Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Mechanism of mastocytoma-mediated suppression of lymphocyte reactivity.

Previously we have shown that mitomycin-treated P-815 (H-2d) cells (P-815m) inhibit the in vitro response of C57BL/6 spleen cells to mitogens and DBA/2 alloantigens. The present data indicate that P-815m cells also inhibit the response of C57BL/6 spleen cells to AKR (H-2k)-stimulating cells and that the inhibition does not appear to be the result of crowding. Subpopulations of spleen cells obtained by density gradient centrifucation or after renoval of glass-adherent cells are all sensitive to the inhibitory effects of P-815m. We also show that P-815m cells appears to operate both in vitro and in vivo. The experiments characterize the suppression of MLC reactivity by cell-free preparations of sonicated P-815 mastocytoma cells. The results suggest that the inhibition is not removed by ultracentrifugation, ultra-violet irradiation, or dialysis of the sonicate. P-815 sonicates subjected to chloroform extraction, heating at 56 degrees C, 0.1 mu filtration, or treatment with anit-minute virus of mice serum are still inhibitory. This inhibition is resistant to RNAase treatment but destroyed by pronase. P-815 sonicate suppresses the response of C57BL/6 spleen cells to both DBA/2 and AKR-stimulating spleen. Inhibition of MLC reactivity is only seen if P-815 sonicate is added within the first 48 hr after the initiation of culture. These results indicate that the inhibition of MLC by sonicates of P-815 cells is due to a nondialyzable protein whose effects are not H-2 restricted and are acting on the early phase of sensitization.     

Immunologic unresponsiveness of mouse spleen sensitized to allogenic tumors.

CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is $\text{1}\text{1}\text{2}\text{–2}\text{1}\text{2}$ months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.     

Humoral mechanisms of in vitro inhibition of mouse lymphocyte immunoreactivity by mastocytoma P815 cells

The inhibitory capacity of mastocytoma cell line P815 and its cultural supernatant (CS) was studied in the reaction of blast transformation (RBT) and mixed lymphocyte culture (MLC). An addition of both P815 cells and CS resulted in dose-dependent inhibition of lymphocyte proliferation in RBT and MLC. The treatment of DBA/2 spleen cells with CS for 2 h at 37 degrees C resulted in a significant decrease in proliferative activity and induction of supressor cells.     

T-cell mediated suppression in the MRL mouse

MRL/lpr mice possess an autosomal recessive gene, lpr, which is associated with lymphoproliferation and acceleration of autoimmune disease. Lymphoproliferation has been ascribed to a single gene defect predominantly affecting the T-lymphocyte component of the immune system. MRL/++ mice do not possess the lpr autosomal recessive mutation and do not develop early lymphadenopathy. T-lymphocyte functional activity was studied in these mice using the polyclonal T-cell mitogens PHA and Con A. Our results indicated a significant suppression of the spleen and lymph node response of MRL/lpr mice to these polyclonal mitogens as compared to the MRL/++ response noted as early as 6 weeks of age. In addition, there was a progressive decline in the MRL/lpr spleen and lymph node cell mitogenic responses with increasing age. Spleen and lymph node cells from 20-week MRL/lpr mice were also relatively unresponsive in the mixed lymphocyte culture reaction as compared to cells from MRL/ ++ or BALB/c mice. The in vitro proliferative response of the MRL mice was further examined with respect to possible accessory cell modulation by both macrophages and T cells. It was found that in 20-week MRL/lpr lymph nodes a significant degree of suppression of lymphocyte proliferation could be mediated by the MRL/lpr T cell. Increased lymphocyte proliferation to a mitogenic signal could only be demonstrated in those MRL/lpr mice 3 weeks of age.     

Effect of cell-free murine liver extract on lymphocyte blastogenesis in vitro.

The effect of cell-free liver extract (LE) on the proliferation of spleen cells in vitro was examined using [³H]thymidine incorporation. LE inhibited the blastogenic response of murine lymphocytes stimulated with plant mitogens, phytohemagglutinin, and concanavalin A and in the mixed lymphocyte reaction (MLR). Suppression of cell proliferation occurred whether the LE was syngeneic or allogeneic to the responding cells. This effect was observed only when LE was present in cultures, as preincubation of cells with LE did not impair their capacity to respond to stimulation. Profound suppression of proliferation was achieved with the addition of LE to the culture up to 48 hr after the onset of stimulation. However, the inhibitory effect was readily reversible upon removal of LE from the culture. Furthermore, although LE was capable of suppressing the generation of cytotoxic lymphocytes, LE did not interfere with their capacity for cytolysis. These findings indicate the presence of a potent inhibitor of lymphocyte proliferation in a cell-free extract of murine liver.     

The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

Trichinella spiralis: correlates in vitro of altered immune responsiveness in mice.

