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Biomedical subjects

T Blankenstein

Publications and source records attributed to T Blankenstein.

At least 55 records · Page 3Linked to original sources
PubMed1995

Tumor growth inhibition mediated by lymphotoxin: evidence of B lymphocyte involvement in the antitumor response.

The antitumor effect of lymphotoxin (LT) and the underlying cellular mechanism were analyzed. To achieve an increased local concentration of LT at the site of tumor growth, which mimics the physiological fashion of cytokine action, we transfected the murine plasmacytoma J558L cells with a human LT expression plasmid and selected several clones that produce varying levels of LT for analysis of their tumorigenicity. The LT produced by the transfected J558L cells effectively suppressed tumor growth in syngeneic BALB/c mice without any obvious side effects. This antitumor function is indirect and LT specific, because the tumor cells did not show altered growth kinetics after the gene transfer in vitro, and tumor growth inhibition in vivo could partially be reversed by an anti-LT mAb. In nude mice, LT producing tumors were initially suppressed, but most mice developed a tumor at the end of the study. However, the requirement of T cells for complete tumor rejection could be compensated for by higher amounts of LT secretion. Furthermore, the antitumor activity of LT seems to involve B lymphocytes in the absence of functional T lymphocytes since a significant difference existed between tumor growth of J558-LT cells in nude and in SCID mice. LT-producing tumors but not parental tumors were massively infiltrated by B220+ cells in nude mice. The secretion of LT by tumor cells also induced a heavy infiltration of Mac-1+ and Mac-3+ cells and a moderate infiltration of Gr-1+ cells, both in nude and in SCID mice. Together, LT-producing J558L cells are rejected by a complex immunological mechanism, which seems to involve T as well as B and other cells. This distinguishes LT from a number of other cytokines analyzed in analogous experiments.

Animals↗
PubMed1995

IL-10 converts mouse lymphoma cells to a CTL-resistant, NK-sensitive phenotype with low but peptide-inducible MHC class I expression.

IL-10 has a variety of effects including: inhibition of monocyte MHC class II-dependent Ag presentation, Th1 cytokine production, and inhibition of T cell proliferation. Recently we have shown that IL-10 inhibits Ag presentation to human tumor-specific and allospecific CTL. In the present study we showed that transfection of the mouse lymphoma RMA (H-2b) with the IL-10 gene induced conversion to a RMA-S-like phenotype. The changes included an inhibition of lysis by minor histocompatibility or tumor Ag-specific CTLs and, conversely, a dramatic increase in susceptibility to lysis by NK cells. The RMA-10 transfectants showed levels of H-2 expression as low or even lower than those found on RMA-S. The levels of tested adhesion molecules were unaltered. Treatment of RMA with rIL-10 gave a less pronounced change in phenotype. In addition, relative to untreated target cells, IL-10 pretreated cells or IL-10 transfectants were unaltered in their capacity to affect cytotoxicity by cold target inhibition, arguing against the possibility that the observed effect could be a direct effect of IL-10 on the CTL. The expression of H-2 was partially restored by coculturing RMA-10 transfectants with class I-binding peptides. Taken together, these results indicate that IL-10 exerts a post-transcriptional effect on H-2 expression, compatible with an induced decrease in the access of peptides to the MHC class I complex. IL-10 is the first cytokine reported to have this effect and also the first factor shown to induce NK sensitivity and reduced sensitivity to CTL, an effect that may be of physiologic relevance.

Animals↗
PubMed1995

Human lymphotoxin has at least equal antitumor activity in comparison to human tumor necrosis factor but is less toxic in mice.

