Macrophage requirement for in vitro generation of specific, secondary cell-mediated cytotoxicity against SV 40-induced tumor-associated antigens in mice

The role of adherent phagocytic cells in an in vitro secondary cytotoxic response against Simian virus 40 (SV 40)-induced tumor-associated antigens was investigated. Spleen cells (responder cells), from mice primed with syngeneic SV 40-transformed cells, extensively depleted of macrophages by filtration through a Sephadex G-10 column followed by iron carbonyl treatment, had a markedly decreased capacity to generate in vitro secondary cytotoxic reactivity against syngeneic SV 40-transformed cells when cultured with the relevant stimulator cells. The secondary response was restored by the addition of adherent peritoneal cells from normal mice syngeneic to those immunized with the antigen. Within a certain dose range, small numbers of peritoneal cells completely reconstituted the response, whereas large numbers inhibited the reactivity. The restored cultures maintained specific cytotoxic reactivity against SV 40-induced tumor-associated antigens which was mediated by effector T cells as shown by sensitivity to anti-Thy-1.2 antiserum and complement. These results suggested a requirement for adherent phagocytic cells (accessory cells) in in vitro generation of a secondary, cytotoxic response to tumor-associated antigens.     

Effect of rifamycin SV on the in vitro induced immune response

Mouse peritoneal cells producing antibodies against sheep red cells were used to study the effect of Rifamycin SV on an in vitro induced immunological response by the localized hemolysis technique. At a concentration of 100 μg/ml, Rifamycin SV has a greater inhibiting effect on lymphocytes than on macrophages. Lymphocytes are still clearly inhibited by 10 μg/ml, but the same concentration acting on macrophages causes an increase in the number of plaque-forming cells. One thousand μg/ml Rifamycin SV completely suppresses antibody production, whereas 1 μg/ml has no detectable action. The effect of Rifamycin SV on incorporation of radioactive amino-acids and uridine was also studied. We found that it has a weak effect on protein synthesis but inhibits synthesis of RNA; this inhibition is stronger in the absence of antigen. Furthermore, protein synthesis by these cells seems to be controlled by long-lived ribonucleic acids.     

Enhancing T lymphocytes from tumor-bearing mice suppress host resistance to a syngeneic tumor

The cell-mediated immune response of animals to a lethal syngeneic tumor was investigated by inoculating C57BL mice with Lewis lung carcinoma (3LL) cells T lymphocytes, obtained from the enlarged spleens of the tumor-bearing mice were found to be cytotoxic to 3LL target cells in vitro. However, we found that such spleen cells enhanced tumor growth in vivo when mice were injected with a mixture of spleen cells and tumor cells. Removal of T lymphocytes by treatment of the spleen cells with anti-Θ serum plus complement reduced the enhancement of tumor growth. Hence, the tumor enhancing cells, like the cytotoxic cells, appeared to be T lymphocytes. Removal of T lymphocytes from normal mice by adult thymectomy before tumor inoculation led to a reduction in the number of tumor metastases. Thus, enhancing T lymphocytes appear to exist in normal as well as in tumor-bearing mice. Investigation of this mechanism of tumor enhancement suggested that the enhancing T lymphocytes act as suppressor T cells inhibiting natural immune resistance to tumor growth.     

Role of macrophages in tumour immunity: III. Co-operation between macrophages and lymphoid factors in an in vitro allograft situation*

Normal mouse peritoneal macrophages were rendered cytotoxic towards target cells in vitro by incubation with immune C57B1 spleen cells taken early (7 days) and late (21 days) after a single injection of allogeneic DBA/2 lymphoma cells (L5178Y). This process, termed arming, was shown to be immunologically specific. X-irradiation of late immune spleen cells reduced their arming potential, but not that of early cells. Late immune spleen cells only were found to release spontaneously a factor which rendered macrophages weakly cytotoxic in vitro, and this release was not affected by irradiation. Both early and late immune spleen cells could be stimulated to release an arming factor by incubating them with irradiated lymphoma cells and this factor rendered macrophages strongly cytotoxic. Allo-immune serum also armed normal macrophages. Peritoneal macrophages harvested at 7 and 21 days from immunized mice were strongly cytotoxic towards the target cells. The results suggest pathways by which macrophages might become specifically cytotoxic in vivo and also integrated into the general immune response seen during rejection of the allogeneic L5178Y lymphoma.     

