IL-2 cDNA Research Articles

AUTOCRINE IL-6 PRODUCTION BY HUMAN TRANSITIONAL CARCINOMA CELLS UPREGULATES EXPRESSION OF THE α5β1 FIBRONECTIN RECEPTOR

Purpose: Studies have demonstrated elevated expression and secretion of IL-6 by transitional cell carcinomas (TCC) following bacillus Calmette-Guerin (BCG) therapy. At present, the role of IL-6 on the biology of TCC is poorly understood. This study evaluated the influence of IL-6 expression on a critical variable regulating BCG-tumor interaction, the tumor expression of α5β1 integrin. Materials and Methods: A human TCC cell line (253J) was transfected with an expression vector containing the full-length IL-6 cDNA sequence. Overexpression of IL-6 mRNA and protein was confirmed by Northern analysis and ELISA, respectively. Clones found to overexpress IL-6 were then assayed for α5β1 expression using Northern analysis and flow cytometry. The effect of alterations in α5β1 expression on tumor adherence to fibronectin (FN), and BCG adherence to tumor cells was determined using specific adherence assays. Results: mRNAs for both the α5 and β1 subunits of the FN receptor were increased an average of 9.4 fold and 125.7 fold respectively in the IL-6 overexpressing transfectants relative to the parental 253J cells. Increased mRNA of α5 and β1 was associated with increased cell surface expression of both proteins. Increased protein expression resulted in greater FN substrate binding affinity and increased adherence of BCG to tumor cells. Conclusions: Autocrine expression of IL-6 upregulates the expression of FN receptor subunits in TCC. Increased α5β1 expression increases cellular adherence to FN, and BCG adherence to tumor cells. These results suggest a role for IL-6 in mediating the antitumor activity of BCG by influencing BCG's adherence to TCC.

AUTOCRINE IL-6 PRODUCTION BY HUMAN TRANSITIONAL CARCINOMA CELLS UPREGULATES EXPRESSION OF THE ??5??1 FIBRONECTIN RECEPTOR

Studies have demonstrated elevated expression and secretion of IL-6 by transitional cell carcinomas (TCC) following bacillus Calmette-Guerin (BCG) therapy. At present, the role of IL-6 on the biology of TCC is poorly understood. This study evaluated the influence of IL-6 expression on a critical variable regulating BCG-tumor interaction, the tumor expression of alpha5beta1 integrin. A human TCC cell line (253J) was transfected with an expression vector containing the full-length IL-6 cDNA sequence. Overexpression of IL-6 mRNA and protein was confirmed by Northern analysis and ELISA, respectively. Clones found to overexpress IL-6 were then assayed for alpha5beta1 expression using Northern analysis and flow cytometry. The effect of alterations in alpha5beta1 expression on tumor adherence to fibronectin (FN), and BCG adherence to tumor cells was determined using specific adherence assays. mRNAs for both the alpha5 and beta1 subunits of the FN receptor were increased an average of 9.4 fold and 125.7 fold respectively in the IL-6 overexpressing transfectants relative to the parental 253J cells. Increased mRNA of alpha5 and beta1 was associated with increased cell surface expression of both proteins. Increased protein expression resulted in greater FN substrate binding affinity and increased adherence of BCG to tumor cells. Autocrine expression of IL-6 upregulates the expression of FN receptor subunits in TCC. Increased alpha5beta1 expression increases cellular adherence to FN, and BCG adherence to tumor cells. These results suggest a role for IL-6 in mediating the antitumor activity of BCG by influencing BCG's adherence to TCC.

Coimmunization with IFN-gamma or IL-2, but not IL-13 or IL-4 cDNA can enhance Th1-type DNA vaccine-induced immune responses in vivo.

