Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine: I. Activation of natural killer cells in mice.

We reported recently that a novel immunomodulator, 7-thia-8-oxoguanosine (7T8OG)2 inhibited formation of pulmonary melanoma metastases (1), prevented against viral infection in mice (2) and potentiated the efficacy of a weakly immunogenic leukemia vaccine (3). Since certain tumor metastases and virus infected cells are targets to natural killer cells (NK cells), we now investigated whether 7T8OG is capable of activating NK cells in mice using NK cell sensitive YAC-1 and B16 and NK cell insensitive P815 targets. CBA/CaJ spleen cells incubated in vitro with 7T8OG at concentrations ranging from 0.005 to 0.5 mM responded with increased NK cell activity (32-62%) compared to controls (4-8%) to YAC-1 targets. Similar levels of augmentation in NK cell activity were observed when 40-168 mg/kg of 7T8OG was administered in vivo. In addition to the spleen, 7T8OG activated NK cells in the bone marrow (BM), the lungs, the liver, and in peritoneal exudate cells (PE). Although 7T8OG elicited activation of NK cells was observed as early as three hours after treatment, the maximal activity was observed after 24 h in the spleen; 12 h in the BM; 48 h in the lungs, and 72 h in PE. Administration of the drug by s.c., i.v., and i.p. routes all induced activation of NK cells in spleen, BM and PE. 7T8OG was found to activate NK cells in seven inbred and an outbred mouse strain, suggesting that the induced cytotoxicity against allogeneic and syngeneic tumor cells is not strain specific as well as independent of MHC restriction. C3H/He, CBA/CaJ and BDF/1 displayed higher levels of increased NK cell activity, whereas AKR mice were low responders. Low concentrations of IL-2 (0.25-5 U/ml) that induce little or no NK cell activity, when used in combination with 7T8OG, elicited significant enhancement of NK cell cytotoxicity. In contrast, IFN and 7T8OG showed no such synergism.     

Cancer immunotherapeutic potential of novel small molecule TLR7 and TLR8 agonists

Toll-like receptor (TLR)-mediated signaling is proposed as an immunotherapeutic target against tumorigenesis. Natural killer (NK) cells play a critical role in host defense against tumors. Specifically, formation of tumor metastasis in various organs can be suppressed by the local activity of NK cells. In this study, we present a novel TLR7 agonist (termed SC-1) that induces pro-inflammatory cytokines in human blood cells, activates NK cell function, and is highly efficient in preventing lung metastases in a pulmonary metastatic Renca model. Furthermore, a second compound (termed SC-2), acting as dual-specific TLR7 and TLR8 agonist, was evaluated with respect to its immunostimulatory and NK cell–activating capacities. The release of pro-inflammatory cytokines was shown to be even more pronounced with this compound. Additional experiments showed a significant up-regulation of activation marker CD69 on NK cells and increased cytolytic activity of peripheral blood cells compared to the effect of a monospecific TLR7 agonist SC-1. Normally, TLR7 and TLR8 are expressed on different immune cell subpopulations. TLR7 expression on antigen-presenting cells is detected in plasmacytoid dendritic cells, CD34⁺-derived dendritic cells, and B-cells, whereas TLR8 is mainly expressed on cells of the myeloid lineage, such as monocytes, macrophages, and myeloid dendritic cells. Therefore, a compound that activates both TLR7 and TLR8 would result in a highly efficient immune system activation and may give rise to an enhanced anti-tumor activity in vivo compared to that elicited by a monospecific TLR7 agonist.     

Immunotherapy of mammary adenocarcinoma metastases in C3H/HeN mice with chronic administration of cyclo-oxygenase inhibitors alone or in combination with IL-2

