The effects of intravenous administration of Methanol Extraction Residue (MER) of tubercle bacilli in the dog

Summary The effects of intravenous administration of the insoluble methanol extraction residue (MER) of tubercle bacilli were studied in six adult Beagle dogs. All animals were closely monitored for clinical signs, alterations in serum biochemistry or hematologic values, and for gross and histologic findings at autopsy. Immune responsiveness was assessed by skin reactivity to test antigens and by ability of spleen and lymph node lymphocytes to produce macrophage activating factor (MAF).Regardless of the dose of MER or frequency of administration, there were no clinical or biochemical alterations detected in the animals. Immediately after injection of MER, a massive but transient granulocytopenia appeared. Skin reactivity failed to demonstrate any alterations in immune responsiveness, but lymphocytes from MER-treated dogs released MAF in response to in vitro stimulation with purified protein derivative (PPD) in contrast to those from nontreated control dogs.Histologic findings were associated with the immune response to MER and were most dramatic in the liver and lungs, where formation of a large number of epithelioid granulomas was observed. MER, intravenously administered in relatively high doses, was well tolerated by dogs, in whom it produced little evidence of toxicity, and seemed to increase some systemic immunologic effects.     

Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.     

Immunoregulation in experimental filariasis. II. Responses to parasite and nonparasite antigens in jirds with Brugia pahangi

Chronic B. pahangi infection (greater than or equal to 5 mo) in the jird, Meriones unguiculatus, leads to the induction of adherent nonspecific suppressor cells that are capable of modulating the in vitro mitogen responsiveness of spleen cells. In the present studies, a correlation between suppression of mitogen responsiveness and lack of reactivity to B. pahangi antigens was observed in vitro with splenic lymphocytes from chronically infected animals. However, the ability of jirds with a chronic B. pahangi infection to develop in vivo humoral responsiveness to SRBC and DTH to DNFB was comparable to that of uninfected controls. Analysis of the relationship between the development of antigen-specific and nonspecific immunoregulatory activity over the course of the infection was undertaken, too. Altered in vitro responsiveness of spleen cells from infected jirds to mitogens and B. pahangi antigens was associated with the onset of microfilaremia (8 wk post-infection). A transient lack of reactivity to SRBC was observed after the development of a patent infection in jirds. However, nonspecific suppressor cells capable of modifying the in vitro mitogen responsiveness of normal lymphocytes were not observed in the spleens of B. pahangi-infected animals exhibiting a lack of reactivity to SRBC. The relationship of antigen-specific suppressor cells to immunoregulation in experimental filariasis is discussed.     

Differential responsiveness of various substrains of inbred strain 2 guinea pigs to immunotherapy with the methanol extraction residue (MER) of BCG

Summary The immunotherapeutic effects of the methanol extraction residue (MER) of BCG were investigated in strain 2 guinea pigs bearing the transplantable line 10 hepatocarcinoma, a tumor originally induced in guinea pigs at the National Institutes of Health (NIH) by ingestion of the carcinogen diethylnitrosamine. MER was more effective in mediating tumor regression in guinea pigs obtained from the Weizmann Institute of Science (WI), Rehovot, Israel, than in animals obtained from the National Institutes of Health (NIH). These differences indicate the dramatic effects which minor histoincompatibilities between cancer cells and animal substrains may have on experimental results, and highlight the need for immunotherapy experiments to be conducted on laboratory tumors grown in their autochthonous hosts. MER was effective only when injected directly into growing tumor nodules and had no effect on tumor development when administered distally. In contrast, all animals which received both MER and tumor cells developed specific cell-mediated anti-tumor immune responsiveness at higher levels than did non-MER-treated tumor-bearing controls as measured by delayed cutaneous hypersensitivity and in vitro lymphocyte reactivity experiments. Furthermore, the results of the latter but not the former studies suggested that guinea pigs which received MER were able to mount such an immune response more rapidly than their non-treated counterparts. This apparent stimulation of anti-tumor immunity was observed in treated animals regardless of substrain or site of MER injection, and could not be correlated with the outcome of immunotherapy.     

