Cytotoxic immune cells with specificity for defined soluble antigens. I. Assay with antigen-coated target cells

An in vitro cytotoxic system is described, in which immune cells specific for a given soluble antigen exert a specific cytotoxic effect on target cells to which this antigen has been covalently linked. The nature of the target cell is important in this system. When antigen-coated P 815-X2 mastocytoma cells and antigen-coated chicken red blood cells were incubated for several hours in culture medium at 37 °C, the presence of membrane-bound antigen could still be demonstrated on the latter, but not on the former target cells. This might be the reason why antigen-specific target cell destruction by specific immune cells was observed only with antigen-coated chicken red blood cells as target cells. The specificity of the cytotoxic effect was controlled in each experiment in a criss-cross way by using two non cross-reacting antigens both as immunogens and for coating the target cells. Specific cytotoxicity was demonstrable with both guinea pig and mouse immune cells and with different kinds of antigens: foreign proteins, hapten-heterologous protein conjugates and hapten-autologous protein conjugates.     

Cytotoxic cells suppress in vitro generation of cellular immunity by stimulator cell lysis

Irradiated BALB/c spleen-cell populations actively cytotoxic to BL/6 alloantigens (modulator cells) were capable of suppression of the in vitro generation of BALB/c anti-BL/6 cellular cytotoxicity. This suppression was abrogated by anti-θ serum plus complement. The suppression was dose dependent on the number of modulator cells and correlated directly with the magnitude of their cytotoxicity. By varying the number of stimulator cells, specific suppression for a relevant stimulator cell and nonspecific suppression for an irrelevant stimulator cell were demonstrated in the same cultures. These data suggest that cytotoxic cells caused specific suppression in mixed lymphocyte culture by lysing stimulator cells although evidence for other nonspecific suppressor factors was seen. A model was proposed suggesting that cell populations possessing high levels of cytotoxicity may feed back negatively on an ongoing immune response by competing with proliferating T cells for cellular antigen.     

Cell-mediated cytotoxicity against syngeneic tumors

Spleen cells from DBA/2 mice bearing the DBA/2 P815X mastocytoma for approximately 2 weeks can be stimulated in vitro by mastocytoma cells to generate cytotoxicity measured as ⁵¹Cr release from mastocytoma cells in a 4-hr assay. These cytotoxic cells will not kill allogeneic cell lines but will kill a series of first transplant generation syngeneic tumors. T cells are involved in that treatment of the responding or the cytotoxic cell populations with either anti-T or anti-theta antibody + complement will abrogate all cytotoxicity. Anti-Ly 2.1 antibody + complement treatment of either responder cells (prior to the in vitro culture with irradiated tumor cells) or effector cells after culture markedly decreases cytotoxicity whereas treatment with anti-Ly 1.1 was more effective prior to culture compared to its effect on cytotoxic cells per se. These T cells are in the small lymphocyte class and occur either singly or in aggregates. Suppression of antisyngeneic tumor cytotoxicity by antibody inhibits preferentially the expression of cytotoxicity in the aggregate fractions.     

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.     

Graft-versus-host reaction-like phenomenon induced by BCG in mice lethally irradiated and transferred with syngeneic bone marrow cells.

Heat-killed BCG in paraffin exerted a lethal effect on CS7BL/6 mice irradiated lethally and transferred with syngeneic bone marrow cells. Such an effect was not detectable when mice were subjected to adult thymectomy and used as the hosts. Lymphoid cells from such nonthymectomized mice exhibited cytotoxicity to syngeneic tumor cells but not to allogeneic tumor cells in an in vivo cytotoxicity test and induced splenomegaly in sublethally irradiated syngeneic recipients after systemic transfer. The cytotoxicity of such lymphoid cells was abolished by a treatment with anti-θ serum and complement. In the bone marrow of mice irradiated and transferred with bone marrow cells, the number of nucleated cells, the ratio of myeloid to erythroid cell series, and the percentage of lymphocytes were increasd by BCG injection. These results suggest the possibility that self-tolerance may be broken by BCG stimulation in the process of reconstitution of lymphoid cells in the irradiated mice.     

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.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.     

