Mitsunobu Matsumoto

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse. I. Generation of suppressor macrophages in spleen cells of BCG-vaccinated mice.

With the aim of clarifying the mechanism of the suppressive action of BCG against insulitis and overt diabetes in NOD mice, we studied the effects of BCG on spleen cell populations and on the in vitro immune responses of spleen cells. The spleen cells of BCG-vaccinated mice showed much lower responsiveness to various mitogens such as Con A, PHA, PWM, and LPS than those of saline-treated mice. Low responsiveness to alloantigens was also observed. Flow cytometric analysis of the spleen cells revealed that Mac-1+ and Mac-2+ cells had increased while T and B cells had decreased in the BCG-vaccinated mice compared with the saline-treated mice at the time when the maximum level of inhibition of mitogen responses of BCG-vaccinated mice was observed. This suggests that the decreased in vitro immune response was due to the increase in macrophages which suppress lymphocyte functions. Support for this interpretation comes from the following two findings: (1) the restoration of mitogen responses of spleen cells when macrophages were eliminated by plastic adhesion or FACS sorting and (2) resuppression of PHA and Con A responses of plastic-nonadherent spleen cells by addition of adherent cells or flow cytometrically sorted Mac-1+ cells obtained from BCG-vaccinated mice. These results indicate the generation of suppressor macrophages after BCG vaccination and suggest that these macrophages prevent the autoimmune pathogenesis leading to diabetes in NOD mice.

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse: II. Suppression of pathogenesis by macrophage transfer from BCG-vaccinated mice

Our previous reports showed that a single injection of live BCG, one of the biological response modifiers, prevents insulitis and overt diabetes in NOD mice and that the suppression could be due to the generation of some type of suppressor cells in the BCG-treated mice. Furthermore, a more recent study has revealed that macrophages suppressive against a variety of lymphocyte functions can be induced by BCG, which suggests that these macrophages are involved in the suppression of the pathogenesis. To obtain valid evidence for this speculation, the effects of transfer of macrophage and T-cell fractions on the pathogenesis were examined in the present study. Transfer of macrophage-enriched spleen cell fraction harvested from the BCG-treated females to young females abolished the occurrence of spontaneous diabetes up to the age of 25 to 30 weeks. Also, macrophage transfer prevented the progress of insulitis. In contrast, transfer of a T-cell-enriched fraction did not suppress insulitis and overt diabetes. From these results, it could be concluded that the suppression of the autoimmune pathogenesis of diabetes by BCG is due to the generation of suppressor macrophages.

Effect of recombinant interleukin 2 (R-IL2) on in vivo growth of murine myeloma X5563

SummaryThe present study deals with the effect of recombinant interleukin 2 (R-IL2) on in vivo growth of murine myeloma X5563. Administration of R-IL2 (5×104 J.U./mouse per day) s.c. starting 1 day after X5563 inoculation i.d. had a marginal effect on the growth of X5563, and all the mice repeatedly given R-IL2 from day 1 to day 17 died. However, daily administration of R-IL2 starting 7 days after the tumor inoculation was highly effective and significantly lengthened survival time compared with the control mice injected with vehicle alone. About 50% of the treated mice were completely cured, and survived for more than a month after the therapy ceased. In a representative experiment, where the growth of X5563 was slow because of the small number of inoculated tumor cells, all the mice (n=6) given R-IL2 from day 11 to day 23 showed complete cure of the established X5563 solid tumor. These mice showed in vivo protective immunity and in vitro cytotoxic T cell responses to X5563 tumor antigens. Histologically, a large number of macrophages and lymphocytes had infiltrated the area around the necrotic X5563 tumor mass in the mice which had received R-IL2 therapy. These results suggest that repeated injections of R-IL2 at the local site after tumor development can augment antitumor immunological responses and subsequently induce tumor regression.

