Diabetes mellitus induced by low-dose interleukin-2

 Interleukin-2 (IL-2) is a potent immunomodulator that has been associated with the clinical development of autoimmune disorders. However, diabetes mellitus has not been reported in patients treated with single-agent IL-2. We conducted a clinical trial of a protracted daily schedule of subcutaneously administered low-dose IL-2. A patient with advanced colorectal cancer, treated with 1.5×10⁶ international units of IL-2 daily, developed insulin-requiring diabetes during therapy. Hyperglycemia improved during treatment interruption and recurred with reinstitution of IL-2. The diabetes in this patient developed in the context of T cell and natural killer cell expansion, and the presence of islet cell autoantibodies was documented. We postulate that, in this patient, IL-2 reversed the anergy of autoreactive T cells that had escaped clonal deletion. It is possible that prolonged daily exposure to immunomodulatory doses of IL-2 will result in the development of autoimmune phenomena not observed with other schedules of administration. Received: 15 April 1996 / Accepted: 1 August 1996     

Somatostatin receptor subtype 1-selective activation reduces cell growth and calcitonin secretion in a human medullary thyroid carcinoma cell line

Medullary thyroid carcinoma (MTC) is a rare and aggressive tumor and so far medical therapy has provided inconclusive results. In the human MTC cell line TT, expressing all somatostatin (SST) receptor subtypes, cell proliferation decreases with SST and SST receptor subtype 2 (sst(2)), but not sst(5), selective agonist treatment, whereas calcitonin (CT) expression and secretion are reduced by SST, but not by sst(2) and sst(5) agonists. The effectiveness of two new SST analogs, BIM-23926 and BIM-23745, selectively interacting with sst(1), was investigated in the TT cell line. DNA synthesis is significantly reduced by BIM-23926 (27-40% at 10(-10)-10(-6)M) and BIM-23745 (32-90% at 10(-8)-10(-6)M). Viable cell number is also significantly reduced by both BIM-23926 (40% at 10(-12)-10(-6)M) and BIM-23745 ( approximately 40% at 10(-10)-10(-6)M). Treatment with sst(1)-selective agonists significantly reduces CT secretion and gene expression, with a reduction of CREB phosphorylation. These findings suggest that potent sst(1)-selective agonists could have a therapeutic role in MTC.     

Inhibition of interleukin-2 receptor (CD25) expression induced on T cells from children with acute lymphoblastic leukemia

 Peripheral blood lymphocytes obtained from children with acute lymphoblastic leukemia (ALL) at onset were studied for the expression of interleukin-2 (IL-2) receptor α-chain (CD25) by two-color flow-cytometric analysis. Stimulated with anti-CD3 monoclonal antibody (mAb) alone, CD25 expression was significantly suppressed in CD4⁺ T cells from 27 of 48 (56.3%) cases and in CD8⁺ T cells from 29 of 48 (60.4%) cases. When stimulated with anti-CD3 mAb plus phorbol 12-myristate 13-acetate (PMA), CD25 expression was clearly restored in certain cases of ALL. When PMA plus ionomycin were used for stimulation of T cells, CD25 was inducible in a majority of cases. Interestingly CD25 expression upon anti-CD3 mAb stimulation was recovered after complete remission had been achieved. These observations suggest the presence in ALL children at onset of an in vitro defect in the signal transduction pathway of the T-cell-receptor/CD3 complex, resulting in inefficient CD25 expression. However, immune-staining analysis indicated that protein kinase C was normally translocated from the cytosol fraction to the cell membrane fraction. The mobilization of cytoplasmic free calcium is also normal. Received: 27 March 1996 / Accepted: 23 December 1996     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Tunnelled central venous catheters yield a low incidence of septicaemia in interleukin-2-treated patients