Effects of the MER tubercle bacillus fraction on the production of antibodies in vitro: I. Effect on the primary response

The effect of the methanol extraction residue (MER) fraction of BCG tubercle bacilli on the primary antibody response in vitro to sheep red blood cells (SRBC), TNP conjugates, and the monovalent hapten DNP-glycine was studied. Addition of MER to whole splenocyte cultures simultaneously with antigen presentation potentiated the antibody response to SRBC and TNP-SRBC, and facilitated reactivity to DNP-glycine; there was no effect on the response to the T-independent entity TNP-LPS (lipopolysaccharide from E. coli 055-B5). Immunopotentiating activity of MER for SRBC and DNP-glycine was also evident in macrophage-depleted cultures. Peritoneal exudate cells (PEC) taken from MER-treated donors were more efficient than PEC from untreated donors in reconstituting antibody formation to SRBC by macrophage-depleted spleen cell populations. The results obtained indicate that activation of both macrophages and of certain lymphocyte population(s) by MER may play a role in the potentiation of antibody responsiveness in vitro by this agent.     

Potentiation and modulation of the immune response of guinea pigs to poorly immunogenic protein-hapten conjugates by pretreatment with the MER fraction of attenuated tubercle bacilli

The MER fraction of attenuated tubercle bacilli of the BCG strain was shown capable of stimulating and modulating the immunological responsiveness of guinea pigs to immunization with DNP conjugates of allogeneic globulin (DNP-GPG) and xenogeneic albumin (DNP-HSA). These antigens are very poorly immunogenic and fail to evoke detectable immune responses following single administration alone.When incorporated in incomplete Freund's adjuvant (IFA) together with the conjugates, MER could substitute for whole tubercle bacilli in the adjuvant mixture, and cause the conjugates to evoke both cellular and humoral reactivity, the former indicated by the development of skin reactions of delayed type (DH) to test injections of the antigens, the latter by the formation of humoral antibodies detected by an indirect hemagglutination (HA) test. When administered in saline together with antigen, MER was ineffective.Pretreatment with MER by any of several different routes 7 or 14 days prior to sensitization enabled a large number of the animals to respond with either DH, circulating antibody formation, or both. Under similar circumstances, pretreatment with Freund's complete adjuvant (FCA) elicited no such preparatory effect. In order to be efficacious in pretreatment, MER had to be given in a saline suspension; activity was lost when it was applied in IFA.MER pretreatment modulated the immune response to subsequent sensitization with the conjugates preferentially towards DH or antibody production, depending on the parameters of treatment and specific immunization. It appeared that when the specific immunogenic stimulus was weak, pretreatment with MER strongly favored DH.     

T cell regulation of polyclonal B cell responsiveness. I. Helper effects of T cells.

Polyclonal activation of murine splenic B lymphocytes to secrete immunoglobulin was shown to be subject to regulation by splenic T cells. By admixture of separated B and T cell populations it was demonstrated that normal fresh splenic T cells were able to augment polyclonal B cell responsiveness to LPS up to several-fold. Optimal collaboration between these two cell types ensued when they were co-cultured in equal numbers. T cell-mediated enhancement of polyclonal B cell responses was dependent upon the ability of T cells to divide and was manifested upon T cell interaction with B cells soon after culture initiation. Originally expounded as a one-signal phenomenon, polyclonal activation of lymphocytes by LPS is, under the circumstances described, attributable instead to two distinct, nonspecific signals acting in concert. The observation that T cells from LPS-nonresponder (C3H/HeJ) mice were deficient in the capacity to enhance polyclonal B cell responsiveness of B cells derived from responder (C3H/HeN) mice implied a direct action of LPS on the involved T cells as well as an active role for the T cell signal in this immunoregulatory event. The novel observation of a functional T cell defect in LPS responsiveness in the C3H/HeJ mouse is discussed in terms of its other cellular defects.     