Macrophage cytotoxicity factor. A product of in vitro sensitized thymus-dependent cells

Peritoneal mouse macrophages are rendered specifically cytotoxic to allogeneic target cells by a cell-free filtered supernatant from in vivo sensitized spleen cells. Pretreatment of sensitized spleen cells with anti-θ serum and complement completely abolishes secretion of the supernatant factor. The factor can also be induced by in vitro sensitization of mouse spleen cells to alloantigens. Furthermore, a cortisone-resistant population of pure thymocytes can be sensitized in vitro and induced to liberate the factor. The macrophage cytotoxicity factor is only effective if bound to macrophages and can be transferred to iso- and allogeneic macrophages without loss of activity.     

Cell-mediated immune responses in vitro. V. A comparative study of in vitro immunogenicity of splenic lymphocytes, neoplastic lymphoid cells and fibroblats

This study compares the effectiveness of mouse lymphocytes, neoplastic lymphoid cells or fibroblasts in stimulating allogeneic cells to embark on an in vitro cytotoxic response, as measured by a ⁵¹Cr release assay. In addition, during ontogeny of mouse spleen cells, their capacity to stimulate in the mixed lymphocyte culture (MLR) was compared to their capacity to stimulate cytotoxic allograft responses. During ontogeny, there was amarked increase in the capacity of mouse spleen cells to stimulate mitotic responses in the MLR. In contrast, the magnitude of cytotoxic allograft responses induced by neonatal mouse spleen cells in the cytotoxic allograft system was comparable in magnitude to that induced by spleen cells of adult mice. The immunogenicity of subpopulations of mouse spleen cells was investigated. Mouse lymphoid cell populations, enriched for B or T lymphocytes or hemopoietic stem cells were equally immunogenic in vitro, as were myeloma or thymoma lymphoid cells. In contrast, mouse fibroblasts were found to elicit poor cytotoxic allograft responses. In fact, lymphoid cells were about 20–40-fold more immunogenic than fibroblasts.     

The thymus origin of the carrier-specific cell in an anti-hapten response in vitro

The primary in vitro response of spleen cells to 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)- hapten was markedly reduced by procedures which depleted the spleen cell population of T cells. One approach was to use adult thymectomized, irradiated, bone marrow restored (TxBM) mice. The anti-NIP response of spleen cells from carrier (SRBC)-primed TxBM mice was significantly reduced compared to that of primed sham-TxBM or primed normal spleen cells, and correlated with a deficient secondary anti-SRBC response in vitro. Two criteria were used for T cell depletion of TxBM mice: their impaired ability to mount a primary response to SRBC in vivo and the lower proportion of their spleen and lymph node cells susceptible to the cytotoxic action of anti-Θ serum. Using a second approach, the anti-NIP response of normal carrier-primed spleen cells was reduced by pretreatment of the cells with anti-Θ serum and complement. The anti-NIP response of anti-Θ treated cells and cells from TxBM mice was enhanced by the addition of irradiated spleen cells from primed normal mice. The requirement for carrier-priming to induce a good primary in vitro antihapten response was not abrogated by injecting donor mice with allogeneic lymphoid cells at various times before the preparation of cell cultures.     

Graft-versus-host reaction in F 1 recipients in the absence of donor (parental) cell proliferation

This study concerns the role of donor cell proliferation in graft-versus-host (GVH) reaction, using in vivo and in vitro systems. We have confirmed the reports of others that treatment of parental cells with mitotic inhibitors prevents the development of GVH splenomegaly in newborn F₁ recipients. However, in an in vitro GVH assay, parental cells treated with mitomycin C at doses as high as 50 μg/ml were shown to cause enlargement of newborn F₁ spleen fragments. This apparent anomaly was partly resolved by showing that mitomycin C-treated parental cells could give rise to splenomegaly in newborn F₁ mice in vivo, provided they were mixed with newborn F₁ spleen cells prior to injection. The possibility that mitotic inhibitors adversely affect the ability of parental cells to migrate to the sites of interaction with recipient cells, rather than inhibiting their actual ability to interact (and hence cause proliferation) is discussed. The nature of this GVH interaction is also considered.     

