Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

Augmentation of Mouse Natural Killer Cell Activity by Lactobacillus casei and Its Surface Antigens

Lactobacillus casei YIT 9018 (LC 9018) augmented the natural killer (NK) cell activity of spleen cells from inbred BALB/c mice injected intravenously with LC 9018 or intraperitoneally with polyinosinate-polycytidylate. Augmentation of this activity by LC 9018 was also observed in male C3H/He, CBA/N, and C57BL/6 mice. The spleen cells exhibited no cytolytic activity against P815, a cell line insensitive to NK cells. The cytolytic activity of the spleen cells increased 2 days after the injection of 250 μg of LC 9018/mouse, peaked on day 3, and gradually declined thereafter. The increase caused by LC 9018 was also observed in normal and Meth A-bearing mice. In vitro treatment with anti-asialo GM1 antibody plus complement completely-abrogated the LC 9018-augmented murine NK cell activity. The NK activity on the 3rd day after LC 9018 injection was reduced by in vitro treatment with anti-Thy 1.2 monoclonal antibody plus complement to half of that observed when treatment was with complement alone. This suggests that there were two populations of NK cells in the spleen cell suspension derived from LC 9018-treated mice. One population was asialo GM1-positive and Thy 1-negative, the other was asialo GM1-positive and Thy 1-positive.     

Expression of a laminin-like substance on the surface of murine natural killer (NK) lymphocytes and its role in NK recognition of tumor target cells

We have identified a structure on the surface of murine NK cells that is immunochemically cross-reactive with laminin. Treatment of normal CBA/J spleen cells with monospecific anti-laminin serum plus complement completely eliminated NK cytolytic activity against YAC-1 or RL male 1 target cells. In the absence of added complement, spleen cells preincubated with anti-laminin serum were also reduced in their cytolytic activity due to a reduced capacity to bind to the target cells. Treatment with anti-asialo GM1 serum plus complement also eliminated NK activity, but pretreatment of NK cells with anti-asialo GM1 in the absence of complement did not reduce cytolytic activity. Thus, anti-laminin and anti-asialo GM1 bind to structures on the surface of NK cells that distinguish functional (laminin) from nonfunctional (asialo GM1) sites. Flow cytometric analysis revealed that approximately 15% of normal nonadherent splenic lymphocytes expressed laminin-like structures, whereas 16% expressed asialo GM1 and 19% expressed the NK alloantigen NK 2.1. Treatment of alloimmune cytotoxic T lymphocytes (CTL) with anti-laminin plus complement did not affect CTL activity. Thus, anti-laminin serum appears to detect a cell surface structure present on the NK subset of lymphocytes.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Metabolic and physiologic studies of nonimmune lymphoid cells cytotoxic for fibroblastic cells in vitro

An in vitro reaction between mouse lymphoid cells and target fibroblastic cells in wells of microtest plates, which appears to simulate the in vivo rejection of hemopoietic allografts, has been analyzed for metabolic and physiologic requirements. Protein synthesis was required for only the first few hours of culture. Inhibition of RNA synthesis and alteration of cell surface charge with various agents were without obvious effects. Metabolic slowing at 4 °C or deviation of the pH of the culture medium suppressed the reaction. Thymus cells, which are not cytotoxic in this system, significantly but not completely inhibited the cytotoxicity of lymph node cells. Antiserum directed against target cells specifically protected them from the cytotoxic lymphoid cells in the absence of complement.Precursors of cytotoxic lymphoid cells were radiosensitive, unlike the cytotoxic cells themselves. BALB/c anti-C57BL/6 spleen cell serum and ⁸⁹Sr both are able to prevent rejection of marrow allografts in vitro. Lymphoid cells incubated with this antiserum plus complement lost much of their cytotoxicity but were still effective at high ratios of aggressor to target cells. Lymphoid cells of mice treated with ⁸⁹Sr were effectively cytotoxic but lost practically all of their cytotoxicity afer incubation with the antiserum plus complement. Thus, it appears that this reaction detects two different cytotoxic lymphoid cells, either of which can function in vitro. Both cell types may need to cooperate in vivo during marrow allograft rejections.     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

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.     