In this study the efficacy of treatment of two cyclo-oxygenase inhibitors, ibuprofen (Ibu) and indomethacin (Indo), are compared in the immunotherapy of metastasis designed to reverse prostaglandin E₂ (PGE₂)mediated inactivation of interleukin-2 (IL-2)-dependent host killer cell lineages. These agents were tested either alone for the prevention of metastasis or in combination with IL-2 for the eradication of established metastasis. C3H/HeN mice were placed on chronic oral Ibu (CIbT; 200 and 600 ,g/ml of water) or Indo (CIT; 10 g/ml) 5 days after s.c. transplantation of 5 × 10⁵ metastatic C3L5 mammary carcinoma for the prevention of spontaneous lung metastases. They showed intolerance to Indo at a dosage of 14 g/ml, which was well tolerated by other mouse strains in previous studies, but tolerated the Ibu dosages used. Control and treated mice were killed on day 30 to score metastatic lung colonies, to evaluate killer activity in splenocytes against natural killer (NK)-sensitive YAC-1 lymphoma or NK-resistant C3L5 adenocarcinoma and 8911 lymphoma targets, and to phenotype the surface markers of killer cells. CIbT and CIT alone at the above dosage significantly reduced the number of lung colonies, retarded local tumor growth and restored NK activity of splenic killer cells expressing AGM-1⁺, Thy-1^–, Lyt-2^– phenotype. To treat established lung metastasis, mice bearing 15-day C3L5 transplants were given CIbT or CIT alone or in combination with two 4-day rounds (days 20–23, 31–34) of IL-2 (15 000 Cetus units, i.p. every 8 h) and were killed on day 35 to score lung colonies and characterize splenic killer cells. CIbT or CIT alone reduced the number of spontaneous lung metastases and restored anti-YAC-1 killer function of splenocytes with NK-like phenotype (AGM-1⁺, Thy-1^–, Lyt-2^–); some anti-C3L5 killer function was also generated in the high dose Ibu group and the killer cell showed AGM-1⁺, Thy-1⁺ and Lyt-2⁺ phenotype. Combined therapies with CIbT or CIT plus IL-2 were more effective in reducing metastases and promoting killer cell function, the best results being achieved with high dose Ibu + IL-2. All killer cells expressed AGM-1 and Thy-1. In addition, C3L5 killer cells also expressed Lyt-2, suggesting T-cell stimulation. PGE₂ synthesis in the host was inhibited by at least 50% in mice subjected to CIbT or CIT. Thus, Ibu proved to be an excellent substitute for Indo in preventing metastasis and NK cell activation when given alone, and also in ameliorating established metastasis and activating lymphokine-activated killer cells when combined with IL-2.     

The establishment of two cell lines from a mouse uterine cervical carcinoma (U14) and their metastatic phenotype changes

This paper studies the heterogeneity of metastatic potential of murine cervical carcinoma (U₁₄). Two cell lines, P_11–90 and L_10–90, were established from a pulmonary metastatic substrain (U₁₄AP₁₁) and a lymphatic metastatic substrain (U₁₄AL₁₀), which were selected from U₁₄ in vivo after 11 and 10 passages, respectively. The biologic differences between the two cell lines are as follows. (1) The cells of the P_11–90 line grow more rapidly compared with the L_10–90 line. From the 40th passage the medium pH was different. (2) The median number of chromosomes in P_11–90 and L_10–90 was 72 and 64, respectively; the rates of gap aberration were 88% and 78%, respectively. (3) The number of T lymphocytes and T helper lymphocytes in the peripheral blood from hosts with P_11–90 were higher than that of hosts transplanted with L_10–90, but the number of B lymphocytes in the latter was larger than that in the former. (4) The metastatic potential of each cell line partially decreased compared to the relative tumor substrain, but their organ preference still remained and the transplant locations, axillary or footpad, had a prominent influence on their metastatic behavior. To observe the effects of metastatic target organs on the metastatic phenotypes of tumor cells, as well as to explore a method for the establishment and maintenance of the metastatic organ preference of tumor cells, conditioned medium (CM) from pulmonary or lymphatic node diploid cells was added to the culture medium of P_11–90 and L_10–90. Two sublines, P + P_11–90 and Ln + Lon10–90, were thus established. Using stereological methods we found that the majority of P + P_11–90 cells became larger and their nuclei also increased in size compared with their parental lines, but the majority of Ln + L_10–90 cells became smaller in size, though the nuclei were enlarged. The pulmonary metastatic rate and lymphatic metastatic rate of P + P_11–90, as well as the lymphatic metastatic rate of Ln + L_10–90,, were restored dramatically. The results suggest that by taking advantage of the interaction between tumor cells and the CM of host cells the metastatic potential of tumor cell lines can be maintainedin vitro. Our work may offer an experimental model for the manipulation of metastasis of cell lines coming from the same parent strain but with different metastatic potentials. Our study demonstrates that although the specific metastatic tendencies of the cell lines decrease after multiple passages, these propensities are restored after passages with CM from normal lung and lymph node tissue.     