Immunological memory function of the T and B cell types: distribution over mouse spleen and lymph nodes

Spleens from LAF₁ mice injected intravenously with sheep erythrocytes (SE) are relatively rich in memory T cells early in the immune response (1 to 3 days) and rich in memory B cells as the response progresses (2 weeks or more). Marked cooperation for the secondary immune response in vitro was obtained by combining 10⁶ spleen cells from LAF₁ mice, taken 2 days after intravenous priming with SE, with 10⁷ spleen cells from day 14 primed mice. The results indicate relative deficiencies in the spleen for B memory cells on days 1 to 2 and for T memory cells on day 14 after priming. Day – 14, but not day – 2, immune lymph node (LN) cells could replace the day – 2 spleen cells (anti-Thy 1.2 sensitive) in the in vitro cooperation with day – 14 immune spleen cells. Immune spleen cells taken 4 to 7 days after priming contain more equivalent numbers of B and T memory cells, but 10 to 7 days after transfer of such immune spleen cells without SE into irradiated recipients the T memory cells were again more prominent in lymph node and the B memory cells in spleen as shown by in vitro cooperation studies. These results suggest that during the second week after intravenous injection of SE relatively more T than B memory cells migrate from spleen to lymph node, resulting in an imbalance in the splenic memory cell population favoring B memory cell function.     

Interleukin-1 synthesis and activity in aged mice

Recent studies have provided evidence that deficient interleukin-2 (IL-2) production by helper T cells contributes to the impaired T-cell-mediated functions observed in aged mice. Since most of these responses depend upon the presence of macrophages, a deficit in the functional capacity or in cell cooperation of macrophages may result in a decrease in immune reactivity. We found in the present study, that in vitro the cytostatic activity of macrophages from aged C57BL/6 (B6) mice is affected only slightly, but that in vivo their number increases with age. The synthesis of IL-1 is reduced when macrophages from aged mice are stimulated in vitro by lipopolysaccharide, but addition of exogenous IL-1 apparently does not restore either the mixed lymphocyte reaction or cytotoxic T lymphocyte generation. Co-cultures of young splenic macrophages with aged T lymphocytes do not restore to normal level the impaired proliferative response to T mitogens of aged B6 mice, but aged splenic macrophages provide a full accessory help for mitogenesis of young T cells. Thus, absorption of IL-1 by phytohemagglutinin-activated T cells is slightly altered in aged mice. IL-2 responsive T cells are not altered since exogenous IL-2 supply in vitro completely reconstitutes cytotoxic T lymphocyte generation after an allogeneic stimulation. Moreover, the number of Lyt 1⁺ cells is not modified in aged B6 mice. These results suggest that the impaired capacity of macrophages to release IL-1 and of blast T cells to bind IL-1 may contribute to the depression of cell-mediated immune reactivity associated with aging but also that the main defect is a functional lesion of IL-2 production by Lyt 1⁺ helper T cells.     

Inhibition of myeloid cell differentiation in cancer: the role of reactive oxygen species

