In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

The mediator of cellular immunity. X. Interaction of macrophages and specifically sensitized lymphocytes.

Specifically sensitized lymphocytes have a focusing influence on mononuclear phagocytes that is expressed in the local accumulation and division of macrophages in bacteria-induced exudates. This was demonstrated by injecting Listeria monocytogenes into the peritoneal cavity of normal rats immediately before the animals were transfused with thoracic duct lymphocytes from either Listeria-immune donors or donors that had been infected with the unrelated parasite, Francisella tularensis. Sensitized lymphocytes originally present in the intravenous inocula were found later in the exudates. The arrival in the inflamed peritoneal cavity of specifically sensitized lymphocytes was associated with an exuberant influx of newly formed host cells and a local proliferative response that involved both immunoblasts and macrophages. These cytokinetic observations provide a plausible explanation of the delayed inflammatory response induced by the parasite, and imply that sensitized lymphocytes contribute to the host's defence by encouraging the prompt and purposeful deployment of monocyte-derived macrophages in centers of infection. In addition to their focusing influence on mononuclear phagocytes, specifically sensitized lymphocytes or their products can enhance the metabolic and microbicidal activity of macrophages. This activation process was revealed in the ability of rats infected with L. monocytogenes or BCG to control the growth of F. tularensis at a challenge site in the testis. But resistance was expressed only when the challenge organisms were injected with killed bacteria against which the recipients had been specifically immunized. The results accord with the view that macrophages are functionally activated by an immunological mechanism, and imply that the process is triggered locally by sensitized lymphocytes which are recruited from the blood.     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Macrophage activation of allogeneic lymphocyte proliferation in the guinea pig mixed leukocyte culture.

The role of the macrophage in the guinea pig mixed leukocyte culture was investigated. Macrophages obtained from oil-induced peritoneal exudates, peritoneal wash-out cells, spleen, and alveolar washings were found to be effective stimulators of allogeneic lymph node and splenic lymphocyte DNA synthesis. The stimulatory properties of macrophages proved radioresistant but viability dependent. Unfractionated lymph node cells or adherence column purified lymph node lymphocytes and thymocytes were only minimally active as stimulators, even in the presence of macrophages syngeneic to the responder lymphocytes. Allogeneic fibroblasts, polymorphonuclear leukocytes, L2C leukemia cells, and xenogeneic (murine) macrophages failed to simulate. These data provide evidence that the macrophage is the predominant stimulator of the mixed leukocyte culture in the guinea pig.     

Identification of the principal cell type yielding immune RNA capable of transferring tumor-specific cellular cytotoxicity

A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC.     

The mediator of cellular immunity: XI. Origin and development of MIF producing lymphocytes

Thoracic duct lymphocytes obtained from rats infected with Listeria monocytogenes were characterized with respect to size, turnover and their capacity to release macrophage migration inhibitory factor (MIF). Cells responsive to Listerial antigens (LMA) in the MIF assay were identified in lymph during the first week of an immunizing infection. These were immunoblasts or large lymphocytes, as evidenced by their sedimentation with S phase lymphocytes at unit gravity. When labeled cells from the lymph of Listeria-infected donors were infused into similarly infected recipients, donor S phase lymphocytes localized rapidly, and in substantial numbers, in peritoneal exudates induced by the unrelated organism, F. tularensis. Within this immigrant population were cells which conferred immunity against L. monocytogenes and released MIF in cultures containing LMA. Exudates harvested 36 hr or 61 hr after stimulation contained labeled lymphocytes that were smaller than the S phase cells recovered during the early post-induction period. The observed shift of radioactivity from large to smaller lymphocytes was parallelled by a shift MIF production to exudate fractions containing smaller cells. The MIF producing cells in exudates of advancing age also exhibited increasing resistance to inhibition by vinblastine. These findings suggest that MIF is released by a family of lymphocytes—large, medium and small. LMA-responsive lymphocytes are delivered to the thoracic duct soon after their formation, at a stage in development when they can be stimulated to release only low levels of MIF. These mediator producing cells circulate briefly in the blood and differentiate fully only after they extravasate into inflammatory foci.     