Synergy between an antibody and CD8+ cells in eliminating an established tumor

We investigated the effector mechanisms operating during the rejection of a transplantable solid lymphoma E.G7 (H-2^b) which expresses the gene encoding chicken ovalbumin (OVA). Anti-OVA cytotoxic T lymphocytes (CTL) completely and specifically protected animals from the onset of, but could not eradicate established, E.G7 tumors. The growth of the same lymphoma was also effectively prevented by the antibody GK1.5, whose target molecule, CD4, was expressed on E.G7 cells in vivo. Furthermore, GK1.5 was able to eradicate established solid E.G7 tumors. GK1.5-mediated tumor elimination was due to its antitumor activity, and not to the elimination of regulatory CD4⁺ cells, based on unimpaired tumor growth in the absence of GK1.5 in animals that genetically lack CD4 T cells. In vitro, GK1.5 did not kill tumor cells: complement activation or apoptosis induction were not evident. In vivo, GK1.5-mediated tumor regression did not depend on natural killer cells, but it absolutely required CD8⁺ cells and intact Fcγ receptor. We conclude that, in the E.G7 model, the collaboration of antibody and CTL immunity was crucial for the successful immunotherapy of established tumors. The mechanism of this collaboration is discussed.     

Immunomodulatory activity of a novel nucleoside, 7-thia-8-oxoguanosine. II. Characterization of induced effector cells and the mechanism of induction.

In a preceding paper we characterized the in vivo and in vitro induction of cytotoxic effector cells elicited by a novel synthetic immuno-stimulator 7-thia-8-oxoguanosine (7T8OG)2. In the present study we further characterized the cells responsible for the induced cytotoxicity and the mechanisms together with the lymphokines mediating the immunological response to 7T8OG. Removal of macrophages from 7T8OG activated spleen cell suspensions by various methods resulted in a significant increase in cytotoxicity to YAC-1 targets. 7T8OG induced effectors did not exert cytotoxic effect on macrophage sensitive P815 target cells. In vivo activated effectors when incubated with anti-asialo-GM1 antibody plus complement lost completely their ability to lyse YAC-1 targets. Together, these findings indicate that the 7T8OG induced effector cells are not macrophage like. Spleen cells from nude mice were readily activated by 7T8OG. The induced effectors were resistant to complement mediated lysis using anti-L3T4, anti-Lyt1 or anti-Lyt2 antibodies. Pretreatment of spleen cells with macrophage depleting agents both, in vitro and in vivo and subsequent activation of cells by 7T8OG resulted in effectors with reduced cytotoxicity. When injected in vivo, 7T8OG induced strong IFN production which paralelled the kinetics of NK cell activation. Furthermore, antibodies to alpha & beta-IFN but not to gamma-IFN diminished the induction of the cytotoxic activity. Although these findings suggest that activation of NK cells by 7T8OG is most likely to be mediated by alpha & beta-IFN involvement of other cytokines can not be ruled out.     

Phenotypical and functional heterogeneity of the large granular lymphocytes increased after various treatments in a patient with combined immunodeficiency

A boy with combined immunodeficiency having low natural killer (NK)-cell activity received thymopoietin pentapeptide (TP-5) treatment, transplanted with T cell-depleted HLA-haploidentical bone marrow (BMT) cells from his father and with thymus tissue from an infant at different times during the first year of life. He showed a marked increase in large granular lymphocytes (LGL) both during the treatment with TP-5 and after BMT. The LGL generated following TP-5 injection had a T3⁺ Leu 11^– surface phenotype and low NK activity. In contrast, the LGL appearing after BMT showed T3^–, Leu7⁺, and/or Leu11⁺ surface phenotypes, had high NK- and K-cell activities, and were lymphokine-activated killer (LAK)-cell precursors. These killer activities were assigned to the Leu7^– Leu11⁺ subset and proved to be of recipient origin. LGL proliferation following BMT was accompanied by neutropenia, which was improved in association with a reduction in the number of LGL and the appearance of T cells of BMT donor origin following thymus transplantation. This suggested the inhibition of granulopoiesis by the LGL and anin vitro study revealed that the Leu7⁺ Leu11^– subset of LGL suppressed the growth of granulocyte/macrophage colony-forming units. These results indicated that phenotypically different LGL could be generated by different treatments and that the LGL showing NK activity were distinct from those regulating granulopoiesis. It was also suggested that the generation of LGL was controlled by T cells.     