As we explore the potential improvements to the current DNA vaccine strategies, it may be desirable to investigate methods to improve the level of resulting immune responses. One strategy is the use of cytokine cDNA as molecular adjuvants for DNA-based vaccines. Codelivery of these molecular adjuvants consisting of expression plasmid encoding for cytokines with DNA vaccine constructs is an effective method to modulate the magnitude and direction (humoral or cellular) of the immune responses. We have previously reported on the immunomodulatory effects of codelivering cDNA for interleukin-2 (IL-2) and IL-4 as molecular adjuvants for DNA-based vaccines. In this report, we extend these finding and compare the immunomodulatory effects of IL-2 and IL-4 with those of cDNA for prototypical Thl-type cytokine interferon-y (IFN-gamma) and Th2-type cytokine IL-13. We observed that distinct antigen-specific immune modulation can be achieved by the coinjection of IFN-gamma or IL-13 genes with DNA immunogen cassettes. We observed that IFN-gamma is a strong driver of Thl immune responses. Furthermore, in contrast to previous reports on their similarities in biologic activities, IL-13 and IL-4 cDNA coimmunizations modulated vaccine-induced immune responses differently in this model. Overall, these results further support the potential utility of this strategy as an important tool for the development of vaccines and immune therapies.

Impaired tumorigenicity of human pancreatic cancer cells retrovirally transduced with interleukin-12 or interleukin-15 gene.

We examined the antitumor effect of locally secreted interleukin (IL)-12 or IL-15 on human pancreatic cancer cells (AsPC-1). We subcutaneously inoculated AsPC-1 cells retrovirally transduced with IL-12 or IL-15 cDNA into nude mice. Tumors derived from these cells showed retarded growth compared with those from wild-type (wt) cells. Nude mice inoculated intraperitoneally with the cytokine producers survived longer than those injected with wt cells. These cytokine producers were also tested for their tumor growth in severe combined immunodeficient mice. The tumor growth of IL-12 producers was similarly suppressed as found in nude mice, but the average tumor volumes of IL-15 producers were not statistically different from those of wt tumors. In nude mice that were administered anti-asialo GM1 antibody before the inoculation of the tumor cells, growth retardation of tumors of IL-12 producers remained the same as in untreated animals, but that of IL-15 producers was markedly reduced. Immunohistochemical analysis revealed that CD11b+ cells migrated into the tumors of cytokine producers and that the number of CD31+ endothelial cells within the tumors was not different between IL-12 producers and wt cells. Taken together with other data, it is possible that granulocytes are candidate cells for the IL-12-mediated antitumor effect, and that natural killer cells and gammadelta T cells are involved in the IL-15-induced antitumor effect. We did not observe synergistic effects of these cytokines to suppress subcutaneous tumors.

Differential roles of interleukin 15 mRNA isoforms generated by alternative splicing in immune responses in vivo.

At least two types of interleukin (IL)-15 mRNA isoforms are generated by alternative splicing at the 5' upstream of exon 5 in mice. To elucidate the potential roles of IL-15 isoforms in immune responses in vivo, we constructed two groups of transgenic mice using originally described IL-15 cDNA with a normal exon 5 (normal IL-15 transgenic [Tg] mice) and IL-15 cDNA with an alternative exon 5 (alternative IL-15 Tg mice) under the control of an MHC class I promoter. Normal IL-15 Tg mice constitutionally produced a significant level of IL-15 protein and had markedly increased numbers of memory type (CD44(high) Ly6C(+)) of CD8(+) T cells in the LN. These mice showed resistance to Salmonella infection accompanied by the enhanced interferon (IFN)-gamma production, but depletion of CD8(+) T cells exaggerated the bacterial growth, suggesting that the IL-15-dependent CD8(+) T cells with a memory phenotype may serve to protect against Salmonella infection in normal IL-15 Tg mice. On the other hand, a large amount of intracellular IL-15 protein was detected but hardly secreted extracellularly in alternative IL-15 Tg mice. Although most of the T cells developed normally in the alternative IL-15 Tg mice, they showed impaired IFN-gamma production upon TCR engagement. The alternative IL-15 transgenic mice were susceptible to Salmonella accompanied by impaired production of endogenous IL-15 and IFN-gamma. Thus, two groups of IL-15 Tg mice may provide information concerning the different roles of IL-15 isoforms in the immune system in vivo.