Preclinical in vivo Antitumor Efficacy of Nedaplatin with Gemcitabine against Human Lung Cancer

The antitumor efficacy of the combination of nedaplatin (NDP) with gemcitabine (GEM) was evaluated. We also compared the antitumor activity of NDP plus GEM with that of cisplatin (CDDP) plus GEM or carboplatin (CBDCA) plus GEM. Ma44, which is a human lung cancer sensitive to GEM, and NCI-H460, which is a human lung cancer refractory to GEM, were used in this study. GEM was injected i.v. once followed by i.v. injection of NDP at an interval of approximately 30 min into tumor-bearing athymic mice. GEM was administered again 3 or 4 days thereafter. Combined dosing of NDP with GEM resulted in synergistically enhanced inhibition of tumor growth in the Ma44 tumor model. NDP plus GEM was also effective against Ma44 cells when given late in the therapy, a model for advanced disease. Potent augmentation of growth inhibition by NDP with GEM was also found with the NCI-H460 tumor model. The combination effect of NDP plus GEM appeared to be superior to that of CDDP plus GEM or CBDCA plus GEM in both tumor models. Toxicity in terms of blood cell numbers was not enhanced by the combination of NDP with GEM. These results suggest the effectiveness of combination of NDP with GEM for clinical therapy.

Immune deficiency of the cts mouse. I. Deficiency of in vitro t cell-mediated immune response

The cataract Shionogi (CTS) mouse characterized by cataracts and microphthalmia is a sister strain of the NOD mouse. We have made the immunological characterization of the CTS mouse by means of in vitro assays. Splenocytes of the CTS mouse were very low in the responsiveness to T cell mitogens such as Con A and PHA but not to a B cell mitogen, LPS. The production of IL 2 and expression of IL 2-receptor of spleen cells after in vitro stimulation with Con A decreased in the CTS mouse, when compared with those in the NOD and the other reference strains. In mixed lymphocyte culture, CTS splenocytes did not proliferate and did not generate cytotoxic T lymphocytes when cocultured with splenocytes of the C3H/He mouse. The NK activity against YAC-1 target cells was lower in the CTS mouse than in the C3H/He mouse, an NK high responder, but higher than in the NOD mouse, a low responder. These results suggest that the CTS mouse is deficient in T cells. Subset analysis of splenic lymphocytes of the CTS mouse using flow cytometry revealed that the percentage of T cells in the CTS mouse was significantly lower than those in the reference strains, which was consistent with the reduced responsiveness to T cell mitogens in the CTS mouse. The deficiency in the Ly-2+ T cell subset was particularly striking. However, the response to PHA of the splenocytes of the CTS mouse was normalized when T cells were enriched by nylon wool-passing and cell-sorting. Therefore, it seems that decreased T cell activity is due to a decrease in T cell number and not to dysfunction of individual T cells.

Combination Therapy of Interleukin-2 and Sorafenib Improves Survival Benefits and Prevents Spontaneous Pulmonary Metastasis in Murine Renal Cell Carcinoma Models

OBJECTIVE The objective of this study was to evaluate the benefits of combination therapy consisting of recombinant human interleukin-2 and sorafenib for survival efficacy and the suppression of metastasis in murine renal cell carcinoma models. METHODS Lung-metastasized renal cell carcinoma mice were treated with various combinations of recombinant human interleukin-2 and sorafenib. Tumor growth was observed using a bioluminescence imaging system. Next, the nephrectomized renal cell carcinoma mice were administered various combinations of recombinant human interleukin-2 and sorafenib, followed by a lung resection in order to examine lung metastasis by bioluminescence imaging. RESULTS The increased life-span ratio in mice receiving combination therapy was 1.45, whereas that in mice treated with sorafenib or recombinant human interleukin-2 alone therapy was 1.28 and 1.07, respectively. The concomitant administration of recombinant human interleukin-2 and sorafenib had a metastasis-inhibitory effect, whereas the other treatments failed. CONCLUSIONS These findings indicate that combination therapy of recombinant human interleukin-2 and sorafenib may offer better outcomes than either monotherapy with recombinant human interleukin-2 or sorafenib with respect to survival benefits and the prevention of pulmonary metastasis in renal cell carcinoma patients.