Multiple phenotypic divergence of mammary adenocarcinoma cell clones.

We have shown that, with in vitro passage, subclones derived from clonal cell populations of 13762NF mammary adenocarcinoma undergo phenotypic drift and diversification in their cellular properties. Here we examine whether phenotypic divergence of 13762NF cell clones extends to therapeutic treatments used in eliminating mammary tumors and whether the apparent rates of phenotypic divergence vary for different treatments. Six subclones isolated from low passage clone MTF7 (T11 l; tissue culture passage 11) cells were compared to a similar number of subclones isolated from high passage clone MTF7 (T35; tissue culture passage 35) cells. Subclones derived from clone MTF7 (TI l) were relatively homogeneous (not significantly different) in their inherent sensitivities to ionizing radiation, extrapolation coefficients and quasithreshold dose values (D_o = 1·61–1·99 Gy; n=0·89–3·42; D_q = 0–2·34). When the MTF7 (Tll) subclones were examined for their sensitivities to 45°C hyperthermic treatment, the inherent sensitivities and dose-response curve parameters (D_o = 5·24–10·05 min; n = 1·08–10·47; D_q = 0·78–12·31) were heterogeneous (significantly different). In addition, the MTF7 (T1 l) subclones were heterogeneous (significantly different) in their sensitivities and dose-response curve parameters to 5-fluoro-2-deoxyuridine (FUdR) treatment (slope= –0·70 to –1·59; y-intercept = 1·31 × 10² to 47·80 × 10²). The LD₅₀ values for FUdR ranged from 14–150 nm for the MTF7 (T11) subclones. At high passage MTF7 (T35) subclones were heterogeneous in their dose-response parameters to ionizing radiation (D_o = 1·17–2·05 Gy; n = 0·80–41·18; D_q = 1·79-\24·94), hyperthermia (D_o = 3·57–6·32 min; n = 2·08–13·54; D_q = 3·68–9·30) and FUdR (slope= –0·77 to –0·93; y-intercept = 4·64 × 10² to 8·83 × 10²; LD₅₀ = 50–160nm). The results indicate that clonal cells diverge for distinct phenotypic properties at differing rates to form heterogeneous cell populations with unique sensitivities to various therapeutic treatments.     

Effect of various doses of infectiveToxocara canis andToxocara cati eggs on the humoral response and distribution of larvae in mice

The effect of 5–2,500 infectiveToxocara canis and 5–1,000T. cati eggs on the humoral immune response and on the distribution of larvae in the organism was studied in paratenic hosts — inbred C57BL6/J mice. With each dose ofT. canis eggs the maximal antibody level was recorded on day 56 post infection and was followed by a moderate decline that lasted until day 154 of the experiment. A correlation between the antibody level and the egg count was observed only with the infective dose of 5–50 eggs. A more rapid occurrence of antibodies was recorded in mice infected with a high dose of eggs. In those given 5 and 7T. cati eggs the antibody level exceeded the extinction threshold value only from day 21 to day 84. Low doses ofT. canis (n=5) andT. cati (n=7) eggs caused a comparable distribution of larvae in mice, and the larval recoveries on day 70 post infection ranged between 10.00% and 25.74%. Following a dose of 500T. cati eggs, 22.28% of the larvae were recovered, although only 1.08% were localized in the brain. A dose of 1,000T. canis eggs yielded, 36.37% of the larvae, with as much as 28.13% being found in the brain.     

The relative contribution of IL-4 and IL-13 to human IgE synthesis induced by activated CD4 or CD8 T cells

The relative contribution of IL-4 and IL-13 to the regulation of IgE synthesis has remained relatively poorly characterized, partially because of lack of suitable animal models. We have studied the roles of IL-4 and IL-13 in human IgE synthesis induced by supernatants derived from activated CD4⁺⁺ or CD8⁺ T cell clones. Neutralizing anti–IL-4 and anti–IL-13 monoclonal antibodies (mAbs) inhibited IgE synthesis induced by anti-CD40 mAbs and supernatants from CD4⁺ T cells by an average 61% and 42%, respectively (n = 25). Recombinant IL-13 had additive effects on IL-4-induced IgE synthesis, but only when IL-4 was present at low concentrations. Accordingly, IL-4 was the dominant IgE synthesis–inducing cytokine derived from highly polarized T helper (TH)₂ cells. However, anti–IL-13 mAbs also significantly inhibited IgE synthesis induced by two of three supernatants derived from allergen-specific T_H2-like cell lines generated from the skin of patients with atopic dermatitis. Furthermore, anti–IL-13 mAbs almost completely inhibited IgE synthesis induced by supernatants from T_H1 cells or CD8⁺ T cell clones. Taken together, these data indicate that IL-13, in addition to IL-4, contributes to IgE synthesis induced by all T helper cell subsets, including allergen-specific T_H2 cells. Moreover, IL-13 appears to be the major IgE synthesis–inducing cytokine derived from T_H1 cells or CD8⁺ T cells. (J Allergy Clin Immunol 1997;100:792-801.)     

