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.     

Functional analysis of mononuclear cells infiltrating into tumors. III. Soluble factors involved in the regulation of T lymphocyte infiltration into tumors

We have analyzed the mechanisms controlling the accumulation of T lymphocytes in tumor tissues. Spleen cells, left or right popliteal lymph node cells, and tumor-infiltrating cells were obtained from tumor-inoculated rats and were cultured for 24 h. Culture supernatants were obtained and assessed for lymphocyte migration factor (LMF) activity with the use of a modified Boyden chamber. We found that tumor-infiltrating cells derived from T-9-sensitized rats produced LMF. Two waves of LMF production were observed. The first wave of LMF production was detected between 6 and 12 h (LMF-a) and the second wave of LMF production was detected between 4 and 6 days (LMF-4d and -6d) after tumor inoculation. The tumor-infiltrating cells consisted of heterogenous cell populations. We found that only tumor-infiltrating neutrophils of T-9-sensitized rats produced LMF-a. Five peaks of LMF (A through E) were detected upon fractionation of LMF-a using Mono Q anion exchange column chromatography. Peak D exhibited the strongest activity. The action of peak D was chemotactic, but not chemokinetic. The m.w. of peak D was 33,000 and 70,000. Only W3/25 (+) (helper/inducer) T cells were found to be sensitive to peak D. The production of LMF-a by purified tumor-infiltrating neutrophils in vitro is in agreement with the histologic observation that the infiltration of neutrophils precedes the appearance of W3/25 (+) T cells in tumor tissues of T-9-sensitized rats. It is thus likely that peak D of LMF-a is responsible for the infiltration of T lymphocytes into tumor tissues.     

Role of interleukin 1 in experimental pulmonary granuloma in mice

Pulmonary granulomas were induced in immunized BALB/c mice by the intratracheal injection of antigen-coated and plain agarose beads. Prominent lesions developed within 24 hr, reached peak intensity within 3 days, and gradually declined in size thereafter. The hypersensitivity granulomas induced in sensitized mice by antigen-coated beads were much larger than the lesions induced by plain beads. Minimal inflammation was produced in unsensitized mice injected with antigen-coated or plain beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in sensitized mice by antigen-coated beads contained high levels of interleukin 1 (IL 1) and migration inhibition factor (MIF) activities. The kinetics of appearance of these mediators were similar. Lower but detectable activity of both mediators was detected in extracts prepared from sensitized mice injected with plain beads. Neither interleukin 2 (IL 2) nor IL 2 neutralizing activities were detected in the extracts. The presence of IL 1 and MIF in extracts prepared from early and peak pulmonary granulomatous lesions suggests that these soluble factors are produced by cells within the lesions, and that they are involved in mediating the expression and/or maintenance of the granulomas.     

Differing time courses of spleen leukocyte MIF synthesis and cytotoxicity during rejection of a murine lymphoma allograft

In vitro spleen leukocyte MIF synthesis and direct cytotoxicity were studied during growth and rejection of the EL-4 murine lymphoma in allogeneic tumor-bearing BALB/c mice to compare the time courses of these events. Rejection of EL-4 tumors occurred between 6 and 8 flays after intraperitoneal inoculation. Spleen leukocyte cytotoxicity measured by isotope release from chromium-51 labeled EL-4 target cells was first detected on day 6 and was maximal between days 11 and 18. In contrast, spleen leukocyte MIF synthesis stimulated by intact EL-4 cells was sometimes observed on day 11 and was maximal between 18 and 25 days after tumor challenge. These results show that maximal spleen cytotoxicity and MIF synthesis occur after completion of, rather than during ip tumor rejection and, in addition, that these two in vitro lymphocyte responses follow independent, significantly different time courses (P < 0.05). This asynchrony of MIF synthesis and cytotoxicity suggests that these in vitro correlates are mediated by distinct lymphocyte subpopulations.     

Macrophage Migration Inhibitory Factor (MIF) Enzymatic Activity and Lung Cancer

The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif ^P1G). Primary tumor growth was significantly attenuated in both Mif-KO and Mif ^P1G mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.     