Presence of Interleukin 4 or Interleukin 10, but Not Both Cytokines, in Pancreatic Tissue of Two Patients with Recently Diagnosed Diabetes Mellitus Type I

Studies in the NOD mouse model suggest that development of diabetes mellitus type I can be prevented and established disease cured by deviation towards a Th2-type response. To obtain insight into whether this approach may be applicable to human disease, we investigated the Thl/Th2 cytokine balance in pancreatic tissue from two patients with diabetes of recent onset (Case 1, accidental death; Case 2, ketoacidosis). Using the polymerase chain reaction to amplify reverse-transcribed cDNA, signals for actin and CD35 confirmed mRNA integrity and the presence of T cells in pancreatic tissue from both patients and from a control. IFN-y cDNA was also amplified from all three tissues. However, IL-4 (but not IL-10) cDNA, was amplified from the pancreas of Case I. Conversely, IL-10 (but not IL-4) cDNA was amplified from the the pancreas of Case 2. The control pancreas yielded specific signals for both IL-4 and IL-10. Our data extend the limited database on Thl and Th2 cytokine expression in human pancreatic tissue from recently diagnosed diabetics. Moreover, together with previous observations, our findings raise the possibility that the lack of both IL-4 and IL-10 may be associated with the development of IDDM in humans

Gene transfer of a secretable form of IL-15 in murine adenocarcinoma cells: Effects on tumorigenicity, metastatic potential and immune response

IL-15 is an immunostimulatory cytokine with IL-2-like activities. To exploit the potential role of IL-15 in cancer immuno-/gene therapy, we engineered murine TS/A cells with different IL-15 cDNA constructs. Significant IL-15 secretion was achieved only by the use of a modified cDNA encoding for an IL-15 pre-protein bearing the IgK light chain signal peptide. Different TS/A clones (TS/A IL-15 C6, C23, C29) producing 390 to 1,600 pg/ml biologically active IL-15 showed reduced tumorigenicity when implanted s.c. in syngeneic mice and significantly reduced metastatic potential by i.v. injection. Tumorigenicity of s.c. TS/A IL-15 was restored in animals depleted of CD8(+) lymphocytes or of natural killer cells and partially in CD4(+)-depleted mice. TS/A IL-15 cells displayed a significantly reduced growth rate by s.c. implant in nude mice. Also, >50% syngeneic animals rejecting TS/A IL-15 were resistant to a subsequent rechallenge with wild-type tumor (TS/Apc), indicating induction of protective immunity against TS/A tumor-associated antigens (TAAs). Cytolytic T lymphocyte (CTL) activity, specifically inhibited by anti-CD3 antibodies, was inducible in the splenocytes of TS/A IL-15-immunized animals by mixed lymphocyte/tumor culture (MLTC), and IFN-gamma was released in the supernatant of MLTC, mainly by CD8(+) cells. Immunohistochemistry of the TS/A IL-15 tumor area revealed the presence of an inflammatory infiltrate with predominant natural killer, macrophage, and granulocyte components and expression of IFN-gamma as a distinctive secondary cytokine. Use of TS/A IL-15 mitomycin-treated cells for therapeutic vaccination in experimental TS/A metastasis was effective in 60% of animals treated; these animals showed no metastatic tumor growth.

349 The immune responses in asthmatic mice with DNA vaccine containing allergen and IL-12 cDNA sequences

349 The immune responses in asthmatic mice with DNA vaccine containing allergen and IL-12 cDNA sequences

346 IL-10 cDNA transfection releives IFN-gamma effects in normal human lung fibroblast cells

346 IL-10 cDNA transfection releives IFN-gamma effects in normal human lung fibroblast cells

Tumor growth inhibition effect of hIL-6 on colon cancer cells transfected with the target gene by retroviral vector.

AIM:To observe the tumor inhibitory effects by transfecting IL-6 cDNA into colon cancer cell line HT-29 with retroviral vector pZIP cDNA.METHODS:Human IL-6 gene was reconstructed in retrovirus vector and transfected into incasing cells PA317 by lipofectamine mediated method, the clones of the cells transferred with hIL-6 were selected by G418,and targeted HT-29 cells were infected with the virus granules secreted from PA317 and also selected by G418.Test gene transcription and expression level by hybridization, ELISA and MTT assay,etc. Analyze tumor inhibitory effects according to the cell growth curve, plating forming rate and tumorigenicity in nude mice.RESULT:Successfully constructed and transfected recombinant expressing vectors pZIPIL-6 cDNA and got positive transfected cell lines. The colon cancer cell line (HT-29 IL-6) transfected with the hIL-6 gene by retroviral vector was estab-lished. The log proliferation period and the doubling time of this cell line was between 4 to 7 days and 2.5 days according to the direct cell count, the cell proliferation was obviously inhibited with MTT assay, the plating inhibitory rate was 50% by plating efficiency test. When HT-29 IL-6 cells were inoculated into the nude mice subcutaneously, carcinogenic activity of the solid tumor was found superior to the control group and the size of tumor was not significantly enlarged. Injection of combination virus fluid containing IL-6 gene into transplantation tumors could inhibit the growth and development of the tumor.CONCLUSION:IL-6 could inhibit the growth and proliferation of colon cancer cells by retroviral vector-mediated transduction.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA.