The effectiveness of cis-platinum,cyclophosphamide and melphalan in treating disseminated tumor cells in mice

Both B16 melanoma and Lewis lung carcinoma growing in C57/B1 mice spontaneously metastasize to the lungs and other organs. When the tumors are grown in the mouse tail to a specific volume and amputated, the spontaneously disseminated tumor cells can then be independently treated. The effects of a single dose of cyclophosphamide (200 mg/kg), cis-platinum (6 mg/kg) and melphalan (10 mg/kg) on the appearance of pulmonary and other metastases were measured. The cis-platinum treatment was shown to reduce the number and incidence of metastases of both tumors at various times after treatment. The antimetastatic effectiveness of cis-platinum against these two tumors was increased when 2·4 mg/kg was administered each day for five consecutive days after amputation of the primary. Cyclophosphamide, when administered at two-thirds maximum tolerated dose, had a small promoting effect on the number and incidence of pulmonary metastases of Lewis lung carcinoma, whereas, applied in the same dose, it had efficacy in the treatment of disseminated B 16 melanoma and inhibited appearance of both pulmonary and lymph-node metastases. When melphalan was administered in single- and multiple-dose regimens, the number and incidence of metastases of both tumors increased at various times after primary tumor amputation. These data suggest that melphalan can promote the growth of disseminated tumor cells in both the lungs and other sites and that some systemic chemotherapies may result in promotion instead of suppresssion of metastatic disease     

Effect of Gadolinium Chloride on the Growth and Metastasizing of Lewis Pulmonary Adenocarcinoma in Mice

A single intraperitoneal injection of GdCl₃ in doses of 14 or 28 mg/kg to mice with intravenously transplanted Lewis pulmonary adenocarcinoma on days 3 or 8 after tumor transplantation reduced the mean number of tumor metastases in the lungs. The effect of GdCl₃ was more pronounced if it was injected at the stage of tumor dissemination (day 8). The positive antimetastatic effect of GdCl₃ was presumably due to its capacity to macrophage depression. The direct (not mediated through mononuclear phagocyte system cells) effect of GdCl₃ on tumor cells cannot also be excluded. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 11, pp. 548–551, November, 2008     

Chemokine receptor CXCR4 expression in breast cancer as a potential predictive marker of isolated tumor cells in bone marrow

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1α are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin–biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK⁺ cells was 236 (range, 20–847) per 5 × 10⁴ enriched BM cells. The presence of CK⁺ cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK⁺ per 5 × 10⁴ enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.     

Cytotoxic activity of lymphocytes isolated from mouse liver involved into tumor process

We studied cytotoxic activity and immunophenotype of mononuclear cells isolated from the liver of mice after implantation of ovarian cancer cells into the liver parenchyma. The isolated cells exhibited higher natural killer cell activity and possessed higher cytotoxic potential against autologous tumor cells compared to spleen lymphocytes. The ratio of CD3⁺ lymphocytes and natural killer cells was high in the liver with tumor metastases. Lymphoid cells were practically absent in the liver of intact animals. Natural killer cells and T cells from liver tumor tissue play an important role in antitumor immunity and can be used for local and regional adjuvant immunotherapy of liver metastases. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 1, pp. 76–79, January, 2006     

Immunotherapy of the rat 13762SC mammary adenocarcinoma by vaccinia virus augmentation of tumor immunity