It is well established that tumor growth is associated with accumulation of immature myeloid cells (ImC). They play an important role in tumor-associated immune suppression. ImC accumulate not only in tumor-bearing hosts but also in immunized, tumor-free hosts or hosts infected with bacterial pathogens. The kinetics of ImC in these mice is different. If in tumor-bearing mice, the number of ImC continues to increase with tumor progression in tumor-free mice after an initial spike, it decreases to the control level. Here, we have investigated the mechanisms of ImC accumulation in tumor-bearing hosts by comparing differentiation of ImC obtained from tumor-free and tumor-bearing mice. In the presence of appropriate growth factors, ImC isolated from tumor-free mice quickly differentiated in vitro into mature dendritic cells (DC), macrophages, and granulocytes. In contrast, differentiation of ImC from tumor-bearing mice was significantly delayed. Similar results were obtained in vivo after adoptive transfer of ImC into na?ve, congeneic mice. ImC transferred into tumor-bearing recipients failed to differentiate into DC or macrophages. ImC from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC obtained from tumor-free mice. Hydrogen peroxide (H(2)O(2)) but not superoxide radical anions was found to be the major part of this increased ROS production. In vitro experiments demonstrated that scavenging of H(2)O(2) with catalase induced differentiation of ImC from tumor-bearing mice into macrophages. Thus, this is a first demonstration that tumors may prevent differentiation of antigen-presenting cells by increasing the level of endogenous H(2)O(2) in immature myeloid cells.     

Vernonia cinerea L. scavenges free radicals and regulates nitric oxide and proinflammatory cytokines profile in carrageenan induced paw edema model

In this study, we evaluated the anti-oxidant and anti-inflammatory activities of the medicinal plant, Vernonia cinerea L (Asteraceae) using in vitro as well as in vivo models. Methanolic extract of Vernonia cinerea was found to scavenge the hydroxyl radical generated by Fenton reaction (IC₅₀130 μg/ml), Superoxide generated by photo reduction of riboflavin (IC₅₀190 μg/ml) and inhibited lipid peroxidation significantly (IC₅₀130.5 μg/ml). The drug also scavenged nitric oxide (IC₅₀210 μg/ml). Intraperitoneal administration of Vernonia cinerea was found to inhibit the PMA induced Superoxide generation in mice peritoneal macrophages. The administration of Vernonia cinerea to mice significantly increased the levels of catalase, superoxide dismutase, glutathione, glutathione peroxidase and glutathione-S transferase in blood and liver, whereas lipid peroxidation activity was significantly decreased. It was also found that Vernonia cinerea extract significantly inhibited carrageenan induced inflammation, compared with control models. Down regulation of pro-inflammatory cytokine level and gene expression were also support the above result.     

In vitro induction of tumor specific immunity: requirements for T lymphocytes and tumor growth inhibition in vivo

Cortisone-resistant thymocytes, spleen cells, thoracic duct lymphocytes, peritoneal exudate cells and peripheral blood lymphocytes of BALB/c mice were immunized in vitro against syngeneic HPC-108 plasma cell tumor cells. Cocultivation of spleen lymphocytes together with HPC-108 cells generated the highest cytotoxic immune response in comparison to other lymphocyte sources. Cytotoxicity was tested in a 6 hour ⁵¹Cr release assay using HPC-108 cells as target cells. The use of AKR anti-θ C3H serum indicated that thymus-derived (T) lymphocytes are essential to the initiation phase of the immune response to plasma cell tumor cells. Furthermore, evidence is presented that the cytotoxic effector cells in the in vitro tumor immune response are T lymphocytes. Spleen cells activated in vitro against HPC-108 tumor cells were shown to specifically prevent tumor growth from simultaneously injected HPC-108 cells in irradiated recipient mice.     

Immune responses to weakly immunogenic virally induced tumors II. Suppressive effects of the in vivo carried tumor YAC

Unprimed spleen cells from A and C57BL/6 mice could not produce cytotoxic responses to their syngeneic tumors: a Moloney virus-induced in vitro subline YAC-1 and a Rauscher virus-induced in vitro subline RBL5, respectively. Spleen cells from A and C57BL/6 mice immunized with YAC-1 or RBL5 (which cross-react serologically) generated significant syngeneic cytotoxicities after cultivation in vitro. The in vivo carried tumor of A mice, unlike the in vitro sublines, could not stimulate a priming effect. In contrast, YAC stimulated the formation of suppressor cells in both A and C57BL/6 mice. The suppressor cells abrogated the priming effect of the syngeneic tumors, but not the priming effect of the allogeneic tumors. Furthermore, YAC did not suppress normal allogeneic anti-tumor responses. The theoretical and the practical implications of these studies are discussed.     