Antitumor Efficacy of Recombinant Human Interleukin-2 Combined with Sorafenib Against Mouse Renal Cell Carcinoma

OBJECTIVE Recombinant human interleukin-2 (rhIL-2) has been clinically used in the treatment of renal cell carcinoma (RCC). Sorafenib, a multi-targeted kinase inhibitor, has been approved for RCC as well as IL-2. The purpose of this study was to evaluate the antitumor efficacy of IL-2 combined with sorafenib in three different murine renal cancer models using Renca cells. METHODS We established the subcutaneous tumor model by inoculating wild-type Renca cells into the backs of BALB/c mice, the pulmonary metastatic tumor model by an intravenous injection of luciferase-expressing Renca cells into the tail vain and the orthotopic tumor model by injecting luciferase-expressing Renca cells into the renal subcapsule. These tumor-bearing mice were treated intra-peritoneally with rhIL-2 and/or per os with sorafenib. The antitumor efficacy was evaluated by measuring the tumor size of the subcutaneous tumor or photon intensity of the pulmonary metastatic tumor and the orthotopic tumor. RESULTS When rhIL-2 was combined with sorafenib, the antitumor efficacy was significantly augmented in comparison with either rhIL-2 or sorafenib alone in all the models. Sorafenib did not inhibit rhIL-2-induced natural killer cell expansion and rhIL-2 had no effect on the anti-angiogenic activity of sorafenib. CONCLUSIONS The results suggest that the combination of rhIL-2 and sorafenib may offer significant potential as a novel therapeutic approach for patients with RCC.

New γ-fluoromethotrexates modified in the pteridine ring: synthesis and in vitro immunosuppressive activity

Our continuing program to develop new antifolate drugs useful against rheumatoid arthritis led us to modify the pteridine ring of gamma-fluoromethotrexate. Pyrrolopyrimidine derivatives 1e and 1t were found to exhibit potent suppressive effects on the responses of both T and B cells to mitogens, although tetrahydropyridopyrimidine derivatives 2e and 2t and quinazoline derivatives 3e, 3t and 4e showed very weak suppressive activities. Thus, conversion of the pteridine ring of gamma-fluoromethotrexate to a pyrrolopyrimidine ring led to a new potential antirheumatic compound.

NY-ESO-1 Protein Cancer Vaccine With Poly-ICLC and OK-432: Rapid and Strong Induction of NY-ESO-1-specific Immune Responses by Poly-ICLC.

We conducted a clinical trial of a cancer vaccine using NY-ESO-1 protein with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) and/or OK-432 against solid tumors. A total of 15 patients were sequentially enrolled in 4 cohorts. Patients in cohort 1 received NY-ESO-1 protein; cohort 2a received NY-ESO-1 protein+OK-432; cohort 2b received NY-ESO-1 protein+poly-ICLC; cohort 3 received NY-ESO-1 protein+OK-432+poly-ICLC with Montanide ISA-51. The endpoints of this trial were safety, NY-ESO-1 immune responses, and clinical response. Vaccine-related adverse events observed were fever and injection-site reaction (grade 1). Two patients showed stable disease after vaccination. NY-ESO-1 antibodies were observed in 4 patients at the baseline (sero-positive) and augmented in all patients after vaccination. Eleven patients showed a conversion of negative antibody responses at baseline to positive after vaccination (seroconversion). The seroconversions were observed in all 11 sero-negative patients by the fourth immunization; in particular, it was observed by the second immunization in patients with poly-ICLC, and these induced antibody responses were stronger than those in patients immunized without poly-ICLC. The number of NY-ESO-1–specific interferon (IFN)&ggr;-producing T cells was increased in patients immunized with poly-ICLC and/or OK-432, and furthermore, the increase of IFN&ggr;-producing CD8 T cells in patients immunized with poly-ICLC was significantly higher than that in patients without poly-ICLC. Nonspecific activations of T-cell or antigen presenting cells were not observed. Our present study showed that poly-ICLC is a promising adjuvant for cancer vaccines.