In vitro studies on the mechanism of acquired resistance to tuberculous infection. II. The effects of the culture supernatants of specifically stimulated-sensitized lymphocytes on the growth of tubercle bacilli within macrophages.

Immune lymph node cells were obtained from mice immunized with bovine gamma globulin (BGG) in complete Freund's adjuvant or allogeneic MH134 tumor cells. They showed the capacity of conferring bactericidal activity on macrophages infected with Mycobacterium tuberculosis, H37Rv, when they were incubated on macrophage monolayers together with the corresponding antigen, i.e., BGG or solubilized cellular antigen of the tumor cells. However, such capacity was lower than that of tubercle bacilli-immune lymph node cells. Culture supernatants were harvested after incubation of tubercle bacilli-immune, BGG-immune or allogeneic tumor-immune lymph node cells with the corresponding antigen for 24 hr. Macrophages were altered so as to suppress intracellular bacillary growth when macrophage monolayers were exposed to the supernatants for more than 2 days. When normal lymph node cells were incubated on normal macrophage monolayers together with a mitogen such as PHA or concanavalin A, growth of tubercle bacilli within the macrophages was slightly but difinitely suppressed. The mechanism of elicitation of cellular immunity to the infection with tubercle bacilli is discussed on the basis of results presented in this and the preceding paper.     

The mediator of cellular immunity. VIII. Effect of mitomycin C on specifically sensitized lymphocytes.

The antimitotic agent, mitomycin C, was used to probe cellular events underlying the expression of acquired resistance to L. monocytogenes in the rat. It was found that thoracic duct lymphocytes from donors primarily immunized with this organism can protect normal recipients against a Listeria challenge and that their ability to do so is inhibited by treatment of the lymphocytes with mitomycin C. Concentrations of the drug which are effective in this regard also inhibit lymphocyte proliferation, but have little if any effect upon RNA or protein synthesis in cells tested at the time of transfer. Yet mitomycin C probably has irreversible effects on lymphocytes in addition to its capacity to block cell replication, for inhibitor-treated immunoblasts fail to localize in peritoneal exudates induced in recipient rats. The inability of mitomycin-treated immunoblasts to extravasate in inflammatory foci is associated with a diminished capacity of specificially sensitized lymphocytes to promote cell division in peritoneal exudates induced by L. monocytogenes. The results illustrate the importance of lymphocyte-macrophage interactions in cellular resistance to infection and give credence to the view that these interactions occur locally at sites of microbial invasion.     

Generation of macrophage chemotactic activity in situ in Listeria-immune rats.

Macrophage chemotactic activity (MCA) is generated in situ in peritoneal inflammatory exudates induced by antigens of the intracellular parasite, Listeria monocytogenes. Chemotactic and chemokinetic activity is formed locally in response to an immunologically specific signal. In rats that have been immunized adoptively with thoracic duct lymphocytes (TDL) from specifically immunized donors, the production of MCA depends upon stimulation by LMA of exudate-seeking S-phase lymphocytes of their progeny. The sequential appearance, increase, and subsequent decline of MCA in the peritoneal cavity parallels the influx of lymphocytes and precedes maximal recruitment of labeled monocytes from the blood. The MCA response in peritoneal exudates induced in adoptively immunized rats correlates with the level of macrophage accumulation in the peritoneal cavity and at sites of LMA injection in the pinna of the ear. The results suggest that MCA is released locally by antigen-activated lymphocytes and imply that the factor(s) concerned has a purposeful role in the host's defense by promoting the rapid deployment and/or retention of monocyte-derived macrophages in centers of infection.     