 A retrospective study on the incidence of catheter-related complications and catheter indwelling time (t _CI) during treatment with continuous interleukin-2 (IL-2) infusion in patients with metastatic renal cell cancer, who were equipped with tunnelled central venous catheters (CVC). A group of 72 patients were treated with IL-2-based immunotherapy. Two induction treatment cycles of 35 days each were used. Treatment consisted of IL-2 as a continuous intravenous infusion (c.i.v.) with lymphokine-activated killer cells and interferon α intramuscularly. A tunnelled CVC was inserted at the start of treatment and was kept in place for the duration of the therapy or until the occurrence of complications. Out of 72 CVC, 30 (42%) functioned uneventfully for a median t _CI of 64 days. In another 12 clinically uncomplicated cases (16%), catheter tips were positive in routine culture after a median t _CI of 33 days. In 18 patients (25%), CVC-related infections were noted, including 8 (11%) local tunnel infections and 10 (14%) septic episodes. These complications occurred at a median t _CI of 28 and 20 days respectively. In 15 (83%) of these 18 catheter infections, Staphylococcus aureus was isolated, whereas in the remaining 3 (17%) Staphylococcus epidermidis was found. Subclavian vein thrombosis was noted in 12 (17%) CVC at a median t _CI of 31 days; 5 (36%) of these were diagnosed in the first 14 patients. This prompted us to administer prophylactic heparin 15 000 IU c.i.v. daily during IL-2 treatment. Thereafter the incidence of thrombosis dropped to 7 (12%) in the subsequent 58 CVC inserted (P = 0.03). In conclusion, in contrast to previous reports on the high incidence of CVC-related septicaemia and thrombosis, we observed a relatively low incidence of these complications, which we ascribe to the use of tunnelled catheters and prophylactic heparin. Received: 9. January 1997 / Accepted: 13. March 1997     

Treatment of chemically induced autochthonous rat mammary and colorectal carcinomas with interleukin-2

Summary The antineoplastic efficacy of human interleukin-2 (IL-2) in autochthonous methylnitrosourea-induced mammary carcinoma and in acetoxymethly-methylnitrosamine-induced colorectal carcinoma of Sprague Dawley rats has been investigated. Under the conditions applied, IL-2 was non-toxic. In the mammary carcinoma IL-2 was therapeutically inactive. In the colorectal carcinoma, 1200 U IL-2/day exhibited significant antitumour activity in established tumours as well as in tumours treated prophylactically before their manifestation (P <0.05). The effect of IL-2 seemed to be more pronounced when given before manifestation of colorectal tumours (T/C = 8.7% vs 17.8% in established tumours). The differential sensitivity of the autochthonous mammary and colorectal carcinoma may be explained by differences in their proliferation rates and differences in volumes at the beginning of IL-2 therapy. IL-2 seems to be preferentially active in small tumours with a low proliferation rate, a feature typical of colon tumours     

Increased proliferation of a human breast carcinoma cell line by recombinant interleukin-2

Two adenocarcinoma cell lines, Breast M25-SF and Breast M, were established from tumor tissue resected surgically from a patient with breast cancer. One, Breast M25-SF, expresses interleukin-2 receptor (IL-2R) on the cell surface and the other, Breast M, does not. The effects of recombinant inteleukin-2 (rIL-2) on the proliferation of these cell lines were investigated. The growth of Breast M25-SF was significantly promoted by rIL-2 ranging from 1,25 U/ml to 640 U/ml. Anti-CD25 (Tac) antibody, significantly blocked the growth enhancement of Breast M25-SF by rIL-2. Breast M, however, did not respond to rIL-2. To confirm more directly the promotion of Breast M25-SF growth by rIL-2, cloning of IL-2 responders from parent Breast M25-SF cells was carried out by limiting dilution without feeder cells in 96-well microplates. No colony formation was found in 24 wells without rIL-2. Eleven, 13 and 6 clones were established from groups of 24 wells containing rIL-2 at 200, 20 and 2 U/ml respectively. All of the clones expressed IL-2R and respond to rIL-2. By using a sensitive polymerase chain reaction technique, we demonstrated that Breast M25-SF but not Breast M expressed IL-2 mRNA, and IL-2 secretion from Breast M25-SF but not Breast M was also confirmed by radioimmunoassay. These findings suggest a role for IL-2 in autocrine support of Breast M25-SF growth. IL-2 may play an important role in the growth control of breast carcinoma cells.     