Because of the severe toxicity of systemically applied tumor necrosis factor (TNF) in cancer patients, considerable efforts have been made to construct mutant TNF molecules, which retain antitumor activity, but display less toxicity. We compared tumor suppression in relation to the toxic effects of human TNF and human lymphotoxin (LT) in mice. The genes for these two cytokines were expressed in Chinese hamster ovary (CHO) cells. Intraperitoneal injection of parental and gene modified CHO cell lines producing similar amounts of biologically active TNF or LT, respectively, into nude mice showed that CHO-TNF cells killed the mice more rapidly than parental cells, but that CHO-LT tumor bearing mice lived significantly longer than mice injected with parental cells. Injection of the cells subcutaneously into severe combined immunodeficiency (SCID) mice allowed direct comparison of tumor suppression and toxic effects of the two cytokines. Both TNF and LT produced by the tumor effectively suppressed tumor growth by an indirect mechanism, LT being at least as effective as TNF. However, mice bearing CHO-TNF cells either died rapidly or developed cachexia, as shown by weight loss. In contrast, mice injected with CHO-LT cells never rapidly died and became cachectic much later than CHO-TNF cell injected animals, though serum levels of LT were higher than those of TNF. Analysis of soluble forms of TNF receptors (TNF-R1 and TNF-R2) in sera of tumor bearing mice showed that soluble TNF-R1 was downregulated in both CHO-TNF and CHO-LT, in comparison with CHO-neo cell injected mice and to normal SCID mice. The soluble form of TNF-R2 was induced by CHO cell lines. In CHO-TNF cell injected SCID mice, serum levels were significantly increased, whereas in mice injected with CHO-LT cells, serum levels of soluble TNF-R2 were decreased. Together, our results show a higher therapeutic index of LT compared with TNF.

Animals↗
PubMed1995

Tumor cells cotransfected with interleukin-7 and B7.1 genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaccines.

Interleukin-7 (IL-7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL-7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL-7-transfected sublines to improve vaccine efficacy. Expression of IL-7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL-7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A-IL-7/B7.1 cells retained their full tumorigenic potential in T cell-deficient mice. Analysis of tumor-infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL-7 and IL-7/B7 transfected compared to parental tumors. In IL-7/B7 transfected tumors, T cell numbers were not further increased compared to that in single-gene-transfected tumors. However, T cells in B7 and IL-7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28+ and CD25-, while in IL-7 transfected tumors, T cells were mainly CD28- and CD25+. In IL-7/B7 cotransfected tumors, the majority of T cells was CD28+ and CD25+. Thus, IL-7 and B7 induced an anti-tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL-7/B7 co-expressing cells induced the most strongly protective immunity, which is superior to that induced by single-gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL-7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity.

Animals↗
PubMed1995

The rat interleukin 4 receptor: coevolution of ligand and receptor.

A rat interleukin 4 receptor (IL-4R) cDNA was cloned by polymerase chain reaction (PCR) using RNA of Con A activated T cells and primers deduced from mouse and human IL-4R sequences. Sequence analysis revealed an open reading frame for a putative membrane protein of 800 amino acids in length. It comprises an overall identity of 52 and 78% to its human and mouse homologues, respectively. The extracellular part of the rat IL-4R contains a number of residues including cysteines and a WSXWS motif typical for the cytokine receptor superfamily. Analysis of amino acid exchanges between rat and mouse IL-4 receptors deciphered for replacement (R) or silent (S) mutations suggested different types of selective pressure acting on the extracellular and intracellular domains. A high R/S value that indicates selective pressure for amino acid exchanges was found for the extracellular domain and a low R/S value for the intracellular part of the IL-4R. Since we previously found a similar high R/S value in the rat IL-4 gene encoding the ligand for the IL-4R, the high amino acid exchange rate can best be explained by coevolution between IL-4 and the ligand binding domain of the IL-4R to improve or retain affinity.

Amino Acid Sequence↗
PubMed1995

The thymidine kinase/ganciclovir-mediated "suicide" effect is variable in different tumor cells.