In vitro and in vivo studies on the mechanism of experimental autoimmune thyroiditis in guinea pigs

Thyroid explants of inbred strain 13 guinea pigs were grown in a semisynthetic medium containing 0.3 IU of thyroid-stimulating hormone. The monolayer retained the capacity in vitro to form thyroglobulin. Sensitized lymphocytes from animals with autoimmune thyroiditis could specifically lyse these thyroid target cells in vitro in the presence of an appropriate amount of specific antigen. This cytotoxicity was not observed in thyroid epithelial cells which had been incubated (a) with normal lymphocytes or (b) with purified macrophages either from normal animals or from animals with autoimmune thyroiditis. When thyroid cells were incubated with hyperimmune antithyroglobulin serum, cytolysis did not occur, whether or not complement was added. The cytopathic effect of sensitized lymphocytes was further demonstrated to be caused by a soluble cellular product, termed thyroid cytotoxic factor, or TCF, which was released from sensitized lymphocytes under the stimulation of specific antigen, thyroglobulin, and could exert a cytotoxic effect directly on the target cells. Direct cell-to-cell contact was not required in this type of cell-mediated cytolysis.     

Changes in the capacity of macrophages and T cells to produce interleukins during marine malaria infection

Interleukin 1 (Il-1) produced by activated macrophages and interleukin 2 (Il-2) released by a subset of T lymphocytes upon antigen or mitogen stimulation are the soluble mediators involved in the mechanism of T-cell activation, proliferation, and differentiation. Since these T-cell responses are depressed during malaria infection, the capacity of macrophages to produce Il-1 following lipopolysaccharide (LPS) stimulation and that of lymphocytes to release Il-2 upon stimulation with concanavalin A (Con-A) in mice infected with either nonlethal Plasmodium yoelii (NLPY) or lethal Plasmodium berghei (PB) malaria parasites was analyzed. The results show that while adherent cells from spleen or peritoneal exudates of infected mice were able to produce Il-1, although to a different extent in the two infections, splenic lymphocytes were unable to produce Il-2, but capable of responding to it. This suggests that the diminished T-cell responses in malaria might be due to a defect of Il-2 synthesis.     

Tolerance induction to a thymus-dependent antigen in vitro: treatment of nonadherent cells with tolerogen biologically filtered in vitro

Highly tolerogenic bovine gamma globulin (BGG), a thymus-dependent antigen, was prepared by biologic filtraration in vitro. It readily induced tolerance in vivo in BALB/c mice and also rendered their nonadherent lymph node cells tolerant after in vitro incubation. Biologic filtration in vitro was carried out by incubating 2.5 × 10⁷ lymph node cells with 10 mg of nontolerogenic BGG in 10 ml of Eagle's medium containing 2% normal mouse serum at 37 °C for 6 hr. The BGG-containing medium was clarified by centrifugation and was used without further dilution.For tolerance induction in vitro, lymph node cells were separated into adherent and nonadherent populations on Falcon plastic. These cells were incubated for 0–18 hr at 37 °C with biologically filtered BGG (bBGG). After incubation, the cells were washed three times and (2–2.5) × 10⁷ nonadherent or 4 × 10⁶ adherent cells were injected iv with their untreated counterpart into lethally irradiated mice which had received 10⁶ bone marrow cells. The recipients were then challenged with 300 μg of aggregated BGG, and tolerance was assayed by the elimination of labeled BGG, rosette formation, and passive hemagglutination. Spleen cells were similarly treated for comparison. Our findings show that tolerance was not induced in vitro in adherent lymph node cells. However, in the nonadherent populations, those from the lymph node but not the spleen were rendered tolerant. The acquisition of tolerance in vitro was gradual. It was dependent upon the length of exposure to bBGG and required at least 6 hr.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. IV. Synergy of lymphocytes in the formation of clusters.