Combination immunotherapy with OK-432, recombinant granulocyte-colony-stimulating factor and recombinant interleukin-2 for human hepatocellular carcinoma

 The antitumor effects of immunotherapy using streptococcal preparations (OK-432), recombinant granulocyte-colony-stimulating factor (rG-CSF) and recombinant interleukin-2 (rIL-2) were examined for human hepatocellular carcinoma (HCC). Following subcutaneous injection of OK-432 (2 KE) and rG-CSF (50 – 60 μg), low-dose intratumoral administration of OK-432 (3 – 12 KE) was performed. Thereafter, 2×10⁵ JRU of rIL-2 was subcutaneously injected. This therapeutic regimen was repeated twice. Serum α-fetoprotein levels were markedly decreased in three of seven patients with HCC by this treatment. Post-therapeutic histological examination revealed that trabecular cords or pseudoglandular arrangements of tumor cells were completely disordered in all cases and that extensive infiltration of lymphocytes into the tumor stroma was present in five cases. The number of CD4- and CD57-positive cells among tumor-infiltrating lymphocytes after immunotherapy was significantly higher than that in patients without immunotherapy (P <0.01). These findings suggest that even a small intratumoral injection of OK-432 can induce extensive infiltration of helper/inducer and natural killer cells into the tumor stroma when combined with subcutaneous injection of OK-432, rG-CSF and rIL-2 and that these cells might play important roles in tumor cytotoxicity. Received: 30 December 1994 / Accepted: 6 November 1995     

Immune modulation by interleukin-12 in tumor-bearing mice receiving vitamin D3 treatments to block induction of immunosuppressive granulocyte/macrophage progenitor cells

 Lewis lung carcinoma (LLC-LN7) tumors stimulate myelopoiesis and increase the presence of granulocyte/macrophage (GM) progenitor cells having natural suppressor activity. Treatment of these tumor-bearing mice with interleukin-12 (IL-12) resulted in minimal immune modulation. The objective of this study was to determine whether eliminating natural suppressor activity would allow for immune stimulation by IL-12. Treatment of LLC-LN7 tumor-bearing mice with vitamin D₃ eliminated natural suppressor activity. In mice that were first treated with vitamin D₃ and then also with IL-12, there was stimulation of splenic T cell proliferation in response to immobilized anti-CD3 plus IL-2. In addition, spleen and lymph node cells from vitamin-D₃/IL-12-treated tumor-bearing mice became stimulated in response to autologous tumor to produce interferon γ (IFNγ), although IL-2 production was not stimulated. A prominent effect of the combined vitamin-D₃/IL-12 treatment regimen was the synergistic augmentation of autologous tumor-specific cytolytic activity within the regional lymph nodes. The generation of these tumor-specific effector cells required the presence of the tumor mass since such activity was not elicited in the lymph nodes of mice from which the tumors had been surgically excised. The results of this study show that, after treatment of tumor bearers with vitamin D₃ to eliminate GM-suppressor cells, IL-12 can induce select regional antitumor immune responses, particularly IFNγ production and cytolysis by regional lymph node cells of autologous tumor. Received: 15 December 1995 / Accepted: 22 March 1996     

Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing

 Natural killer (NK) cells may be expanded in vivo with a prolonged course of daily subcutaneous interleukin-2 (IL-2). However, cellular activation requires higher concentrations of IL-2 than are achieved with low-dose therapy. The objective of the current trial was to determine the toxicity and immunological effects of periodic subcutaneous intermediate-dose IL-2 pulses in patients receiving daily low-dose therapy. A group of 19 patients were treated with daily subcutaneous low-dose IL-2 at 1.25×10⁶ International Units (1.25 MIU) m^–2 day^–1. After 4–6 weeks, patients received escalating 3-day intermediate-dose IL-2 pulses administered as single daily subcutaneous injections, repeated at 2-week intervals. The maximum tolerated pulse dose was 15 MIU m^–2 day^–1, with transient hypotension, fatigue, and nausea/vomiting dose-limiting. Subcutaneous IL-2 resulted in in vivo expansion of CD56⁺ NK cells (796±210%) and CD56^bright natural killer (NK) cells (3247±1382%). Expanded NK cells coexpressed CD16, and showed lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity in vitro. Intermediate-dose pulsing resulted in serum IL-2 concentrations above 100 pM. Cellular activation was suggested by rapid margination of NK cells following pulsing, coincident with peak IL-2 levels, with return to baseline by 24 h. In addition, interferon γ production in response to lipopolysaccharide was augmented. Subcutaneous daily low-dose IL-2 with intermediate-dose pulsing is a well-tolerated outpatient regimen that results in in vivo expansion and potential activation of NK cells, with possible application in the treatment of malignancy and immunodeficiency. Received: 31 December 1997 / Accepted: 20 April 1998     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995     