GENETIC CONTROL OF PERIPHERAL T-CELL DEFICIENCY IN THE CATARACT SHIONOGI (CTS) MOUSE LINKED TO CHROMOSOME 7

The cataract Shionogi (CTS) mouse characterized by cataract and microphthalmia has been established at Shionogi Aburahi Laboratories as an inbred strain from a population of outbred ICR mice (Ohotori et al. 1968). This is a strain related to the NOD mouse which is widely used as an animal model of human Type I diabetes mellitus, having the same class II major histocompatibility complex (MHC) and class I D-end as those of the NOD mouse (Makino et al. 1980; Ikegami et al. 1988). In previous studies on CTS mice, we demonstrated a marked decrease of T cells in blood and peripheral lymphatic tissues and a resultant deficiency of T-cell-mediated in vitro and in vivo immune responses (Yagi et al. 1990a, b). Our recent study revealed that numerous CD4 single positive T cells are accumulated in the CTS thymus, suggesting that peripheral T-cell deficiency can be attributed to the defect of migration of mature T cells from the thymus (Yagi et al. 1996). These findings led us to investigate the inheritance mode of this unique immunological abnormality and to determine the locus of the responsible gene(s). The CTS and C3H/He strains bred at Shionogi Aburahi Laboratories were used as parent strains and their reciprocal matings gave F1 hybrids. F1 hybrids were then backcrossed to CTS and C3H/He mice to produce the BC1 and BC2 generations, respectively. The F2 offspring were obtained by mating F1 hybrids. All the mice were bred under specific pathogen-free conditions and used for the following analysis at about 8 weeks of age. To examine the phenotype of T-cell deficiency, CD3+ cells in peripheral blood were analyzed by flow-cytometry. Approximately 0.3 ml of blood was removed from the ophthalmic plexus of each mouse. Blood lymphocytes were stained with FITC-labeled mouse CD3-specific monoclonal antibody (1:50 dilution of ascites of Clone 145-2C11; PharMingen, San Diego, Calif.) and analyzed by using FACScan (Becton Dickinson, Mountain View, Calif.). Mice showing CD3+ cell percentages 515% were taken as the T-cell-deficient (CTS) phenotype, and the remaining mice as the normal (C3H/He) phenotype (details are described below). The rates of phenotype segregation at the BC1 and F2 generations were statistically tested for matching with the Mendelian laws by the c2 test. The percentage of CD3+ cells in peripheral blood differed greatly between the parent strains, being low (510%) in CTS mice (n = 29) and high (70% to 90%) in C3H/He mice (n = 38). A high percentage (averaged 64.4%) was also obtained with F1 hybrids (n = 48), although it was distributed over a relatively wide range from 45% to 85%. Similarly, all the BC2 mice (n = 102) showed high percentages ranging from 55% to 95% (averaged 78.3%). In contrast, segregation of low and high percentages occurred in BC1 mice: 54 of 109 mice displayed low values (510%), and the remaining mice over 15% (averaged 43.3% and ranged from 15% to 85%). Segregation was also observed in F2 mice: 53 of 248 mice displayed low percentages (515%), and the remaining mice over 20% (averaged 64.5% and ranged from 20% to 90%). Thus, the incidence of T-cell deficiency showing the CD3+ cell percentage below the arbitrarily determined criterion, i.e., 15%, was 49.5% in BC1 mice and 21.4% in F2 mice. Statistical examination of these results by the c2 test revealed that the segregation rates matched well the value (50% and 25% in the BC1 and normal F2 generations, respectively) expected from the hypothesis of one-recessive gene regulation (Table 1), which also agreed well with our preliminary test results (Yagi et al. 1996). We decided to tentatively call the present locus ptcd (peripheral T-cell deficiency). Since the average percentage of the normal S. Kimura ( ) ? K. Nakamura ? M. Harada Developmental Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561, Japan