We studied whether vaccinia virus (VV) functioned as an immunogenic carrier in augmenting anti-tumor immunity in rats bearing a syngeneic metastatic tumor. The primary tumor was induced by injecting 10⁶ 13762SC mammary adenocarcinoma cells subcutaneously into the right hind footpad of Fischer 344 rats. A concomitant anti-tumor response is induced by the tumor as demonstrated by the inhibited growth of a second tumor challenge given in the contralateral footpad 3–15 days later. Attempts were made to increase the concomitant immunity by injecting tumor-bearing animals intramuscularly with irradiated, VV-infected or uninfected 13762SC cells without adjuvant. Provided the immunotherapy was done within 5 days of the tumor challenge, administration of 10⁶–10⁷ irradiated, VV-infected 13762SC cells resulted in significantly slower tumor growth, or led to complete tumor regression, compared to control animals given no treatment. In contrast, tumor growth in animals given only VV or given irradiated, uninfected 13762SC cells, alone or mixed with VV, was the same as that in control animals. Kinetics of early primary tumor growth were predictive of a longer-term anti-tumor effect. Rechallenge of 13762SC tumor-cured animals with either the homologous or with a heterologous syngeneic mammary adenocarcinoma showed the animals to be specifically 13762SC tumor-resistant, since only rats challenged with the heterologous mammary adenocarcinoma developed progressive tumors. We interpret these results to mean that early immunotherapy with irradiated, VV-infected 13762SC cells enhances an on-going anti-tumor immune response sufficiently to cause rejection of the primary tumor and any metastases that have occurred. We also believe that later immunotherapy with irradiated, VV-infected cells has no effect due to tumor-induced immunosuppression becoming paramount.To whom correspondence should be addressed.     

Association of αvβ3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

Introduction. Expression of adhesion molecules such as α _v β ₃ integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether α _v β ₃ expression correlated with the metastatic potential of human osteosarcoma cells. Materials and methods. We developed a series of sublines (LM2–LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6–8 weeks. We quantified α _v β ₃ integrin expression using flow cytometry. Results. α _v β ₃ expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of α _v β ₃. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that α _v β ₃ was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. Conclusions. α _v β ₃ integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. α _v β ₃ integrin may therefore be a potential new target for osteosarcoma.This revised version was published online in August 2005 with a corrected cover date.     

Workshop R: Tumor Immunology, Abstract R1 bis R30

R. 1 Serum anti-p53 Ig-autoantibodies in patients with advanced gastrointestinal cancerR. 2 T cells expressing a chimeric anti-CD20/CD3ζR. 3 Characterization of tumor infiltrating immune cells in pancreatic carcinomaR. 4 Adoptive tumor therapy with T lymphocytes enriched by an IFN-γ capture assayR. 5 Tumor growth despite induction of potent tumor specific CD8 T cellsR. 6 Eradication of adenocarcinoma with CpG-DNA-activated CD4⁺ T cells from non-transgenic mice is strictly dependent on a Th1 phenotypeR. 7 Naturally occurring T cell response against mutated p21 ras protein in pancreatic carcinomaR. 8 A novel immune escape mechanism of renal cell carcinoma attributable to abnormal HLA-G expressionR. 9 Functional and structural analysis of tumor-specific T cellsR. 10 Identification of renal cell carcinoma associated genes and antigensR. 11 Combination of T cell therapy and trigger of inflammation induces remodeling of the vasculature and tumor eradicationR. 12 Immunotherapy directed against α-fetoprotein results in autoimmune liver disease during liver regenerationR. 13 Inhibition of metalloproteinases enhances the internalization of anti-CD30 antibody Ki-3 and the cytotoxic activity of Ki-3 immunotoxinR. 14 Upregulation of the chemokine receptor CCR7 in classical but not in lymphocyte predominant Hodgkin's disease correlates with distinct dissemination of neoplastic cells in lymphoid organsR. 15 Expression and recognition of HLA-G in a β₂-microglobulin-negative tumor cell lineR. 16 Unexpected induction of unresponsiveness by vaccination with transformed Salmonella typhimuriumR. 17 Chemokine receptor expression on neoplastic and non-neoplastic T cells in mycosis fungoidesR. 18 Identification of prostate stem cell antigen-derived peptides as specific targets for CD8⁺ cytotoxic T cells in prostate cancer patientsR. 19 Heterogeneous expression of MAGE-A genes in occult disseminated tumor cells: A novel multimarker RT-PCR for diagnosis of micrometastatic diseaseR. 20 Tumor MHC downregulation can lead to T cell-dependent immunityR. 21 Ras-dependent inducible and constitutive expression of T cell death associated gene 51R. 22 P-glycoprotein positive multi-drug resistant ovarian carcinoma cells exert increased complement resistanceR. 23 EBNA1-specific CD4⁺ T cells recognize EBV associated malignancies of latency I and IIIR. 24 Apoptotic tumor cells induce a protective tumor immune response in vivoR. 25 Migration inhibitory factor participates in the tumorigenesis of malignant human gliomasR. 26 Analysis of interleukin-4-mediated cellular interactions during the generation of anti-tumor cytotoxic T lymphocytesR. 27 Complementary methods for the identification of tumor-specific T cell epitopesR. 28 Immunotherapy of experimental tumors by glycodendronsR. 29 Protective effects of gene therapy with interleukin-12p40 in a murine tumor modelR. 30 Targeting of human gamma-glutamyltransferase with mAb 138H11 in a new renal cell carcinoma mouse model     