Prognostic markers for survival in patients with metastatic renal cell carcinoma treated with interleukin-2

 Interleukin-2 (IL-2)-based immunotherapy can induce antitumor responses in about 25% of patients with metastatic renal cell carcinoma (RCC). The limited effect and the severe side-effects of IL-2 have led us to perform a prognostic factor analysis. Twenty-four patients with metastatic RCC were treated with IL-2. Flow cytometry and immunohistology were used to determine DNA ploidy, HLA-II expression on tumor cells, and the presence of macrophages in the primary tumor. These variables were examined in relation to survival. The 4-year overall survival rate was 38%. Forty-six percent of the primary tumors were aneuploid. All tumors, except one, showed HLA-II expression and macrophage presence. A statistically significant correlation (r = 0.66, P = 0.002) was found between HLA-II expression and macrophage presence. Patients with high HLA-II expression had a lower 4-year survival (22% compared to 50%), as had patients with high macrophage presence (20% compared to 42%). Of note, patients characterized by both high HLA-II and high macrophage expression had the worst survival (13% compared to 50%). We concluded that DNA ploidy was not predictive for survival, whereas HLA-II expression and macrophage presence may represent valuable prognostic factors related to survival. The present data suggest that more of the patients with no or moderate HLA-II expression and/or no or moderate macrophage presence in the primary tumor could survive with persistance of their malignant disease after having received IL-2 immunotherapy, as compared to patients with both high HLA-II and high macrophage expression. Received: 2 April 1996 / Accepted: 15 October 1996     

Restoration of interleukin-2 production in tumor-bearing rats through reducing tumor-derived transforming growth factor &;b by treatment with bleomycin

 We studied mechanisms of immunosuppression caused by tumor-derived transforming growth factor-β (TGFβ) and restoration of the immune response by treatment with bleomycin in rats bearing KDH-8 hepatoma. Interleukin-2 (IL-2) production from splenocytes of KDH-8-tumor-bearing rats progressively decreased as the KDH-8 tumor grew. IL-2 production from concanavalin-A-stimulated normal rat splenocytes was signficiantly inhibited by in vitro cultured KDH-8-tumor-cell-conditioned medium; this inhibition could be blocked by neutralizing the conditioned medium with anti-TGFβ antibody. TGFβ activities were found in KDH-8-tumor-tissue-conditioned medium without acid treatment and were found in tumor-cell-conditioned medium after acid treatment; TGFβ mRNA and TGFβ protein were found in cultured KDH-8 tumor cells. These results suggested that the KDH-8-tumor-derived TGFβ might be involved in the inhibition of IL-2 production from splenocytes. To determine whether bleomycin chemotherapy could reduce tumor-derived TGFβ and restore the immune responses, we treated KDH-8 tumor-bearing rats with bleomycin (5 mg/kg, one shot) at an appropriate time (before the occurrence of immunosuppression) resulting in a significiant reduction of TGFβ activity in KDH-8 tumor tissues and restoration of IL-2 production from splenocytes of tumor-bearing rats; KDH-8 tumor growth ultimately regressed. In vitro experiments also showed that TGFβ activity, mRNA expression, and protein synthesis in KDH-8 tumor cells were reduced by bleomycin treatment, and that bleomycin-treated-KDH-8-tumor-cell-conditioned medium did not inhibit IL-2 production from normal rat splenocytes. These results suggest that bleomycin treatment restored IL-2 production in tumor-bearing rats through reducing the tumor-derived TGFβ. Received: 12 June 1995 / Accepted: 3 November 1995     