Induction by an immunogenic immunomodulating agent of nonspecific T cell suppression of lymphocyte responsiveness in MLR but not of antibody production

Summary Spleen cells derived from BALB/c mice that had been repeatedly immunized with the methanol extraction residue (MER) fraction of tubercle bacilli exhibited a depressed capacity to act as responder cells in allogeneic and syngeneic mixed lymphocyte reactions (MLR). Previously reported studies revealed that such spleen cells are also defective in the in vitro generation of antibodies. In order to determine the nature of the cells responsible for the depressed MLR reactivity, purified populations of splenic macrophages, B lymphocytes, T lymphocytes originating from normal and from MER-immunized mice, and cell culture supernatants were added to MLR mixtures consisting of normal mouse splenocytes. Macrophages originating from MER-immunized mice and their culture supernatants exerted a significantly higher suppressive effect on MLR than that of corresponding preparation from normal mice. Splenic T cells originating from MER-immunized mice and their supernatants also significantly suppressed the MLR response. However, the same T cell populations that were inhibitory in MLR failed to suppress the in vitro generation of antibodies against sheep red blood cells in the presence of either MER or 2-mercaptoethanol. These and previously reported findings indicate that a nonspecific immunomodulating agent, MER, can, under certain conditions of treatment, elicit the induction of nonspecific suppressor T cells for MLR but not for antibody production, and, accordingly, can inhibit cellular and humoral immunological responsiveness by different mechanisms.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4 dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by DNFB skin application. Development of skin CH was suppressed by exposure of the animals after sensitization to the cancer chemotherapeutic drugs cyclophosphamide (CY), sodium methotrexate (MTX), and 5-fluorouracil (5FU). Unresponsiveness to DNFB was also induced in parallel experiments by a single intravenous injection of dinitrobenzenesulfonate (DNBS), either before or concomitant with sensitization. Potentiation of CH skin reactivity was achieved by administration of CY prior to sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of animals exposed to CY, MTX, or 5FU to respond to DNFB sensitization. The agent did not impair the potentiation of CH skin reactivity that could be effected by administration of CY prior to sensitization.MER treatment was not effective in reversing hapten-induced (DNBS) tolerance in mice.These findings favor the assumption that MER, under the conditions tested, stimulates the function of positively reacting T cells and exerts no enhancing or protective action on suppressor T cells.     

Effect of treatment with the MER tubercle bacillus fraction on syngeneic plasma cell tumors of BALB/c mice

Summary MER administered prophylactically prolonged the survival of BALB/c mice challenged with transplants of syngeneic plasmacytomas to a moderate but significant extent. In contrast, MER exerted little therapeutic action when given alone at or after tumor implantation.Combined treatment, with MER introduced prophylactically and cyclophopshamide (CY) after tumor implantation, decreased the incidence of recurrence of one of the tumors tested (MPC-11 NP, a non-myeloma protein producer) and prolonged host survival significantly as compared with animals subjected to CY therapy alone. When, instead, MER was introduced at the time of tumor challenge or thereafter to animals also treated with CY, the therapeutic response was not appreciably different from that of mice under therapy with CY only. With regard to the second plasmacytoma (MPC-11 P, a myeloma protein producer), mice treated with CY and given MER prior to or after challenge showed similar responses to animals given chemotherapy only; when MER was injected at the time of challenge and CY thereafter, the chemoimmunotherapy was somewhat inferior to chemotherapy alone.This work was supported by US Public Health Service Contract NO1-CM-12127, and by grants from Mrs. J. H. Hazen, Ruth Estrin Goldberg Memorial for Cancer Research, Leukemia Research Foundation, Inc., Concern Foundation, Inc., and Mr. and Mrs. M. Gordon     

Differences in the repertoire expressed by peritoneal and splenic Ly-1 (CD5)+ B cells.

Purified populations of B cells expressing the Ly-1 and/or Mac-1 surface Ag were isolated from normal unmanipulated mice by cell sorting. The number of lymphocytes in each population secreting antibodies reactive with DNA, bromelain-treated mouse RBC, phosphorylcholine and TNP-keyhole limpet hemocyanin was quantitated by ELISA spot assay. The proportion of B cells secreting Ig in vivo and the repertoire of antibodies they produced varied as a function of B cell phenotype and location. Among peritoneal lymphocytes, those that were Ly-1+ or Ly-1- Mac-1+ secreted Ig 10 times more frequently that Mac-1- Ly-1- B cells from the same location. In addition, the former populations expressed repertoires that were significantly skewed toward the production of antibodies reactive with bromelain-treated mouse RBC (p less than 0.001). In contrast, splenic B cells expressing the Ly-1 surface Ag did not differ significantly from splenic Ly-1- B cells in their expressed repertoire or frequency of Ig production. B cells isolated from the spleen and peritoneum tended to differ in antibody specificity from bone marrow and lymph node-derived lymphocytes. For example, B cells from the spleen secreted anti-DNA antibodies two to four times more frequently than B cells from other organs. These results demonstrate that phenotype and microenvironment influence the repertoire of antibodies expressed by B cells in vivo.     