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways.

Regional treatment of hepatic micrometastasis by adenovirus vector-mediated delivery of interleukin-2 and interleukin-12 cDNAs to the hepatic parenchyma.

We hypothesize that adenovirus (Ad) vector-mediated delivery of the human interleukin-2 (IL-2) cDNA (AdIL2) or the murine IL-12 cDNA heterodimer (AdIL12) would produce high concentrations of cytokines in the local hepatic milieu to induce host responses sufficient to inhibit the growth of experimental colon carcinoma-derived hepatic metastases. Ad vectors administered intravenously, which is a route known to deliver >90% of the vector to the hepatic parenchyma, achieved significant levels of each cytokine locally, with minimal levels in the sera. To examine the therapeutic effect, the AdIL2 and AdIL12 vectors were evaluated in a hepatic metastasis model that was established by injecting 3 x 10(4) cells from the poorly immunogenic syngeneic C26 colon carcinoma cell line into the right lobe of the livers of BALB/c mice. Animals received AdIL2, AdIL12, or control virus (10(8) plaque-forming units each) intravenously for 2 days after tumor implantation, and tumor growth was compared with naive controls. The AdNull control tumors measured 116 +/- 25 mm2 at 2 weeks. The control virus showed no significant antitumor effect. In marked contrast, both AdIL2 and AdIL12 vectors that were delivered regionally had significant antitumor effects, with AdIL2-treated animals having an average tumor size of 16 +/- 8 mm2; AdIL12-treated tumors measured 6 +/- 6 mm2 (P < .01, both compared with control). Both the AdIL2 and AdIL12 vectors provided a significant survival advantage by log-rank analysis (P < .01), but only AdIL12 translated into an increase in mean survival from 27 (naive control) to 37 days. To evaluate whether these antitumor effects were T-cell-mediated, splenocytes from AdIL2-treated, AdIL12-treated, and naive control groups were stimulated in vitro with gamma-irradiated C26 tumor cells for 5 days and tested for C26 tumor cell cytolysis by an in vitro cytotoxicity assay. Splenocytes from both AdIL2- and AdIL12-treated animals showed a dose-dependent, T-cell-mediated, specific cytolysis of CT26 cells. AdIL12 and to a lesser extent AdIL2 induced natural killer cell activity, as determined by a dose-dependent increase in lysis of the natural killer-specific target cell YAC-1. Overall, these data suggest that regional Ad-mediated delivery of IL-2 and IL-12 cDNAs may be useful for local tumor control and may warrant further investigation as a potentially useful adjuvant for the treatment of hepatic micrometastasis.

Use of a human minichromosome as a cloning and expression vector for mammalian cells.

A natural human minichromosome (MC1) derived from human chromosome 1 was shown to be linear and to have a size of 5.5 Mb. Human IL-2 cDNA and the neo gene were co-transfected into a MC1-containing human-CHO hybrid cell line. Integration of the foreign genes was directed to the pericentromeric region of MC1 by co-transfection of chromosome 1-specific satellite 2 DNA. A number of G418-resistant transfectants were obtained and expression of IL-2 was determined. FISH analysis demonstrated co-localization in the minichromosome of the IL-2 gene and of the satellite 2 DNA. An IL-2-producing clone was used in cell fusion experiments with IL-2-dependent murine CTLL cells to generate CTLL-human hybrids containing the modified minichromosome (MC1- IL2 ). The hybrids were able to grow in medium lacking IL-2 for 17 mean population doublings (MPD), indicating that expression of the cytokine was sufficient to relieve the IL-2 dependence of CTLL proliferation. Endogenous IL-2 production delayed the onset of apoptosis in the IL-2-dependent CTLL cells. Mitotic stability was shown to be 100% in the human-CHO hybrids and 97% per MPD in CTLL cells. These results demonstrate that a natural human minichromosome can be utilized as a cloning and expression vector for mammalian cells and that the MC1 minichromosome can be engineered to deliver IL-2 to two types of cells, fibroblasts and lymphocytes.