The herpes simplex virus thymidine kinase (HSV-TK) converts ganciclovir (GCV) into a toxic product and allows selective elimination of TK+ cells in vitro and in vivo. It is currently being used in clinical gene therapy trials as a therapeutic gene or as a safety marker. We have analyzed the susceptibility of different tumor cell lines to the TK/GCV-mediated "suicide" effect. Therefore, tumor cells TSA, J558L, EB, and ESB and, as a control, NIH-3T3 cells were infected with a retrovirus containing a hygromycin/TK fusion gene. All cell lines were sensitive to GCV in vitro; however, the concentration of GCV and the time needed to eliminate tumor cells completely considerably varied between different tumor cell lines. TSA-TK cells were completely eliminated within 10 days in 1 microg/ml GCV, whereas ESB-TK cells required 22 days in 10 microg/ml GCV. When two cell lines were examined, the differing sensitivity to GCV in vitro correlated with the ability to eradicate TK+ tumors in vivo. TSA-TK tumors could be eliminated in almost all animals by systemic GCV administration, whereas ESB-TK tumors were completely resistant. Different sensitivity to GCV was not due to different TK expression levels because the cells were similarly resistant to hygromycin, and Western blot analysis with an anti-TK antiserum revealed similar protein amounts in TSA/TK and ESB-TK cells. Together, the results demonstrate that tumor cells are highly different concerning the susceptibility to the TK/GCV effect, which, however, may be tested for in vitro.

3T3 Cells↗
PubMed1994

Expression of interleukin 10 in human melanoma.

The expression of interleukin 10 (IL-10) mRNA in human malignant melanoma was investigated by reverse transcriptase polymerase chain reaction analysis. Selective expression of IL-10 mRNA in tissues of primary melanomas and melanoma metastases was found in comparison with normal skin. In addition, strong expression of IL-10 mRNA and of biologically active IL-10 was detected in 3 out of 13 melanoma cell lines. Normal melanocytes consistently expressed low levels of IL-10 mRNA but did not produce detectable IL-10 protein, nor did keratinocytes or fibroblasts. The production of biologically active IL-10 by melanoma cell lines suggests that IL-10 mRNA in melanoma lesions may derive at least in part from the tumour cells themselves. Tumour-infiltrating cells, however, could also be a source of IL-10 in melanoma tissues. The presence of IL-10 in melanoma lesions may contribute to the postulated 'paralysis' of an anti-melanoma immune response.

Gene Expression↗
PubMed1994

Observations with tumour necrosis factor gene-transfected tumours.

Tumour necrosis factor (TNF) produced by genetically engineered tumour cells can lead to very effective tumour rejection. Tumour suppression does not result from a direct effect of TNF on tumour cells but rather is mediated by the induction of an anti-tumour immune response. It requires a local and continuous presence of TNF at the tumour site. Tumour rejection induced by TNF is dose-dependent and even tumour cell-derived TNF in amounts which cause complete tumour eradication must not be accompanied by toxic side effects. A complex pattern of tumour infiltrating cells has been observed in TNF-producing tumours consisting of macrophages, CD4+ and CD8+ T cells. For tumour suppression macrophages and CD8+ T cells are needed whereas CD4+ T cells seem to reflect innocent bystander cells. Consistently, TNF is active in T cell deficient mice but in most cases T cells are needed for complete tumour elimination. TNF was active in a number of different tumour models but recent experiments also showed that local TNF failed to induce an anti-tumour response in certain tumour models. Moreover, either depending on the tumour cell line used or on the level of TNF secreted by the tumour, systemic toxicity has been observed, leading to cachexia or wasting of the mice. In one case it has been shown that a TNF gene-transfected tumour showed no tumour growth inhibition in vivo but that TNF augmented metastasis of these cells. Experiments conducted to demonstrate systemic protective immunity by TNF-producing tumours used as a vaccine have not yet been successful.

Animals↗
PubMed1993

Interleukin 10 transfected into Chinese hamster ovary cells prevents tumor growth and macrophage infiltration.