T-lymphocyte-enriched lymph node lymphocytes from guinea pigs immunized with Mycobacterium tuberculosis produce clusters with macrophages when cultivated on monolayers of syngeneic purified protein derivative of tuberculin (PPD)-pulsed peritoneal macrophages. The clusters consist of a macrophage with a central lymphocyte attached to it, and several peripheral lymphocytes attached to the central one. By mechanical manipulation immune lymphocytes incubated on monolayers of PPD-pulsed macrophages were separated into those which adhered firmly to the macrophages after 4 hr of culture and those which did not adhere. While neither of the two populations was able to produce significant numbers of clusters alone, they did so in combination. The number of macrophage-lymphocyte clusters which are produced in a culture depends not only on the absolute number of immune lymphocytes in the culture, but also on the concentration of lymphocytes per area of the macrophage monolayer, with high concentrations resulting in high numbers of clusters. Autoradiographic studies showed that the DNA-synthesizing lymphocytes physically associated with macrophages were located mainly inside the clusters in cultures with high concentrations of lymphocytes but mainly outside the clusters in cultures with low concentrations of lymphocytes.     

Avian delayed-type hypersensitivity: adaptation of the migration-inhibition assay

In this study White Leghorn cockerels were sensitized with Mycobacterium tuberculosis and/or bovine gammaglobulin (BGG). The migration of spleen cells from chickens with delayed dermal hypersensitivity to PPD was markedly inhibited in the presence of PPD but not by BGG. When birds were made doubly sensitive to mycobacteria and BGG, the migration of their spleen cells was inhibited by both antigens. Cells from animals immunized to produce high levels of circulating antibody to BGG were not inhibited in the presence of antigen. Sensitive spleen cells incubated with specific antigen elaborated a substance into the medium which inhibited the migration of normal cells. The results of these experiments indicate that the delayed hypersensitive response in birds parallels that of the mammal in that it is antigen specific, reproducible, independent of the antibody response, and transferable to normal cells with a soluble cell-free product.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on the differentiation and functional capacity of macrophages cultured in vitro.

The effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) on the differentiation and functional capacity of macrophages cultured in vitro from various lymphoid tissues was investigated. In cultures of peritoneal cells, the number of macrophages did not change throughout incubation periods of from 1 hr to 3 days, and the addition of CPS-K had no affect. It appears therefore that CPS-K does not exhibit cytotoxic effects on macrophages. In cultures of spleen cells, only a small number of macrophages appeared within 1 hr, but the number of macrophages increased during further incubation. The addition of CPS-K to cultures of spleen cells at the start of incubation suppressed markedly the increase in the numbers of macrophage. This finding indicates that CPS-K blocks the process of the generation of macrophages, probably from their precursor cells in cultures of spleen cells. Only a small number of macrophages appeared in cultures of thymocytes or lymph node cells either with or without CPS-K. The phagocytic capacity of either peritoneal macrophages or macrophages generated in cultures of spleen cells was activated during incubation in vitro. Macrophages cultured in the presence of CPS-K for 24 hr or longer appeared to have an enhanced phagocytic activity, although the enhancement of their phagocytic activity by the addition of CPS-K was less marked in cultures of spleen cells than in those of peritoneal macrophages. Morphologically, macrophages in both cultures of peritoneal cells and spleen cells incubated in the presence of CPS-K for 4 days possessed much longer cytoplasmic processes than those incubated in the absence of CPS-K. From the present study, it appears that CPS-K exhibits dual effects on macrophage precursor cells and macrophages, a blocking effect on the differentiation from the former to the latter and an enhancing effect on the functional capacity of the latter.     

In Vitro Studies on the Mechanism of Acquired Resistance to Tuberculous Infection

The relationship between lymphocytes and macrophages in cellular immunity against tuberculous infection was studied by means of an in vitro cell culture system without addition of streptomycin. The peritoneal macrophages were obtained from normal mice or mice immunized with heat-killed tubercle bacilli in paraffin oil, boosted with live BCG and infected with H37Rv cells in vitro. The infected monolayers of macrophages were cultivated for 48 hr with immune lymphoid cells obtained from immunized mice. The intracellular growth of H37Rv cells 3,5 and 7 days after infection was examined by counting tubercle bacilli within infected macrophages under a microscope. 1) The increase of bacilli within macrophages derived from immunized mice was slightly smaller than that in normal macrophages. 2) The addition of immune lymph node cells to the macrophage monolayers resulted in a marked decrease in the number of bacilli within both normal and “immune” macrophages. Conversely, normal lymph node cells exhibited an enhancing effect on the intracellular bacillary growth. 3) Immune lymph node cells showed a higher capacity to cause macrophages to suppress intracellular growth of bacilli than that of splenic lymphoid cells or thymocytes after addition to macrophage monolayers. 4) The treatment of lymphoid cells with inhibitors of protein synthesis, cycloheximide or streptovitacin A, resulted in a remarkable reduction of the ability of sensitized lymphocytes to cause macrophages to suppress multiplication of intracellular bacilli.     

Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.     

Inhibition of rat mixed lymphocyte cultures by suppressor macrophages.

Normal rat spleens contain suppressor cells which can inhibit proliferative and cytotoxic responses of lymphocytes to alloantigens in vitro. The suppressor cells are adherent, phagocytic, resistant to treatment with ATS and C, radioresistant, resistant to treatment with mitomycin C, apparently absent from the thymus, and found in very high concentrations in peritoneal exudates. These characteristics indicate that the suppressor cell is a macrophages and not a T cell. When suppressor cells were removed from spleen cell suspensions, strong in vitro proliferative and cytotoxic responses to alloantigens could consistently be observed.     

Primary and secondary antibody responses of rabbits to bacteriophage T2: kinetic and quantitative analysis and suppression by antimacrophage globulin.

Antibody synthetic capacity of popliteal lymph node cells removed from rabbits at various times after immunization with bacteriophage T2 was assayed by radioimmunoassay of tissue culture fluid after incubation with ¹⁴C-leucine. Antibody synthesis began on day 2; IgM synthesis peaked on day 3; IgG synthesis peaked on day 5 and again on day 14. Reinjection of T2 one month later elicited an enhanced response which peaked sharply on day 2. The primary and secondary responses, but not priming for the secondary response, were suppressed by injection of goat antimacrophage globulin (AMG), but only when AMG was injected 1 to 3 days before T2. AMG reacted strongly with rabbit peritoneal macrophages and only slightly with rabbit lymphocytes or erythrocytes. Thus, macrophages appear to participate in the induction of antibody responses of rabbit lymph nodes to T2 and their function inhibited by AMG apparently operates only during the early phase of induction.     

Induction of macrophage-mediated cytolysis of neoplastic cells by mycoplasmas

Unexpected cytolysis was encountered when nonactivated murine peritoneal macrophages were cultured with [³H]TdR-prelabeled syngeneic or allogeneic tumor cells at a 10:1 ratio. The level of specific cytolysis reached 70% within 48 hr of cocultivation. Similar killing was observed whether the macrophages were derived from untreated, thioglycollate-treated, or germ-free mice. Cytolytic activity was also demonstrated when bone marrow-derived or peritoneal macrophages from 9- and 5-day in vitro cultures, respectively, were employed rather than freshly harvested peritoneal macrophages. Thus, the macrophage-mediated killing was neither the result of in vivo preactivation nor a consequence of the presence of lymphocytes in the assay. Moreover, macrophages derived from different strains caused similar effects. Our study revealed that the neoplastic target cell cultures susceptible to cytolysis by nonactivated macrophages were contaminated with mycoplasma. A mycoplasma was isolated from the supernatant of a culture of the A9HT fibrosarcoma line, identified as Mycoplasma orale, and cultivated. Addition of viable mycoplasma from that isolate to mixed cultures of thioglycollate-elicited macrophages and [³H]TdR-prelabeled mycoplasma-free target cells resulted in specific cytolysis of transformed A9 cells, but not of normal mouse fibroblasts. The level of macrophage-dependent cytolysis correlated with the number of viable mycoplasma cells added and was higher than that attained by activation with LPS at optimal concentration. Similar specific cytolysis was observed with heat-killed mycoplasmas. Our results demonstrate that mycoplasmas may cause selective macrophage-mediated cytolysis of neoplastic but not of normal target cells, perhaps via activation of the macrophages. It is suggested that undetected infection of experimental systems by mycoplasmas may account for some reports on lysis of neoplastic cells by nonactivated macrophages.