Temporal analysis of cellular cytotoxicity and humoral factors during progession and regression of Rous sarcomas in Japanese quails.

Temporal appearance of cellular cytotoxicity and humoral activities including blocking and arming activities during the entire course of Rous sarcoma development in Japanese quails was examined by microcytotoxicity assay with comparison of animals bearing regressing tumors induced by a moderate dose of virus (regressors) and animals bearing growing tumors induced by a large dose of virus (progressors). Cellular cytotoxicity of the spleen cells in regressors was detected in a biphasic pattern; the first phase being observed as early as 3-5 days post inoculation (p.i.), followed by an eclipse period between 7-10 days p.i. which was the time of active tumor growth, and the second phase occurring after 12 days p.i. when the tumor had attained the maximum size. In progressors, only the first phase was observed. Instead, a stimulatory effect of the spleen cells on growth of target cells was noticed. Arming activity which confers cytotoxic activity on the normal spleen cells was demonstrated in the sera of regressors in the similar biphasic pattern as the cellular cytotoxicity; the early activity being present at 3 days p.i., and the late one after 19 days p.i. The former was detected by pre-incubation of serum with effector cells in microcytotoxicity assay and the latter by pre-incubation with target cells. In progressors, only the early arming activity which reacts with effector cells was demonstrated. Blocking activity which abrogates cellular cytotoxicity was demonstrated in both regressors and progressors but in different patterns of appearance, that is, blocking activity in regressors was only transiently demonstrated only by pre-incubation with effector cells at the time of maximum tumor growth, while the activity in progressors seemed to persist after the tumor reached the maximum size. Since the earlier activity was found to be effective at effector cell level, and the later one at both effector and target cell levels, participation of blocking factors of different types in progressors was also suggested.     

Stimulation of mouse migration inhibitory factor (MIF) production form MSV-immune lymphocytes by soluble tumor-associated antigen: requirement for histocompatible macrophages.

Spleen cells from C57BL/6 mice immunized with murine sarcoma virus (MSV) are capable of producing migration inhibition factor (MIF) in response to stimulation with a specific tumor-associated antigen prepared by solubilization with 3 M KCL. We have previously demonstrated that this response is T cell-dependent. Further investigations into the effector cells involved in the production of MIF have revealed that spleen cells from mice immunized with MSV cannot produce MIF when stimulated with tumor extract if the population has been previously depleted of macrophages. However, the response can be restored by adding nonimmune syngeneic macrophages but not by allogeneic macrophages. The inability of allogeneic macrophages to provide this function was not due to their increased suppressor activity since in mixing experiments they did not interfere with the ability of immune spleen cells to produce MIF. Furthermore, they were not defective since they could supply this "cooperative function" to appropriate F1 mice. The results indicate that macrophages are required for stimulation of MIF by soluble tumor antigens and that for efficient interaction the macrophages and lymphocytes must share some genetic similarities.     

Role of the thymus in regulation of the macrophage migration inhibitory factor production in mice of different genotypes]

The influence of the thymus on the production of the macrophage migration inhibitory factor (MIF) was studied in C57BL and CBA mice thymectomized at 4--6 weeks of age. On the 1--21st day after the operation they were immunized intraperitoneally with complete Freund's adjuvant. MIF production stimulated by tuberculin was determined on the maximum of the immune response. MIF production was abolished in mice of both lines already during the first days. To elucidate a relationship between MIF production and the presence of the thymus the former was investigated in the thymectomized "nude" mice. The mice showed no MIF production. It was found as well that thymectomy can interrupt the immune response in early stages of its development and completely eliminates MIF production the first days after immunization. Moreover, thymectomy in adult mice also changes spontaneous migration of macrophages both in immunized and non-immunized mice. These changes were more pronounced in C57BL mice.     

Splenic regulation of lymphocyte trapping in lymph nodes draining tumor grafts.