Discrimination between macrophage-and NK-type tumoricidal activities via anti-asialo GM1 antibody

The usefulness of asialo GM1, a glycolipid surface marker, to define the effector cell types involved in tumor resistance in vitro and in vivo was assessed. Pretreatment of rat effector cells with anti-asialo GM1 antibody plus complement in vitro either abrogated or markedly diminished NK activity; in contrast, macrophage-type cytocidal activity was not diminished by such pretreatment. Similarly, systemic inoculation of anti-asialo GM1 antibody selectively eliminated NK activity, leaving macrophage-type tumoricidal reactivity intact. Finally, such pretreatment did not diminish host resistance in an in vivo tumor model in which the available evidence suggests a critical role for macrophages. The asialo GM1 marker may thus be useful in delimitating the tumoricidal capacity of cells exhibiting NK activity from that mediated by other cell types.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Flow cytometric analysis reveals the presence of asialo GM1 on the surface membrane of alloimmune cytotoxic T lymphocytes

Previous studies have demonstrated that natural killer (NK) cells express the glycolipid asialo GM1, as evidenced by the sensitivity of NK cells to treatment with anti-asialo GM1 serum and complement. Because alloimmune cytotoxic T lymphocytes (CTL) were found to be insensitive to treatment with anti-asialo GM1 serum and complement, it was concluded that asialo GM1 is expressed by NK but not by CTL. However, fluorescence studies indicated that a significant proportion of peripheral T cells did express asialo GM1. Flow cytometric studies were undertaken to determine the extent to which alloimmune CTL express asialo GM1. Affinity-purified, monospecific IgG anti-asialo GM1 antibodies were used to label cells from mixed lymphocyte cultures. Separation of asialo GM1-positive and -negative fractions by cell sorting revealed that the majority of CTL activity resides in the asialo GM1-positive population. When these studies are compared with similar studies of splenic NK activity, it is apparent that, despite the relative insensitivity of CTL to treatment with anti-asialo GM1 and complement, both CTL and NK activity are enriched in the asialo GM1-positive cell population obtained by cell sorting.     

Inhibition of memory cell-mediated cytotoxic response by systemic administration of Corynebacterium parvum.

Intravenous administration of Corynebacterium parvum to mice during a developing immune response to alloantigens resulted in the marked inhibition of the generation and expression of memory cell-mediated cytotoxic response in the spleen. The inhibition was observed following rechallenge in vivo or by in vitro culturing with the same alloantigen. The impairment in vitro was due, in part, to the generation of regulatory cells which were non-T phagocytic cells, probably macrophages activated by C. parvum administration. These suppressor macrophages appear to act by inhibiting proliferation and clonal expansion of memory cytotoxic cells.     

Immunosuppressive Activities of Peritoneal and Splenic Macrophages in Murine Leprosy: Effect on Lymphocyte Transformation and Tumor Growth

The ability of peritoneal macrophages (PM) and splenic macrophages (SM) to suppress tumor growth and lymphocyte transformation in vitro was studied in infected mice with Mycobacterium lepraemurium (MLM). Both PM and SM of leprous mice showed cytostatic activity against tumor cells in vitro. However, such cells showed significantly less cytostatic activity on a per cell basis than highly activated macrophages obtained from Corynebaclerium parvum-immunized mice. Furthermore, this cytostatic activity declined as the infection progressed. Mitogen-induced transformation of splenic lymphocytes was also suppressed in the presence of adherent PM and SM from leprous mice. PM from leprous mice showed significantly less activity than PM from C. parvum-immunized mice in terms of suppression of lymphocyte transformation. Moreover, PM from leprous mice treated with C. parvum or sodium thioglycollate broth demonstrated significantly less ability to suppress lymphocyte transformation than did PM from similarly treated normal mice or untreated leprous mice. These findings demonstrated that MLM infection stimulates the mononuclear phagocyte system but does not activated it to the extent that it confers enhanced resistance to MLM on the host.     

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.     

Mechanism for Macrophage Activation against Corynebacterium parvum—Participation of T Cells and Its Lymphokines

It is well known that Corynebacterium parvum activates macrophages to produce tumor necrosis factor (TNF). It is suspected that the activation of macrophages by C. parvum requires T-cell participation. The purpose of this study was to confirm that T cells participate in the activation of macrophages by C. parvum. TNF production in vitro from the spleen cells of BALB/c- + / + mice was abrogated completely by the pre-treatment of spleen cells with anti-Ia antiserum and complement, indicating that Ia+ cells are the source of TNF. TNF production was not elicited at all in BALB/c-nu/nu mice. However, there was an increase in the number of Ia+ cells as well as an increase in the weight of spleen and liver. Supernatant from a culture of spleen cells stimulated with phytohemagglutinin-P (a PHA-induced lymphokine) made it possible for BALB/c-nu/nu mice to produce TNF, associated with an induction of Lyt-1⁺ cells and Lyt-2⁺ cells. However, treatment with the lymphokine did not augment the increases of Ia⁺ cells or liver and spleen weights. These results suggest that increasing the number of Ia⁺ cells is not sufficient to bring about TNF production; Ia⁺ cells must also be stimulated by T cells or T-cell lymphokines in order to produce TNF. These results suggest that T cells play an essential role in the activation of Ia⁺ cells against C. parvum.     