Expression of cytokines in tumor cells provides a sensitive modality to analyze the consequences of local cytokines in vivo on tumor infiltrating cells and tumorigenicity. We have transfected Chinese hamster ovary (CHO) cells with an interleukin 10 (IL-10) expression vector. CHO-IL10 cells although unaltered with respect to their in vitro growth lost tumorigenicity, both in nude and in SCID mice and in an IL-10 dose dependent manner. In addition, CHO-IL10 cells suppressed the growth of equal numbers of coinjected but not of contralaterally injected CHO cells. Immunohistology with anti-CR3/Mac-1 and anti-Mac-3 monoclonal antibodies revealed that CHO tumors were substantially infiltrated by macrophages. However, in CHO-IL10 tumors macrophages were virtually absent within the tumor tissue. Our results suggest that IL-10 indirectly suppresses tumor growth of certain tumors by inhibiting infiltration of macrophages which may provide tumor growth promoting activity.

Animals↗
PubMed1993

Expression of tumor necrosis factor by different tumor cell lines results either in tumor suppression or augmented metastasis.

Tumor necrosis factor (TNF) produced by tumor cells after gene transfer can effectively suppress the growth of locally growing tumors. We wanted to test the effects of "local" TNF on the growth of a highly metastatic cell line. Therefore, a recombinant retrovirus allowing expression of the TNF gene by the beta-actin promotor has been constructed and used to infect the two tumor cell lines EB and ESB, which grow as solid tumor or metastasize, respectively. Expression of TNF by EB cells resulted in their rapid and dose-dependent rejection. In sharp contrast, mice injected with ESB cells producing similar amounts of TNF showed no signs of tumor suppression, but rather had reduced survival rates that correlated with enhanced hepatic metastases. The accelerated formation of liver metastases by ESB TNF cells could be reversed by an anti-TNF mAb. These results demonstrate the opposite effects TNF may have on tumor growth: suppression of a locally growing tumor and promotion of metastasis formation.

Animals↗
PubMed1993

Mechanisms of rejection induced by tumor cell-targeted gene transfer of interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or interferon gamma.

Interleukin (IL)-2, IL-4, IL-7, tumor necrosis factor (TNF), or interferon-gamma (IFN-gamma) has been shown to be able to induce tumor rejection if produced locally by the tumor cells after gene transfer. To analyze whether the cellular rejection mechanisms are different or redundant we have expressed the cytokines in the same tumor cell line (J558L). Cell depletion experiments revealed that all cytokines required CD8+ T cells for complete long-term tumor eradication, although effective but transient host-dependent tumor suppression was also observed in the complete absence of CD8+ T cells. The transient tumor suppression induced by IL-2, IL-4, TNF, or IFN-gamma was also operative in nude and severe combined immunodeficient mice, whereas only tumor suppression induced by IL-7 was dependent on the presence of CD4+ T cells and was not evident in nude mice. The T-cell-independent effector arm of IL-2 and IFN-gamma but not IL-4 and TNF was mediated in part by natural killer cells. The transience of tumor suppression in the absence of T cells reflected loss of cytokine production in the case of TNF, IL-2, and IL-4 but not IFN-gamma. Immunohistologic analysis revealed all cytokine-producing tumors to be heavily infiltrated by macrophages. IL-4 and IL-7 tumors additionally contained eosinophils. The infiltration by T cells did not necessarily reflect their contribution to tumor rejection. Thus, the different cytokines activate heterogeneous transient tumor-suppressive mechanisms but always require CD8+ T cells for complete tumor rejection.

Animals↗
PubMed1993

Vaccinations with tumor cells genetically engineered to produce different cytokines: effectivity not superior to a classical adjuvant.

The potential of tumor cells (J558L) engineered to produce one of 5 different cytokines (interleukin 2, interleukin 4, interleukin 7, tumor necrosis factor, or gamma-interferon) to give rise to systemic immunity protective against a contralateral challenge with the parental cells was analyzed. The rejection of all cytokine-producing cells appeared to induce some systemic response capable of mediating the rejection of low numbers of subsequently contralaterally injected cells, but the effect was much less obvious with higher cell numbers. The injection of any possible combination of two of the cytokine producers did not reveal any synergistic effects. The cytokine gene-transfected tumor cells were not superior to the parental cells admixed with the adjuvant Corynebacterium parvum with respect to their potential as immunogens to induce immunity.

Animals↗