We measured the trapping of 51chromium-labeled splenic lymphocytes in the lymph nodes and spleens of tumor-inoculated mice at various intervals after inoculation. Both histocompatible and incompatible tumors were studied. Significant trapping occurred in the draining lymph nodes over the entire course of the experiments. The splenic trapping response commenced very early after transplantation and abated after a few days. The magnitude of the lymph node trapping response correlated with the magnitude of antigenic difference between tumor and host. Splenectomy before tumor inoculation had bidirectional effects; it caused a marked augmentation of the trapping response in the early period after tumor inoculation, which declined with time so that late after tumor inoculation, splenectomized animals trapped less well than intact controls. Tumor resection always led to a rapid fall in the trapping response in normal mice. However, tumor resection at the time of the peak trapping response of splenectomized mice produced no change. These results support the notion that lymphocyte trapping is a valid and useful monitor of the immune response to tumors. They also serve to reemphasize the important regulatory role of the spleen plays in the immune response to tumor-associated antigens by demonstrating a new parameter of splenic control; regulation of lymphocyte traffic in response to antigenic stimuli.     

Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus

The relationship between virus-specific CD8(+)-T-cell responses and viral persistence was studied in mice by using Hantaan virus (HTNV). We first established a simple method for measuring levels of virus-specific CD8(+) T cells by flow cytometry. Next, to produce a mouse model of persistent HTNV infection, newborn mice were inoculated subcutaneously within 24 h of birth with 1 or 0.1 50% newborn mouse lethal dose of HTNV. All mice that escaped lethal infection were persistently infected with HTNV until at least 30 days after virus inoculation and had no virus-specific CD8(+) T cells producing gamma interferon (IFN-gamma). Subsequently, the virus was eliminated from some of the mice, depending on the appearance of functional virus-specific CD8(+) T cells, which have the ability to produce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) and have cytotoxic activity. Neutralizing antibodies were detected in all mice, regardless of the presence or absence of virus. In the acute phase, which occurs within 30 days of infection, IFN-gamma-producing HTNV-specific CD8(+) T cells were detected on day 15 after virus inoculation. However, TNF-alpha production and the cytotoxic activity of these specific CD8(+) T cells were impaired and HTNV was not removed. Almost all of these specific CD8(+) T cells disappeared by day 18. These results suggest that functional HTNV-specific CD8(+) T cells are important for clearance of HTNV.     

Role of macrophage migration inhibitory factor in bleomycin-induced lung injury and fibrosis in mice

Macrophage migration inhibitory factor (MIF) is a unique cytokine that reportedly overrides the anti-inflammatory effect of endogenous glucocorticoids. MIF has been demonstrated to be involved in a variety of inflammatory diseases. In this study, we examined the role of MIF in bleomycin (BLM)-induced lung injury and fibrosis. The levels of MIF in lung tissues and bronchoalveolar lavage fluids were significantly increased in the period 5-10 days after intratracheal administration of BLM. Treatment with the anti-MIF antibody significantly reduced the mortality at 14 days and the histopathological lung injury score at 10 days. These effects were accompanied with significant suppression of the accumulation of inflammatory cells in the alveolar space and tumor necrosis factor-alpha in the lungs at 7 days. However, the anti-MIF antibody did not affect either the content of lung hydroxyproline or the histopathological lung fibrosis score at 21 days after BLM. These data provide further evidence for the crucial role of MIF in acute lung inflammation but do not support the involvement of MIF in lung fibrosis induced by BLM in mice.     

Regulation of the CTL response by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) has been shown to be a pivotal cytokine that mediates host inflammatory and immune responses. Recently, immunoneutralization of MIF has been found to inhibit tumor growth in mice; however, the contributing mechanisms underlying this effect have not been well defined. We investigated whether MIF plays a regulatory role in the expression of CTL activity. In a mouse model of the CTL response using the OVA-transfected tumor cell line EL4 (EG.7), we found that cultures of splenocytes obtained from EG.7-primed mice secrete high levels of MIF following Ag stimulation in vitro. Notably, parallel splenocyte cultures treated with neutralizing anti-MIF mAb showed a significant increase in the CTL response directed against EG.7 cells compared with control mAb-treated cultures. This effect was accompanied by elevated expression of IFN-gamma. Histological examination of the EG. 7 tumors from anti-MIF-treated animals showed a prominent increase in both CD4(+) and CD8(+) T cells as well as apoptotic tumor cells, consistent with the observed augmentation of CTL activity in vivo by anti-MIF. This increased CTL activity was associated with enhanced expression of the common gamma(c)-chain of the IL-2R that mediates CD8(+) T cell survival. Finally, CD8(+) T lymphocytes obtained from the spleens of anti-MIF-treated EG.7 tumor-bearing mice, when transferred into recipient tumor-bearing mice, showed increased accumulation in the tumor tissue. These data provide the first evidence of an important role for MIF in the regulation and trafficking of anti-tumor T lymphocytes in vivo.     

