on the emigration of antigen-specific cells from the thymus.

Mature T cells found in the lymph nodes and spleen have the capacity to become activated and to proliferate in response to foreign antigens. The response of the thymus to such immunization is less well understood. We have examined one aspect of the thymic response by determining the effect of peripheral immunization upon cell emigration from the thymus. BALB/c (Mls-1b) mice were injected with spleen cells from DBA/2 (Mls-1a) mice, and V beta 6+ (Mls-1a-reactive) thymic emigrants were identified 3-30 days after immunization. Neither the rate of total cell migration from the thymus nor the proportion of V beta 6+ cells was altered, even though the immunizing spleen cells elicited an immune response in the draining (parathymic) lymph nodes. The same immunogen caused deletion of V beta 6+ cells in both the thymus and lymph nodes after intraperitoneal injection into the neonate. The inability of DBA/2 splenocytes to modify the development of adult thymocytes after intrathymic injection of the cells precluded the lack of entry into the thymus as the reason for the lack of any observed effect in the adult. Our results, therefore, indicate that the development of adult thymocytes is not modified by immunization, and suggest that the differing thymic response of mice injected as adults or neonates is related to changes in the intrathymic antigen presentation capacity associated with age.     

Evidence for two distinct populations of suppressor cells in the spleens of Mycobacterium bovis BCG-Sensitized mice.

Spleen cells from female C57BL/6 mice infected intravenously with 1 mg (about 10(7) viable units) of bacillus Calmette-Guérin (BCG) were shown to suppress the blastogenic responses induced by the T-cell mitogens phytohemagglutinin and concanavalin A and by the B-cell mitogen lipopolysaccharide in spleen cells from normal syngeneic mice. By using various separation procedures or cellular treatments, evidence was found for two distinct populations of splenic suppressor cells. One population belonged to the monocyte-macrophage lineage on the basis of their adherence to plastic surfaces, their removal after treatment with carbonyl iron, and their resistance to gamma irradiation. The other population of suppressor cells belonged to the T lymphocytes due to their sensitivity to an anti-Thy 1 antiserum and complement and to gamma irradiation. After separation on nylon wool columns, inhibitory activity was found in both the nonadherent and the adherent spleen cell populations. Both populations of suppressor cells were present in the spleens 14 days after BCG inoculation and persisted for at least 40 days after infection.     

In vitro induction of suppressor T-cells in delayed-type hypersensitivity to BCG and an essential role of I-J positive accessory cells

Antigen-specific suppressor T-cells in delayed-type hypersensitivity (DTH) to BCG were induced in vitro. Normal spleen cells of C3H/He mice were incubated with 50 micrograms of PPD per ml for 4 days at 37 degrees C, and the non-adherent cells in the culture were transferred intravenously into cyclophosphamide (CY)-treated syngeneic recipients. The recipients were immunized to BCG immediately after the cell transfer, and DTH was measured by the footpad reaction to PPD two weeks later. Footpad reaction to PPD was positive in CY-treated C3H/He mice immunized to BCG, while it was suppressed by the transfer of the in vitro induced suppressor cells. When the suppressor cells were treated with anti-thy-1.2 antiserum and complement before transfer, the suppression was abrogated. Next, the spleen cells were separated into plastic adherent and non-adherent fractions. After treatment with anti-thy-1.2 and complement, the adherent cells were treated with either anti-I-Jk or anti-I-Ak antiserum and complement. Then, they were reconstituted with the non-adherent cells and cultured with PPD. Treatment of the adherent cells with anti-I-Jk antiserum and complement abrogated the suppressor cell induction, while the treatment with anti-I-Ak had no effect. These facts indicate that I-J positive non-T-adherent cells play an essential role in the induction of suppressor cells in DTH.     

Antigen-specific suppressor cells in hapten-specific carrier-determined tolerance.

Tolerance to the hapten 2,4-dinitrophenyl (DNP) induced by the injection of DNP coupled to isologous IgG (carrier-determined tolerance) is associated with a receptor blockade of antigen-binding lymphocytes. In the present study, hapten-specific suppressor cells were detected in the spleens of mice made tolerant by intravenous injection of 20 microgram DNP-IgG. When spleen cells from mice rendered tolerant to DNP were co-cultured with normal spleen cells in Marbrook cultures, the response to DNP-Ficoll was suppressed, while the response to sheep red blood cells was not altered. Depletion of T cells from these spleens restored the normal anti-DNP response. The suppressor cells were not detectable in the spleen lymphocyte population of mice in the early stages of tolerance but were present on day 7 after injection of tolerogen, and disappeared by day 14. Mice injected with larger doses of 1 mg or four weekly doses of 200 microgram DNP-IgG did not have detectable suppressor cells. Thus, it appears that a short-lived suppressor T cell is generated in carrier-determined tolerance. This cell most likely plays a minor role in the mechanism of carrier-determined tolerance and may be associated with the receptor blockade which is seen early in tolerance.     

Development of suppressor T cells in mice heavily infected with mycobacteria

Specific pathogen-free B6D2 mice were infected intravenously with 10(8) viable BCG, M. habana or M. simiae and the level of tuberculin hypersensitivity to 2.5 micrograms PPD or cytoplasmic protein antigens (CPA) prepared from the other organisms was determined using the footpad swelling test with increasing time after infection. This was correlated with the growth or persistence of mycobacterial populations within the liver. Spleen cells were removed from these infected mice and the level of blast transformation following exposure to PHA, PPD or M. habana or M. simiae CPA was measured in vitro. Early in the mycobacterial infections (day 14) thymidine incorporation by the spleen cells was significantly enchanced followed by a profound depression in incorporation rates as the infection progressed. The mechanism of this depressed response involved the production of suppressor T cells in the spleen. In the case of the M. simiae or M. habana infection, cells capable of mediating suppression were still present even after 12 months of infection. In the BCG infection, suppressor T cells declined with time so that by 4 months incorporation rates were back to normal and suppressor cells were no longer detectable in the spleens of the infected animals.