IL-10 Transgenic Mice Present a Defect in T Cell Development Reminiscent to SCID Patients

AbstractTo analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-αβ T cells as the absolute number of thymic dendritic, TCR-γδ and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.

Immunogene therapy of murine fibrosarcoma using IL-15 gene with high translation efficiency.

We have recently found that translational efficiency is up-regulated by an alternative exon in IL-15 mRNA in mice. In a malignancy model using BALB/c mice and syngeneic Meth A fibrosarcoma (Meth A), we successfully applied immunological gene therapy with IL-15 protein using alternative IL-15 cDNA with high translational efficiency. Two expression vectors carrying the murine IL-15 gene were constructed for use in tumor immunotherapy, one utilizing IL-15 cDNA with alternative exon 5 and the second utilizing IL-15 cDNA with normal exon 5. The first vector induced the production of a large amount of IL-15 protein in Meth A cells, whereas tumor cells transfected by the second vector produced only a marginal level of IL-15 protein. Although cell growth of both transfectants in vitro remained unchanged, inoculation of clones transfected with normal IL-15 cDNA resulted in progressive tumor growth, while clones transfected with alternative IL-15 cDNA led to the rejection of the tumor. The clone producing high levels of IL-15 grew progressively in nude mice and mice treated with anti-CD4 monoclonal antibodies (mAb), whereas the growth of the transfectants was retarded in anti-CD8 mAb- or anti-asialo GM1 antibody-treated mice. Cured mice were shown to have generated immunity against a subsequent challenge with the wild type of Meth A but not against Meth 1 tumor cells, another type of fibrosarcoma derived from BALB/c mice. Thus, tumor therapy based on IL-15 gene transfection was effective against Meth A tumor cells, suggesting a possible application to human neoplasms.

IL-12 Gene as a DNA Vaccine Adjuvant in a Herpes Mouse Model: IL-12 Enhances Th1-Type CD4+ T Cell-Mediated Protective Immunity Against Herpes Simplex Virus-2 Challenge

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.

Molecular and functional characterization of chickenIL-15

A cDNA encoding chicken interleukin-15 was cloned from a CD4 + T cellhybridoma expression library by screening with a rabbit antibody against a protein fraction ofconditioned medium containing T cell growth promoting activity. The chicken IL-15 cDNAcontains an open reading frame of 143 amino acids with a single potential N-linked glycosylationsite. The predicted m.w. of the encoded protein [16]matched the size of animmunoreactive band on Western blots of E. coli expressing the recombinant IL-15.Amino acid and nucleotide sequence analyses of chicken IL-15 revealed 31% and 46% identitywith bovine IL-15 respectively and lesser homologies to other mammalian IL-15s. Chicken IL-15contained all 4 highly conserved cysteine residues present in mammalian IL-15 sequences.RT-PCR demonstrated that the chicken IL-15 gene is expressed in many tissues including spleen,intestine, and muscle and in established macrophage, T lymphoma and fibroblast cell lines.Activation of spleen cells with Con A enhanced the expression of IL-15 gene transcripts in atime-dependent manner. CHO-K1 cells transfected with the chicken IL-15 cDNA secreted abiologically active protein supporting the growth of Con A activated spleen lymphocytes.Continuous culture of spleen Con A lymphoblasts with chicken IL-15 over two months resulted inan enriched T lymphocyte population expressing the gdTCR, CD8a, and CD3 cell surfaceantigens.

Interleukin-6 transduction of a rat T9 glioma clone results in attenuated tumorigenicity and induces glioma immunity in Fischer F344 rats.