Study of the immune responses to nucleated cells. I. In vitro functional evaluation of the immune effectors responsible for complement-dependent cellular cytotoxicity

A modification of the ⁵¹Cr cytotoxic test has made it possible to assess under the same conditions not only cytotoxic serum antibodies, and cell-mediated immunity, but also cells releasing cytotoxic antibody. The measurement of these antibody-releasing cells was carried out with nucleated target cells, both normal and leukemic, across θ or H-2 antigenic differences. This test was found to be specific. The release of ⁵¹Cr from the labeled target cells was proportional to the ratio of immune cells to target cells, and for a given ratio to the incubation time, 60 min usually being the optimum time at ratios of 50–100 to 1. The test was not affected by treatment of the effector cells with an anti-θ serum; however, pretreatment of these cells with an anti-IgM serum, even without complement, inhibited the test with cells taken during primary responses. Both cytotoxic IgM and IgG antibodies were detected by the assay directly without the addition of enhancing serum; discrimination between these two γ-globulins can be made by suppressing the cytotoxicity due to either Ig class consequent to the addition of the appropriate specific anti-globulin serum during the incubation.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Further evidence for autonomy of T cells mediating specific in vitro cytotoxicity: efficiency of very small amounts of highly purified T cells

Cytotoxic educated thymus cells were either incubated in a plastic flask or in the presence of iron with subsequent magnetic removal of iron-containing cells or subjected to a combination of both treatments. Acridine orange staining showed that the resulting purified cells contained about one macrophage per 5 × 10⁴ cells. More generally, their content of non-T cells was less than 1%, as judged by the proportion of cells resistant to anti-θ antiserum in the presence of complement. Improvements in the ⁵¹Cr-release Cytotoxicity test allowed us to demonstrate a significant cytotoxicity of as few as 10³ of these highly purified T cells. Under these conditions, any participation by macrophages or other non-T cells in T cell-mediated cytotoxicity could be excluded.     

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro. The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro, one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.     

Mechanism and specificity of macrophage-mediated cytotoxity.

The cytotoxic effect of macrophages derived from alloimmunized mice (immune macrophages) was found to be immunologically specific. The immune macrophages killed only target macrophages carrying the alloantigens used for immunization in mixed macrophage cultures (MMC) under optimal conditions of contact between effector and target cells. T-sensitized lymphocytes, but not B cells, were capable of arming nonimmune macrophages and conferring upon them cytotoxic activity; the arming factor, which seemed to be a T mediator or T-cell receptor (membrane component) was removable by trypsin. Frequent rinsing or addition of hydrocortisone significantly decreased the cytotoxicity of the MMC. Pretreatment of peritoneal cells with anti-θ antisera and complement markedly decreased immune macrophage cytotoxic activity. It is suggested that the presence of a very small number of T-sensitized lymphocytes is required for strong cytotoxic activity to be manifested by the macrophages.     

Specificity of Anti-θ Sera in Cytotoxicity and Functional Tests on T Lymphocytes

THE theta (θ) alloantigen is a marker for thymus-dependent (T) lymphocytes in mice^1–4 and anti-θ serum has been used to study the distribution, differentiation and function of T lymphocytes⁵. We report here studies which indicate that anti-θ sera raised in conventional inbred mice are usually contaminated with a number of antibodies directed against surface antigenic determinants other than θ, at least one of which is present on thymus-independent B lymphocytes. These contaminating antibodies have been demonstrated by cytotoxicity tests with rabbit complement and by rosette-forming cell (r.f.c.) inhibition studies. In the presence of guinea-pig complement, however, most anti-θ sera can be used selectively to kill and/or inhibit T cells.     

Dialyzable leukocyte extract induces mast cell secretion

Semiallogeneic somatic hybrid cells (AB2) derived from fusion of a C57B1/6 chemically induced fibrosarcoma (MCB6-1) and a fibroblastic cell (A9) of C3H origin were used to immunize C57B1/6 mice against the parental MCB6-1 tumor cells. In vitro immune lymphocytes were directly cytotoxic against AB2 hybrid cells and A9 allogeneic parental cells, but could not lyse the syngeneic MCB6-1 parental tumor cells. Nevertheless, after a 4-day culture of these immune lymphocytes, a cytotoxic activity against the syngeneic MCB6-1 tumor cells appeared; expression of such a cytotoxic activity did not require the presence of stimulator cells (mitomycin-treated MCB6-1 tumor cells) during the culture. This cytotoxicity is mediated by T cells, as it was completely abrogated by treatment with anti-Thy 1–2 antiserum and complement. These results suggest that a maturation or a differentiation of immune T lymphocytes occurs during in vitro culture, and is necessary for the expression of antitumor cytotoxicity.     

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.     

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.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.     

Immune responses in vitro. VII. Loss of susceptibility of functional theta-bearing cells to cytotoxic action of anti-theta serum and complement in vitro

θ-Bearing lymphocytes become progressively less susceptible to cytolytic effects of specific anti-θ serum and complement with incubation in tissue culture. Thymus-derived cell function, in terms of “helper cell” capacity for antibody responses and DNA synthetic responses to phytomitogens, however, remains intact. The data indicate that the loss of susceptibility to cytoloytic effects of anti-θ serum and complement is due to a decrease in the amount of θ antigen on these cells. This phenomenon hampers attempts to determine the length of time that collaborative or “helper” influences of thymusderived cells are required for development of optimal antibody responses in vitro.