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes

 Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans. Received: 4 April 1996 / Accepted: 31 May 1996     

A rat monoclonal anti-(human CD2) andL-leucine methyl ester impacts on human/SCID mouse graft and B lymphoproliferative syndrome

 The transfer of human peripheral blood mononuclear cells (hu-PBMC) from adult Epstein-Barr- virus(EBV)-seropositive donors in SCID (severe combined immunodeficiency) mice frequently leads to the development of a human B lymphoproliferative syndrome (hu-BLPS). Therefore, as 90% of adult potential donors are EBV-seropositive, efforts have to be made to avoid the occurrence of this B lymphoproliferative disorder. McCune et al. [Science 241:1632 (1988)] used human fetal organs for a human SCID graft. This system does not give rise to hu-BLPS but human fetal organs are much less available than peripheral blood leucocytes. The experiments reported in this paper show how crucial is the presence of functional T lymphocytes for a graft to take and for development of hu-BLPS in hu-PBMC-reconstituted SCID mice, since inhibition of T lymphocyte by a rat anti-(human CD2) monoclonal antibody (LO-CD2a) during the first 10 days of the graft prevents successful engraftment of human normal lymphocytes as well as hu-BLPS in SCID mice. The transfer of B cells alone or B cells plus monocytes in SCID mice does not permit either long-term engraftment or development of hu-BLPS. We also demonstrate that hu-PBMC treated with L-leucine methyl ester are less susceptible to the development of hu-BLPS after engraftment in SCID mice than are untreated hu-PBMC. The mechanism of action of L-leucine methyl ester on these cells is discussed. Received: 12 December 1994 / Accepted: 20 March 1995     

Adoptive immunotherapy of advanced melanoma patients with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes selected in vitro with low doses of IL-2

Freshly isolated tumor-infiltrating lymphocytes (TIL) from stage IV melanoma patients were cultured for 2 weeks with low doses of interleukin-2 (IL-2; 120 IU/ml), to select potentially for tumor-specific lymphocytes present in the neoplastic lesion, followed by high doses (6000 IU/ml) to achieve lymphocyte expansion. TIL were serially analyzed for their expansion, phenotype and cytotoxic activity against autologous and allogeneic tumor cells. A preferential lysis of autologous melanoma cells was obtained in long-term cultures of 7/13 cases (54%), while the remaining ones showed a major-histocompatibility-complex-unrestricted, lymphokine-activated-killer(LAK)-like activity at the time of in vivo injection. Sixteen patients with metastatic melanoma were infused with TIL (mean number: 6.8×10⁹, range: 0.35 × 10⁹–20 × 10⁹) and IL-2 (mean dose: 130 × 10⁶ IU, range: 28.8 × 10⁶–231 × 10⁶ IU); 1 complete and 3 partial responses were observed in 12 evaluable patients (response rate 33%). In all responding patients, injected TIL showed an in vitro preferential lysis of autologous tumor cells, while in no cases were TIL with LAK-like activity associated with a clinical response. The mean autologous tumor cytotoxic activity of TIL at the time of in vivo injection was significantly higher in responding patients in comparison to nonresponding ones, suggesting that a marked and preferential cytolysis of autologous tumor cells is associated with the therapeutic efficacy of TIL.     

MiR-129-5p is down-regulated and involved in the growth,apoptosis and migration of medullary thyroid carcinoma cells through targeting RET

Dysregulation of the REarranged during Transfection proto-oncogene (RET) pathway and microRNA (miRNAs) are crucial for the development of medullary thyroid carcinomas (MTC). Here we demonstrate that miR-129-5p is down-regulated in MTC tissues and cell lines and inhibits RET expression by directly binding its 3′ untranslated regions. Ectopic expression of miR-129-5p significantly decreases cell growth, induces apoptosis and suppresses migration ability in MTC cells through decreasing the phosphorylated AKT, thus functioning as a tumor suppressor. These findings give new clues for understanding MTC carcinogenesis and may help in developing a therapeutic approach for the treatment of RET-activated MTC.