Concanavalin A-induced resistance to Listeria monocytogenes and activation of macrophages: defect in mice treated with 89Sr

⁸⁹Sr-treated mice injected with concanavalin A (Con A) 24 h prior to infection with Listeria monocytogenes (LM) could not enhance the clearance of LM from the spleen. Adoptive transfer of normal syngeneic spleen cells together with Con A rendered these animals more resistant. Spleen cells of ⁸⁹Sr-treated or age-matched control mice were stimulated with Con A for 24 h, and supernatant fluids were assessed for macrophage-activating factor (MAF), i.e. the ability to activate resident peritoneal macrophages to kill LM intracellularly in vitro. A defective MAF production by spleen cells was observed in ⁸⁹Sr-treated, 2 week-old, and athymic nude mice. Also, treatment of spleen cells with anti-Thy-1.2 antiserum plus complement inhibited MAF production. Synergism between spleen cells from ⁸⁹Sr-treated and nude mice did not occur. The cells required for MAF production were relatively resistant to gamma irradiation. Nylon wool filtration did not modify the ability of spleen cells to make MAF. ⁸⁹Sr-treated mice possess macrophages responsive to MAF derived from normal spleen cells. The data suggest that the failure of ⁸⁹Sr-treated mice to develop an anti-LM response observed in this system could be due to a defective capacity to produce protective humoral factors and/or cells in response to Con A.     

Immunization of mice with the N-terminal (1–272) fragment of simian virus 40 large T antigen (without adjuvants) specifically primes cytotoxic T lymphocytes

Immunization of C57BL/6 (B6) mice (H-2^b) with the “large tumor antigen” (T-Ag) of simian virus 40 (SV40) in its soluble form without adjuvants primed CD8⁺ cytotoxic T lymphocytes (CTL) in vivo. CD8⁺ CTL primed in vivo by this non-structural 708-amino acid (aa) viral protein, and specifically restimulated in vitro, lysed H-2^b target cells, either transfected with an SV40 T-Ag-encoding vector, or transformed by SV40 infection. H-2^b RMA-S transfectants expressing the complete 708 aa T-Ag (which fail to transport peptides through the endoplasmic reticulum membranes) were not lysed. CTL were also efficiently primed in vivo by injection of the N-terminal 272 aa fragment of the T-Ag. Hence, this fragment contains the structure (s) required for a soluble protein to enter the “endogenous” class I-restricted antigen processing and presentation pathway for CD8⁺ CTL activation. In soluble form, the complete T-Ag or the N-terminal T-Ag fragment sensitized in vitro RBL5 cells for lysis by T-Ag-specific CTL lines and clones. This in vitro sensitization was blocked by brefeldin A. In contrast, specific recognition of RBL5 cells pulsed in vitro with synthetic, immunogenic nonapetides (derived from N-terminal T-Ag epitopes) by CTL lines was insensitive to brefeldin A. Hence, T-Ag and its 272-aa N-terminal fragment can enter the “endogenous” processing pathway and prime CD8⁺ CTL in vivo and in vitro.     

Cell surface binding affinity of Simian virus 40 T-antigen

Simian virus 40-transformed cells are characterized by a virus-induced tumor transplantation antigen (SV40 TSTA) defined in vivo by the rejection of tumorigenic SV40-transformed cells in SV40-immunized mice and in vitro by SV40 tumor cell-specific cytotoxic T cells. Several experimental findings support the notion that SV40-infected and -transformed cells express SV40 large tumor antigen (TAg) or closely related antigens on the cell surface (surface T). In this report, evidence is presented for a cell surface binding affinity of SV40 TAg solubilized and extracted by disruption of SV40-transformed and SV40-infected cells in growth medium. Incubation of various transformed and nontransformed living monolayer cells in situ with these extracts led to a significant uptake of TAg to the cell surface (called “externally bound TAg”) up to two to five times higher amounts in comparison to native surface T on SV40-transformed cells. This was demonstrated by the highly sensitive ¹²⁵I-protein A assay using rabbit antisera directed against purified SV40 TAg. Serological analysis of TAg in cellular extracts and of externally bound TAg revealed no apparent differences suggesting the cell surface binding affinity as a new property of SV40 TAg. We interpret our results as an indication that this property enables purified TAg to initiate the cellular immune response necessary for the SV40-tumor rejection in mice.     