Induction of interferon production in mouse spleen cell cultures by corynebacterium parvum.

Spleen cells of CS7BL/6 mice produced considerable amounts of interferon (IF) in vitro when tested 5 to 20 days after injection of killed Corynebacterium parvum. Interferon was also produced when C. parvum was added in vitro to spleen cell cultures of previously untreated mice. High levels were detected after 1 day of culture with some increment during subsequent days. In a number of experiments IF was also produced in untreated control cultures but only after prolonged cultivation and not after 1 day. The highest levels of IF were usually obtained when spleen cells of C. parvum-treated mice were challenged with additional C. parvum in vitro. The IF induced by C. parvum shared certain physicochemical properties with a tested immune IF and was not neutralized by an antiserum raised against a type I IF. Spleen cells of nu/nu mice and spleen cells treated by anti-θ serum plus complement did not differ from their respective controls, indicating that production of IF did not require mature T lymphocytes. Removal of B lymphocytes by nylon wool columns abolished the capacity of spleen cells to produce IF. When spleen cells were freed of adherent cells by the use of plastic surfaces, they no longer produced IF. Peritoneal exudate macrophages (PEC), which by themselves did not produce IF, in small numbers reconstituted nonadherent spleen cells. Nylon column-treated spleen cells, however, could not be restored by PEC. It is concluded that IF upon challenge with C. parvum is produced by B lymphocytes and requires the help of macrophages.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

Systemic administration of Corynebacterium parvum during sensitization to tumor alloantigen-modified response to rechallenge

Summary Intravenous administration of Corynebacterium parvum (C. parvum) to mice during a primary immune response against tumor alloantigens impairs their ability to generate memory cell-mediated cytotoxicity (CMC) in response to an intraperitoneal rechallenge with the same tumor alloantigens. Decreased CMC was observed in spleen and mesenteric lymph nodes, whereas CMC of lymphoid populations from the peritoneal cavity was merely delayed, reaching comparable levels to those found in control animals by day 5. Serum levels of cytotoxic antibody were unaffected, indicating that C. parvum administered during a primary immune response has selective effects on the cytotoxic memory response.     

Participation of three lymphoid cell types in the in vitro activation of cell-mediated immunity to a syngeneic gross virus-induced lymphoma in rats.

W/Fu rats inoculated with the syngeneic Gross-virus induced lymphoma, (C58NT)D, had transient lymphocyte-mediated specific cytotoxicity against the tumor cells at 7–15 days after tumor injection. Spleen cells 40 days after immunization (spleen 40) were unreactive by a direct 4-hr ⁵¹Cr release assay, but activity appeared after in vitro culture of these spleen eclls by themselves for 18–24 hr. The nature of the cells involved in the activation of cytotoxicity and the characteristics of the effector cells themselves were studied. Significant differences were seen in the cell types involved in the activation phase and the effector phase. Activation appeared to require the cooperation of three cell types. Induction of activity was lost by treatment of cells with ATS plus complement, by passage over an EAC-column, or by treatment with carbonyl iron. Thus, T cells, CRL and macrophages were necessary for full activation of cytotoxicity in spleen 40. In contrast, after activation, only CRL seemed to be required for cytotoxicity, and treatment wih ATS or carbonyl iron had little effect. The effector cell detected after in vitro activation was quite distinct from that seen in the direct cytotoxicity assay with spleen cells at 10 days after tumor cell inoculation. The early, direct cytotoxic reactivity was dependent on T cells, being eliminated by treatment with ATS and complement but not by EAC columns or carbonyl iron. It appears therefore that the in vitro activation is a separate mechanism for cytotoxicity against tumor cells, rather than a simple recovery of T cells from in vivo inhibition.     

Heterogenous populations of cytotoxic cells in the peritoneal cavity of BALB/c mice immunized with allogeneic EL4 leukemia cells

Adherent cells, presumably macrophages, obtained from the peritoneal cavity shortly after rejection of the allogeneic leukemia EL4, produced effective cell-mediated cytotoxicity (CMC) in vitro. These cytotoxic cells were sensitive to anti-macrophage serum and resistant to anti-thymocyte serum and 10,000 roentgen irradiation. In contrast, a second population of specifically cytotoxic cells were nonadherent, sensitive to x-rays and anti-thymocyte serum, but not to anti-macrophage serum.The two cell populations had a cooperative cytotoxic effect in vitro against allogeneic tumor cells.