Production and properties of immune interferon from spleen cell cultures of Toxoplasma-infected mice

In response to antigenic stimulation, spleen cells from Toxoplasma-infected mice produce a factor showing inhibitory activity against vesicular stomatitis virus infection in L cell cultures. When BALB/C and ICR mice were inoculated intraperitoneally with the low-virulent S-273 strain of T. gondii, such activity was first detected in 4 and 7 days and reached maximum levels at 10 and 14 days respectively, and retained these levels for at least three weeks. However, BALB/C mice, which are considerably more sensitive to Toxoplasma infection than ICR mice, produced significantly smaller amounts of interferon (IF) after challenge with the high virulent strain. The IF produced in this system possessed certain known properties of immune (type II) IF and was not neutralized by rabbit antiserum against mouse type I IF. The immune IF preparation also inhibited multiplication of Toxoplasma within nonphagocytic L cells in an IF-like fashion, whereas Newcastle disease virus-induced (type I) IF had no effect on this parasite. The antiviral and anti-Toxoplasma activity in immune IF preparations could not be distinguished solely on the bases of their molecular weight and isoelectric point. The experiments with anti-theta serum plus complement and with nylon wool column effluent cells strongly suggest that immune IF was produced by T lymphocytes and required the assistance of macrophages.     

Characterization of Effector Cells against B16 Melanoma in Mice Inoculated with Allogeneic Spleen Cells

Inbred C57BL/6 (B6) mice which had received an inoculation of allogeneic spleen cells showed remarkable antitumor activity against syngeneic tumor challenge with B16 melanoma cells 3 days after the allogeneic cell inoculation. This antitumor activity was not specific to the inoculated alloantigen, since the challenging B16 cells are syngeneic to B6 mice and since it was induced by BALB/c spleen cells as well as C3H/He spleen cells. The antitumor activity was sensitive to an in vivo treatment with anti-asialo GM1 (AGM1) antiserum or anti-Thy.1 monoclonal antibody (mAb) just before the tumor challenge and was resistant to an in vivo treatment with anti-CD8 (Ly. 2) mAb. These results suggest that AGM1+Thy.1+CD8– activated natural killer (NK) cells were generated by alloantigen inoculation and took an important part in the antitumor effect of the alloantigen inoculation.     

The allogeneic effect on tumor growth. II. Suppression of both ascitic and solid MOPC 315 plasmacytoma by the graft-vs-host reaction, with pathologic correlation.

The growth of an ascitic murine plasmacytoma, MOPC 315, can be retarded in CAF1 hybrid host mice by the i.p. injection of donor lymphoid cells. The graft-vs-host reaction can be established by a variety of donor cells, including parental BALB/c and A/J and congenic inbred B10.D2 which share the major histocompatibility locus with BALB/c(H-2d). Optimal results are consistently obtained when parental BALB/c spleen cells are injected before tumor inoculation, and a second dose of donor spleen cells injected 1 week later. This aloogeneic effect on tumor growth is manifested by delayed appearance of the tumor and prolonged host survival. Pathologic studies on the ascites tumor indicated that the allogeneic effect suppresses the initial appearance and early growth of the plasmacytoma. However, once established, MOPC 315 grows rapidly and fatally in both control mice and recipients of donor lymphoid cells. Further, a subcutaneous implant of MOPC 315 is suppressed by an allogeneic effect established either i.v. with BALB/c spleen cells before tumor inoculation or by BALB/c spleen cells administered subcutaneously at the time of MOPC 315 implant. Thirty percent of mice treated by i.v. or subcutaneous donor lymphoid cells were tumor free at 150 days after tumor inoculation.     