Mixed lymphocyte reactions and in vitro antibody responses to dinitrophenol (DNP) after immunization with DNP-Ficoll were measured in spleen cells from mice following infection with 200 Trichinella spiralis larvae. A depression of the mixed lymphocyte reaction was observed at 14 through 84 days after infection. A reduced response to concanavalin A stimulation was demonstrated over a similar time period, 7 through 63 days of infection. The addition of mitomycin C-treated spleen cells from mice infected with T. spiralis to cultures of normal splenocytes suppressed the mixed lymphocyte reaction by 28% to 65%. The antibody response to DNP-Ficoll immunization was enhanced 20 days after infection, a time when the T-dependent antibody response to sheep erythrocytes was depressed.     

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.     

A fluorescence study on the mobility of surface antigens of untreated tumor cells and of tumor cells undergoing cell-mediated lysis

Heterologous (rabbit) antibodies were raised against murine P-815 mastocytoma cells of DBA/2 origin. Antisera and IgG preparations were highly cytotoxic, whereas Fab fragments thereof lost all activity. Fab fragments also showed a much lower avidity than IgG, both for tumor and normal DBA/2 and C57 spleen cells as measured by the release of iodinated Fab and IgG. Both preparations bound specifically to P-815 cells since they were capable of inhibiting T cell-mediated target cell lysis. The binding of IgG and monovalent Fab fragments was studied by fluorescence. Rhodamine-coupled IgG bound homogeneously in the cold and quickly formed patches upon warming but did not form caps even after prolonged incubation at 37 degrees C. Rhodamine-coupled Fab fragments also bound homogeneously. Their distribution was unaltered after incubation at 37 degrees C even when tumor cells formed uropod-like tails. Fab fragments, however, could be induced to cap with a second and third antibody layer. P-815 cells labeled with rhodamine-coupled Fab fragments were incubated with cytolytic T cells (CTL). The conjugates formed between CTL and fluorescent target cells were observed. No gross redistribution of surface antigens on target cells was observed even at late stages of the lytic process. CTL, therefore, do not seem to operate via a redistribution of surface antigens.     

"Physiological interaction" does not explain the H-2 requirement for recognition of virus-infected cells by cytotoxic T cells.

B10.A (H-2Kk, H-2Dd) ectromelia-immune T cells from secondary responses in vitro were protent killers of both infected L929 (H-2Kk H-2Dk) and infected P-815 (H-2Kd, H-2Db) target cells. Specific competition with unlabelled targets showed that two separate T cell subsets were responsible for lysis of infected L929 and infected P-815 cells. One hypothesis to account for this (a form of "physiological interaction") is that T cells which kill one target e.g. infected L929) display only one out of two possible self-complementary recognition structures, in this example the H-2Kk alloantigen, not H-2Dd, whereas T cells that lyse infected P-815 targets display only H-2Dd, not H-2Kk. This hypothesis was tested and seems untenable because of the following results: A.TH (H-2Ks, H-2Dd) ectromelia-immune, secondary cytotoxic T cells which killed infected SJL/J (H-2Ks, H-2Ds) targets were themselves inactivated by pre-incubation with SJL/J cytotoxic T cells generated in one-way mixed lymphocyte reaction (MLR) against BALB/c (H-2Kd, H-2Dd). A.TL (H-2Ks, H-2Dd) ectromelia-immune secondary cytotoxic T cells which killed infected BALB/c targets were themselves inactivated by BALB/c cytotoxic T cells generated in MLR against SJL/J. Thus, virus-immune T cells which lyse infected targets by virtue of shared H-2K are also displaying H-2D alloantigen, and vice versa.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Macrophage activation during experimental murine brucellosis: II. Inhibition of in vitro lymphocyte proliferation by brucella-activated macrophages

During infection of CBA mice with Brucella abortus strain 19, there is a massive accumulation of macrophage-like cells in the spleen with resultant gross splenomegaly. In vitro cultures of cells from these spleens show a reduced proliferative response to brucellin and to other mitogens (phytohemagglutinin, concanavalin A, and lipopolysaccharide). The effect could be overcome by the addition of high concentrations of mitogen. Removal of adherent cells from spleen populations derived from 20-day infected mice abrogated the suppressive effect. Conversely, adherent cells from the spleens of 20-day infected mice inhibited proliferation of normal spleen cell cultures. Inhibition of responsiveness of normal spleen cells by cells from the spleens of infected mice occurred even when the two populations were separated by dialysis membranes. Although proliferation was measured by uptake of tritiated thymidine, inhibition in this system was not due to the release of unlabeled thymidine from macrophages.     

Interactions between neuropeptides and dexamethasone on the mitogenic response of rabbit spleen lymphocytes.

The interactions between vasoactive intestinal peptide (VIP), substance P (SP), a somatostatin analog (SMS 201-995) and dexamethasone have been investigated on the Con A mitogenic response of rabbit spleen cells. The neuropeptide regulatory effects appeared to be time dependent: when added with the Con A mitogen, they inhibited (VIP) or did not modulate (SMS and SP) the rabbit lymphocyte proliferation and did not change the inhibitory effect induced by a dexamethasone preincubation. When added 18 h before the mitogen, they all induced an increase of the proliferative response at high concentration. The mitogenic response observed when adding dexamethasone to lymphocytes previously preincubated in the presence of neuropeptides was not different from control response except with SMS 10(-10) M. The similar lymphocyte responses obtained whatever the neuropeptide suggested that the immunomodulatory effect induced by a neuropeptide preincubation might be mediated by the induction of common effector(s).     