Cellular immunity in the mouse. I. In vitro lymphocyte reactivity

The mixed lymphocyte culture (MLC) and antigen-mediated proliferative response represent important correlates to the in vivo phenomena of allograft rejection and delayed hypersensitivity. This study defines an in vitro model to measure mouse lymphocyte responsiveness to allogeneic cells, antigen (tuberculoprotein), and nonspecific mitogens. Results describe optimal cells concentration, time and conditions of culture. Optimal conditions include the use of high cell concentration, flat-bottomed vials, RPMI-1640 medium, and fresh human serum. Peripheral blood lymphocytes demonstrated greater proliferation than lymph node lymphocytes, which in turn demonstrated greater activity than splenic lymphocytes. Significant proliferation occurred in serum-free media, dialyzed against fresh serum and supplemented with hydrocortisone and carrier protein. The MLC response in the mouse appears dependent on multiple subpopulations of cells and on soluble substances produced by them.     

Effect of methanol extraction residue tubercle bacillus fraction treatment in vivo and in vitro on the release and activity of suppressor factor inhibiting the efferent phase of contact sensitivity in mice

BALB/c mice subjected to injection of dinitrobenzenesulfonate (DNBSO3) and skin painting with dinitrofluorobenzene (DNFB) produce splenic T-suppressor (Ts) cells that, when transferred to DNFB-sensitized recipients, suppress the efferent (eff) phase of contact sensitivity (CS). Splenocyte populations containing such Ts-eff cells release a specific soluble suppressor factor (SSF) in vitro that similarly suppresses CS in sensitized recipient mice. Treatment with the methanol extraction residue (MER) tubercle bacillus fraction, a known immunomodulating agent, of animals exposed to DNBSO3 and DNFB ("DNBSO3-DNFB" donors) prevented release of SSF by their spleen cells at in vitro incubation. Incubation of DNBSO3-DNFB donor splenocytes with MER in vitro also abolished SSF release, and treatment with MER of test animals prior to DNFB sensitization prevented the suppressive action of subsequently administered SSF. The observations are discussed with regard to the potentiating capacities of MER on cellular immunity and states of resistance.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

In vitro immune blastogenesis during contact sensitivity in tumor-bearing mice: I. Description of progressive impairment and demonstration of splenic suppressor cells

T-cell responsiveness was measured by the DNA response of disassociated spleen and lymph node cells when exposed to antigen in vitro. Sensitized splenic lymphocytes from fibrosarcoma-bearing mice immunized with 2,4-dinitro-1,5-difluorobenzene (DN2FB) demonstrated a progressive decrease in T-cell responsiveness to the haptenprotein conjugate DNP-BSA. Hyporesponsiveness to the dinitrophenylated-protein conjugate appeared in the spleens but not lymph nodes of tumorous animals. Normal host lymph node cells (LNC) responded strongly 24 to 48 h after sensitization and subsequently declined with a corresponding increase in responsiveness in the spleen. Tumor-bearing hosts (TBH) had similar LNC kinetics during immunization, however, spleen cells were significantly suppressed when compared to normal BALB/c mice sensitization kinetics. Spleen cells from TBH were also capable of suppressing the in vitro response of normal primed lymphocytes to DNP-BSA when admixed. Results from these experiments suggest that in vitro measurement of contact sensitivity was affected by suppressor cells/products existing in the spleens but not lymph nodes of fibrosarcoma-bearing mice.     

Antisense RNA of the latent period gene (MER5) inhibits the differentiation of murine erythroleukemia cells

The MER5 cDNA was cloned from RNA preferentially synthesized in murine erythroleukemia (MEL) cells during the early period of MEL cell differentiation. To understand the role of the MER5 gene in the differentiation, we have transferred the MER5 cDNA into MEL cells in both sense and antisense orientations under control of the promoter of the human metallothionein gene. Only in the transformants with the antisense MER5 cDNA, did their elevated expression inhibit differentiation. The result suggests that the MER5 gene product may promote early events in the differentiation of MEL cells.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