Disaggregation and invasion of ovarian carcinoma ascites spheroids

Background

In vivo studies have recently demonstrated that interleukin 21 (IL-21) enhances the anti-tumor function of T-cells and NK cells in murine tumor models, and the combined use of IL-21 and IL-15 has resulted in prolonged tumor regression and survival in mice with previously established tumors. However, the combined anti-tumor effects of IL-21 and low dose IL-2 have not been studied even though IL-2 has been approved for human use, and, at low dose administration, stimulates the proliferation of memory T cells, and does not significantly increase antigen-induced apoptosis or regulatory T cell (Treg) expansion. This study examined whether recombinant IL-21 alone or in combination with low-dose IL-2 could improve the in vivo anti-tumor function of naïve, tumor-antigen specific CD8⁺ T cells in a gp100_25–33 T cell receptor transgenic pmel murine melanoma model.

Methods

Congenic C57BL/6 (Ly5.2) mice bearing subcutaneous B16F10 melanoma tumors were sublethally irradiated to induce lymphopenia. After irradiation naive pmel splenocytes were adoptively transferred, and mice were immunized with bone marrow-derived dendritic cells pulsed with human gp100_25–33 (hgp100_25–33). Seven days after vaccination groups of mice received 5 consecutive days of intraperitoneal administration of IL-2 alone (20 × 10³ IU), IL-21 alone (20 μg) or IL-21 and IL-2. Control animals received no cytokine therapy.

Results

IL-21 alone and IL-2 alone both delayed tumor progression, but only IL-21 significantly augmented long-term survival (20%) compared to the control group. However, combination therapy with IL-21 and IL-2 resulted in the highest long-term (>150 days) tumor-free survival frequency of 46%. Animals that were tumor-free for > 150 days demonstrated tumor-specific protection after rechallenge with B16F10 melanoma cells. At peak expansion (21 days post vaccination), the combination of IL-21 plus IL-2 resulted in a 2- to 3-fold higher absolute number of circulating tumor antigen-specific pmel CD8⁺ T cells than was stimulated by IL-2 or IL-21 alone. Pmel CD8⁺ T cells were predominantly partitioned into central memory (CD62L⁺/CD127⁺) or effector-memory (CD62L^-/CD127⁺) phenotypes by day 28-post vaccination in IL-21 + IL-2 treated mice.

Conclusion

These observations support the potential use of IL-21 and low-dose IL-2 therapy in combination with a tumor-antigen vaccine and lymphopenic conditioning in future cancer clinical trials to maintain high numbers of anti-tumor memory CD8⁺ T cells with the potential to sustain long term tumor regression and survival.     

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5–30 ng/ml per 10⁵ cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8⁺, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell pro liferatiowhich was not only due to IL-2 but was depen dent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.Abbreviations IL Interleukin - LAK Lymphokine-activated killer - PBMC Peripheral blood mononuclear cells     

Efferent enhancement of a methylcholanthrene-induced Sarcoma transplantable in strain 13 guinea pigs

The immunobiology of an antigenic methylcholanthrene-induced sarcoma (MC-D) of inbred strain 13 guinea pigs has been investigated. The induction of concomitant immunity by growing MC-D tumors was indicated by the suppression of small tumor inocula in the presence of a large tumor cell dose and by the regression of intradermal tumor nodules. Furthermore, it was demonstrated that this tumor was coated with antibody in vivo. Previous studies showed that MC-D tumors were infiltrated with killer T cells which were capable of complete tumor destruction in vitro, but could never induce spontaneous regression in vivo. On the basis of all these facts, antibody-mediated efferent enhancement is proposed to be the major escape mechanism of this tumor.