Lactate dehydrogenase-elevating agent is responsible for interferon induction and enhancement of natural killer cell activity by inoculation of Ehrlich ascites carcinoma cells into mice

Inoculation of Ehrlich ascites carcinoma cells (EAC) into the peritoneal cavities of outbred ddY mice induced interferon (IFN) in the circulation. The maximum titer (1,280 U) was obtained at 24 hr after inoculation. This induced IFN had the characteristics of type I IFN, i.e., stability at pH2 and lability at 56 C. An increase in natural killer cell (NK) activity was also observed for the first 3 days after inoculation. In addition, plasma lactate dehydrogenase (LDH) activity was elevated in these mice. Inoculation of ascitic fluid or serum of EAC-bearing mice into normal mice increased plasma LDH activity six- to sevenfold over normal levels and elevated activities persisted throughout the life of the mice. These results suggest that the LDH-elevating agent was responsible for IFN induction and for enhancing NK activity. Because lactate dehydrogenase-elevating virus (LDV) can be eliminated from tumor cells by passage in vitro, we attempted to grow EAC in tissue culture for several months and re-examined whether the inoculation of such cells could elevate plasma LDH activity induce IFN and enhance NK activity. The results showed that inoculation of the passaged cells had no effect on these activities in normal mice. Therefore, we concluded that the IFN inducer was LDV which contaminated the EAC and then enhanced the NK activity. N-tropic murine leukemia virus also contaminated EAC, but this virus was not responsible because cultured cells of EAC still shed this virus.     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.     

An antibody for macrophage migration inhibitory factor suppresses tumour growth and inhibits tumour-associated angiogenesis

To verify the role of macrophage migration inhibitory factor (MIF) in tumourigenesis, we examined the effect of an anti-MIF antibody on tumour growth and angiogenesis. We inoculated murine colon adenocarcinoma cell line colon 26 cells subcutaneously into the flank in BALB/c mice. After nine days, we treated tumour-bearing mice with an anti-rat MIF antibody by intraperitoneal injection on days 9, 11, 13, 15, 17, 19 and 21. We found significant inhibition of tumour growth by this treatment from day 15 to day 22. Next, we implanted a chamber filled with colon 26 cells, which only passes soluble factors, in the subcutaneous fascia of the flank, and treated mice with the anti-rat MIF antibody at days 1, 3 and 5. By histological examination at day 6, angiogenesis within the subcutaneous fascia in contact with the chamber was markedly suppressed. In vitro, we added an anti-human MIF antibody to human umbilical vein endothelial cells (HUVEC) to evaluate its effect on cell growth by measurement of [3H]thymidine incorporation. We observed that the anti-MIF antibody significantly suppressed [3H]thymidine uptake by HUVEC. These results suggest the possibility that MIF is involved in tumourigenesis via promotion of angiogenesis.     

Antibody formation and transient immune complex glomerulopathy in A-Strain mice with C1300 neuroblastoma tumors

One to three-month-old A-strain mice, inoculated subcutaneously with 2 x 10(6) viable syngeneic C1300 neuroblastoma cells (clone NB9R) developed a palpable tumor within 9-12 days and died within 28-30 days. A transient glomerulopathy developed after 16-24 days. Despite a normal histologic appearance, the nephropathy was clearly demonstrated by electron microscopy and was classified as a focal mesangiopathic glomerulonephritis. Deposits of host 7S-G immunoglobulins and C3 complement fragments were detected in these same kidneys by immunofluorescence. Radioimmunoprecipitin determinations on sera obtained from mice at different intervals from tumor cell inoculation, revealed that untreated mice contained circulating antibodies capable of reacting with 125I-labeled gp69-71 glycoprotein from Gross murine leukemia virus (MuLV). Antibodies to p30 MuLV antigen and to crude membrane antigen (s) (CMA) solubilized from NB9R cells were found in sera only after tumor cell inoculation. Circulating immune complexes formed by host 7S-G immunoglobulins were clearly detected from day 16 to 22. Antibodies eluted from kidneys with nephropathy were shown to react with NB9R cells in vitro and to react specifically with CMA and the p30 MuLV antigen.