We transduced a highly tumorigenic T9 clone (T9.F), isolated from the rat T9 glioblastoma cell line, with a retroviral expression vector containing the human IL-6 cDNA and investigated the effects of IL-6 secretion on glioma formation in the syngeneic Fischer rat. Two subclones producing high and low levels (35 and 3.5 ng/10(6) cells/48 h) of IL-6 were identified and were termed T9.F/IL6/hi and T9.F/IL6/lo, respectively. Subcutaneous (SC) injection of 1 x 10(6) parental T9.F cells resulted in 100% tumor formation and progression. When 1 x 10(6) IL-6 secreting T9.F cells were injected SC, a small palpable tumor formed which sometimes regressed. In this regard, no tumors were detected after 30 days in 76% (13/17) of animals injected with T9.F/IL6/hi cells, whereas only 10% (1/10) of the rats injected with T9.F/IL6/lo cells completely rejected their tumors within this time frame. The addition of an IL-6 neutralizing antibody to the T9.F/IL6/hi SC inoculum followed by an intratumoral injection of the IL-6 neutralizing antibody, seven days later, abrogated the anti-tumor effects. Animals that rejected the IL-6 secreting tumors were 100% protected from subsequent intracranial (IC) challenges with the parental T9.F glioma as well as the original T9 glioblastoma; partially protected from an IC challenge with the unrelated, syngeneic RT-2 glioma; but were not protected from an IC challenge with the syngeneic MadB106 adenocarcinoma. When 1 x 10(4) cells were injected in the brain of naive animals, survival time was significantly increased for those rats implanted with T9.F/IL6/hi cells, but not T9.F/IL6/lo cells, as compared to animals implanted with T9.F parental cells (p = 0.003). This study demonstrates that IL-6 secretion attenuates SC and IC glioma growth and SC rejection of IL-6 secreting T9.F cells induces long-term glioma immunity which is effective in the brain.

Modulation of alloimmunity to major histocompatibility complex class I by cotransfer of cytokine genes in vivo

Major histocompatibility complex (MHC) class I antigen is a potent stimulus for alloimmune responses and is the principal immunologic target mediating acute cellular rejection of allografts. Using a method of direct in vivo gene transfer of cDNA encoding donor type MHC class I, we showed in a rat model that recipient muscle could express the transferred MHC class I cDNA, resulting in alloimmunization of the recipient. This was most graphically demonstrated by accelerated rejection of cardiac allografts expressing the same MHC class I as encoded by the immunizing cDNA. We now report the use of the particle-mediated gene transfer via a gene gun (Geneva, Middleton, WI, USA) to transfer MHC class I, as well as cytokine gene expression vectors, into rat skin. Compared to intramuscular injection, gene gun transfer to skin resulted in more efficient immunization. Donor-specific cytotoxic T lymphocyte (CTL) responsiveness and antibody levels increased. Furthermore, coexpression of certain cytokine genes with the MHC class I cDNA modulated the immune response. Specifically, coimmunization with IL-10 cDNA abrogated immunity to allo-MHC class I, while coimmunization with GM-CSF cDNA enhanced it. The influence of expression of these genes in skin was demonstrated by alteration of donor cardiac allograft survival. This model is useful for induction and modulation of alloimmune responses and may be used to develop gene therapy strategies to modify them.

Expression of interleukin-2 gene in rat brain in normal and stress conditions

Recently the presence of cytokines in rat brain cells was discovered. It is well known now that cytokines play important roll in communication between the nervous and the immune systems. In our experiments we studied gene expression of IL-2 gene by in situ hybridisation under normal and different stress conditions. The rotation stress and several noxious stimuli (injection of 5% mustard in plant oil, plant oil and prick in left paw of rats) were used. In in situ hybridisation experiments the digoxigenin labelling IL-2 cDNA were used. In control animals the expression of IL-2 gene was very low and was detected only in cerebral cortex. Within 4 h after rotation stress application, IL-2 mRNA cells were detected in internal capsule of thalamus. After injection of plant oil we observed localisation of IL-2 mRNA synthesis in thalamic and hypothalamic structures. After administration of 5% mustard in plant oil, the labelled cells were also shown in thalamic and hypothalamic structures, but in greater amount. In the thalamus the labelled cells localised, mainly, in paraventricular, ventramedial and dorsomedial nuclei. Thus, the expression of IL-2 gene was firstly localised in different structures of rat brain and influence of diverse stimuli on IL-2 mRNA synthesis was shown.