Study of the action of tumor cell immunosuppressive factors on the production of cytokines by lymphocytes and macrophages and the mitogenic activity of interleukin-2

The effect of immunosuppressive factors (ISF) derived from tumor cell lines (mastocytoma P-815, leukosis EL-4 and melanoma B16) on the production of interleukin-1 (IL-1) by macrophages and of interleukin-2 (IL-2) by splenocytes of BALB/C mice was investigated. We could show that treatment of above cells by the tumor products resulted in strong decrease of IL-2 production but did not affect IL-1 secretion. ISF inhibited the proliferation of BALB/C lymphoblasts caused by recombinant IL-2. Thus, one of the significant mechanisms of ISF action seems to be disturbance of monolymphokine cascade of lymphocyte activation.     

Studies on the mechanism of lymphocyte- mediated cytolysis. VIII. The use of con A to delineate a distinctive killer T cell subpopulation.

Alloimmune spleen cells (C57BL/6 anti P815), but not normal spleen cells, lyse syngeneic (EL4) target cells in the presence of Con A. Con A dependent cytotoxicity was mediated by T cells and required the continued presence of lectin. Cytolysis in the presence of a succinylated derivative was equivalent to that seen with the parent Con A molecule. In contrast to previous reports of Con A dependent cytolysis, however, we conclude that lysis is not primarily caused by directly cytotoxic T cells. The reasons for this conclusion are: 1. Removal of directly cytotoxic cells by adsorption on P815 monolayers did not alter the Con A dependent cytolysis of EL4 cells; 2. Populations in which no direct T killers were demonstrable (e.g., spleen cells harvested 5 days after alloimmunization) lysed both P815 and EL4 cells in the presence of Con A; and 3. Con A dependent cytolysis, but not direct cytotoxicity, could be induced by culturing normal C57BL/6 spleen cells for 4 days with a sonicated extract of P815 cells. We hypothesize that the cell "activated" to lyse targets in the presence of Con A is a T cell which has differentiated lytic potential following alloantigenic stimulation, but has either insufficient density or affinity of antigen receptors to serve as a directly cytotoxic cell. The role of Con A is viewed as 2-fold: i) to "bridge" killer and target cell, and ii) to "activate" the effector.     

Isolation of a low molecular weight inhibitor of lymphocyte proliferation from tumorous ascites

Intraperitoneal growth of P-815 mastocytoma cells in syngeneic DBA/2 mice produces ascites fluid which strongly inhibits mitogen-stimulated lymphocyte proliferation. The less than 10,000 m.w. fraction from gel filtration chromatography of tumorous ascites on Sephadex G-150 showed no inhibition of proliferation when eluted under physiologic conditions but was inhibitory when eluted with a high ionic strength, acidic buffer. The organic phase of a chloroform/methanol extract of the low m.w. fraction contained all the inhibitory activity. Purification of the inhibitor to relative homogeneity was achieved by reverse phase, HPLC with a gradient of acetonitrile in dilute acetate buffer. Inhibitory activity eluted between 30 and 35% acetonitrile. The active fraction contained less than 30 pg/ml PGE by RIA which was insufficient to inhibit proliferation and may actually have been stimulatory. Inhibition comparable to that produced by the ascites fraction required greater than 300 pg/ml of PGE. This low m.w. (less than 10,000), lipid-like inhibitor of lymphocyte proliferation is acid stable, not sensitive to proteolytic enzymes, soluble in both aqueous and organic solvents and occurs normally bound to a higher m.w. carrier molecule.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Suppressed proliferative response of spleen T cells from metallothionein null mice

To investigate the role of metal-binding protein, metallothionein (MT), in lymphocyte activation, the mitogen-induced proliferation of freshly isolated spleen cells was compared among MT-I, II null, and control 129/Sv mice. Spleen cells from MT null mice exhibited a markedly reduced proliferation compared with control cells when stimulated by concanavalin A or anti-CD3(epsilon) mAb, but not by lipopolysaccharide, indicating that only the response of T cells to mitogens was suppressed in MT null mice. Flow cytometric analysis of unstimulated spleen cells demonstrated no significant difference in the relative percentages of either B220+ and CD3+ cells or CD4+ and CD8+ cells between the two strains of mice. The production of interleukin (IL)-2 by MT null spleen cells after the stimulation by anti-CD3(epsilon) mAb was lower than that of control spleen cells, especially within 24 hr after the stimulation. The addition of IL-2 recovered the proliferation of MT null spleen cells to the control level. The reduced proliferative response to mitogenic stimulation of MT null T cells was confirmed by using purified splenic T cells. These results suggest that the MT expressed at basal level in the splenocytes plays an important role in T cell mitogen-induced proliferative response, probably by positively regulating the production of IL-2.