Induction of nonspecific cytotoxicity by monoclonal anti-T3 antibodies

The effects of monoclonal anti-T3 antibodies on the effector phase of cytotoxic T lymphocytes (CTL) were studied with respect to antigen-specific and antigen-nonspecific lysis of different target cells. Anti-T3 antibodies inhibited the antigen-specific lysis by CTL generated in mixed lymphocyte cultures (MLC), but they concomitantly augmented the nonspecific killing of third-party cells such as the cell lines Daudi, Raji, and K562. This nonspecific cytotoxicity was induced by various anti-T3 antibodies, whereas antibodies reactive with other antigens expressed on the cytotoxic effector cells lacked any such activity. Anti-T3 antibodies induced nonspecific cytotoxicity only when activated T cells, obtained by primary MLC, by repeated restimulation, or after cloning, were used. The antibodies had no effect on unstimulated peripheral T lymphocytes or thymocytes. The inhibition of the antigen-specific lysis and the induction of nonspecific lysis by anti-T3 was dose dependent, and both effects occurred at the same concentration range of anti-T3. F(ab')2 fragments of anti-T3 inhibited the specific lysis but were not able to induce cytotoxic activity, indicating that this induction is an Fc-dependent process. When different target cells were tested, only Fc receptor-positive cells were susceptible for this nonspecific cytotoxicity. Thus, anti-T3 antibodies have a dual effect on effector CTL: they inhibit antigen-specific lysis and concomitantly induce nonspecific lysis in an Fc-dependent way.     

T and B lymphocytes of irradiated, tumor-bearing mice treated with an immunostimulator, pyran.

The T and B splenic lymphocyte populations of BALB/c mice were determined in Madison lung 109 carcinoma-bearing animals. Concurrently, some groups of tumored mice were exposed to 500 rads of whole body irradiation and treated with one dose of a nonspecific immunostimulating agent, pyran. By indirect immunofluorescence, it was found that the percentage of splenic T lymphocytes were significantly depressed in the tumored irradiated mice. Mitogenic studies revealed that the PHA-sensitive T lymphocytes were more depressed in the tumored irradiated mice than in the corresponding Con A-sensitive T lymphocytes. Pyran was relatively effective in reconstituting the T cell compartment of these splenic T lymphocytes.The B cell compartment of the splenic lymphocytes of the tumored irradiated mice was found to be extremely radiosensitive. Utilizing a specific anti-F(ab')₂ serum, no B lymphocytes were detected throughout the duration of testing. Blastogenic studies using LPS as the mitogenic probe revealed that the incorporation of [³H]TdR of the tumored irradiated mice was just slightly higher than background values. Pyran proved to be relatively ineffective in reconstituting the splenic B cells of the tumored irradiated mice.     

Production of autoantibodies associated with polyclonal activation in Yersinia enterocolitica O: 8-infected mice

Polyclonal lymphocyte stimulation is one of the immunomodulatory mechanisms induced by arthritogenic pathogens. In this study we examined the polyclonal activation potential of a virulent strain of Y. enterocolitica serotype O: 8 (WA 2707(+)) and its plasmidless isogenic pair (WA 2707(-)). SPF Swiss mice were infected intragastrically and spleen cells were obtained on days 7, 14, 21, 28, 35 and 42 after infection. The number of cells secreting nonspecific immunoglobulins of IgG, IgM and IgA isotypes was determined by the ELISPOT technique. The presence of serum-specific antibodies was investigated by ELISA and the presence of autoantibodies by dot-blot assay. Although the patterns of infection of the two bacterial strains were almost the same, only the animals infected with the virulent strain presented clinical anomalies. Neither arthritic nor inflammatory signs were observed in the joints of the infected animals. The greatest activation observed was that of the nonspecific IgM-secreting cells, and their peak of secretion occurred between the 28th and the 42nd day after infection, for both strains of Y. enterocolitica O: 8. Only the animals infected with the virulent strain (WA 2707(+)) produced IgG-specific antibodies in the serum, from the 28th day after infection. The serum of animals infected with either strain showed reactivity to all the autologous constituents tested, mainly on the 28th and 42nd day after infection. It was concluded that infection of mice with either the virulent strain of Y. enterocolitica O: 8 or with its plasmidless isogenic pair resulted in the polyclonal activation of the splenic B lymphocytes including some autoreactive clones.