Phosphorylation of a C-SRC-related protein in macrophages activated in vitro with lymphokine

Treatment of proteose peptone elicited peritoneal macrophages from C3H/HeN mice or the macrophage cell line B6MP102 with a T-cell lymphokine preparation induces cytotoxicity for SV3T3 tumor cells. The Triton X-100 (TX-100) insoluble fractions from activated macrophages possessed kinase activity for an endogenous 53 kDa phosphoprotein (pp53) which was markedly greater than extracts from untreated macrophages. Addition of the tyrosine phosphatase inhibitor, Na₃,VO₄ to the cytotoxicity assay also enhanced tumor cell lysis and Na₃VO₄ treated macrophages showed increased phosphorylation of pp53. Moreover, addition of Na₃VO₄ to the cytoskeleton kinase assay enhanced the phosphorylation of pp53 in a dose dependent manner. Pp53 was immunoprecipitated from the in vitro phosphorylated TX-100 insoluble fraction with monoclonal antibody to pp60^v-src. Anti-pp60^v-src also precipitated a 53 and a 60 kDa phosphoprotein from whole cell extracts and from TX-100 cytoskeleton extracts of macrophages phosphorylated as viable intact cells. Addition of a known tyrosine kinase inhibitor, quercetin, to the macrophage cytoskeleton kinase assay inhibited phosphorylation of pp53, and the in vitro phosphorylated pp53 was resistant to 1 N NaOH hydrolysis, indicating phosphorylation of tyrosine residues. Immune complex kinase assays of anti-pp60^c-src precipitated TX-100 insoluble macrophage fractions revealed strong phosphorylation for α-casein which was inhibited by quercetin. These data suggest that macrophage pp53 is a c-src-related gene product that is inducible by stimuli that activate macrophages to cytotoxicity.     

Population dynamics of natural killer cells in the spleen and bone marrow of normal and leukemic mice during in vivo exposure to interleukin-2.

By quantitative and functional methods, changes were assessed in NK(ASGM-1+) cell numbers and NK cell-mediated lytic function of the spleen and bone marrow of mice bearing a tumor of hemopoietic origin (FLV-induced erythroleukemia) for 9 days +/- simultaneous administration of indomethacin (10 micrograms/ml drinking water) +/- rIL-2 (3x/day, 12 x 10(3) Units/injection) during the last 4 days of tumor-bearing. Recombinant IL-2 alone during the last 4 days of tumor-bearing increased both the NK(ASGM-1+) cell numbers (p less than 0.001) and the functional activity (24-fold) of the spleen. In the bone marrow, however, no change in the numbers of NK(ASGM-1+) cells was observed relative to untreated tumor-bearing mice, but the NK cell-mediated lytic activity of that organ was augmented 30-fold. The continuous presence of indomethacin from the onset of tumor-bearing prior to rIL-2 treatment during the last 4 days of tumor-bearing, further boosted both the already high, rIL-2 driven numbers of NK(ASGM-1+) cells in the spleen (p less than 0.01), as well as splenic NK cell lytic function (2-fold). In the bone marrow, continuous presence of indomethacin prior to and during the terminal 4 days of co-administration with rIL-2 increased 3-fold the numbers of NK(ASGM-1+) cells relative to that of the bone marrow of tumor-bearing mice given rIL-2 alone, and resulted in lytic activity of that organ which was 140% of that of the rIL-2 treated, tumor-bearing mice. The results indicate that under the combined influence of indomethacin and rIL-2, the production of NK(ASGM-1+) cells was augmented in the bone marrow of tumor-bearing mice, export of immature NK(ASGM-1+) cells from the bone marrow was increased, and import of immature NK(ASGM-1+) cells by the spleen was increased. The increased NK(ASGM-1+) cell numbers in each organ was reflected in increased lytic function.