Enhancing the immunogenicity of liposomal hepatitis B surface antigen (HBsAg) by controlling its delivery from polymeric microspheres.

Microencapsulated liposome systems (MELs) were investigated as a potential immunization carrier for a recombinant 22-nm hepatitis B surface antigen (HBsAg) particle. MELs were prepared by first entrapping the HBsAg particles within liposomes composed of phosphatidylcholine:cholesterol (1:1 molar ratio), which were then encapsulated within alginate-poly(L-lysine) (PLL) hydrogel microspheres. The entrapped HBsAg particles retained immunoreactivity, as judged by an enzyme-linked immunosorbent assay (ELISA). Direct imaging of HBsAg particles and HBsAg incorporated into liposomes by cryo-transmission electron microscopy (cryo-TEM) indicated that HBsAg is embedded in the liposomal membrane. The antigenic particles were released from MELs mainly within the context of liposomes. The release rates in vitro and in vivo depended on the molecular weight of PLL used for MEL coating; MELs-214, coated with 214 kDa PLL, released the liposomal HBsAg at much higher rates than MELs-25, which was coated with 25 kDa PLL. Concomitantly, the specific anti-HBsAg titers in mice receiving HBsAg in MELs-214 were higher than those induced by MELs-25. MELs-214 were more efficient than conventional liposomes or alum in eliciting higher and prolonged antibody levels in mice. The ability of MELs to provide an HBsAg depot as well as a sustained release of liposomal HBsAg suggests that these carriers may be an ideal immunoadjuvant.

Macrophage activation for the production of immunostimulatory cytokines by delivering interleukin 1 via biodegradable microspheres.

Interleukin 1alpha (IL-1alpha), a pleiotropic cytokine with multiple anti-tumour activities, has been investigated in our laboratory for its potential to serve as an immunotherapeutic agent. In the present study, an attempt was made to direct IL-1alpha to macrophages, in order to induce their immunoregulatory activities. For that purpose, IL-1alpha was encapsulated within biodegradable poly(lactic/glycolic acid) microspheres, 1-5 microm diameter in size. The microspheres were efficiently taken-up by macrophages in culture and after intraperitoneal injection into mice. In culture, phagocytosis of the microspheres reached saturation within 3 h and there was no apparent effect of polymer type on the extent of uptake. In vivo uptake of human IL-1alpha-microspheres by the macrophages lead to cell activation, as evidenced by the enhanced production of murine IL-1alpha, IL-6 and IL-12. Control microspheres, containing bovine serum albumin, induced only background to low levels of cytokine production. These cytokines, when expressed by or secreted from macrophages, may stimulate in situ diverse immune and inflammatory responses, including T cell-mediated immune responses, such as the development of Th(1)cells and cytotoxic lymphocytes. Thus, directing IL-1alpha into macrophages, via the appropriate microspheres, may serve as a unique mean to activate these cells to participate in anti-tumour immune responses in situ.

Interleukin 1alpha activity of peritoneal and bone marrow macrophages infected with Leishmania major and Leishmania donovani in vitro.

In this study, the pattern of interleukin-1alpha (IL-1alpha) production by both peritoneal (PM) and bone marrow macrophages (BMM) from resistant (C3H/HeJ) and susceptible (BALB/c) mice was investigated, using a bioassay and an IL-1alpha-specific ELISA kit. PM from normal uninfected mice showed either an initial high (C3H/HeJ) or a neglected (BALB/c) level of IL-1alpha activity, respectively, probably due to thioglycollate stimulation. Infection with Leishmania major induced only a marginal effect on IL-1 production by both cells. Normal, uninfected and unstimulated BMM from both mice did not produce IL-1alpha over a 7-day period of cultivation in vitro. Upon stimulation with either lipopolysaccharide (LPS) (BALB/c) or concanavalin A (Con A) (C3H/HeJ), both cell types produced IL-1alpha that peaked within the first 12-24 h following stimulation. BMM from C3H/HeJ and BALB/c mice failed to produce IL-1alpha when infected in vitro with L. major or L. donovani promastigotes. However, infection with these two parasites did not interfere with the capability of the host cell to produce IL-1alpha when stimulated with LPS or Con A. The level of IL-1alpha production was independent of the degree of parasitization of the macrophages. Similar results were observed with IL-1beta and IL-6 production by BMM, even though their levels were generally slightly higher than those obtained with IL-1alpha.

Expression of PKCeta in NIH-3T3 cells promotes production of the pro-inflammatory cytokine interleukin-6.

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. The generation and characterization of NIH-3T3 cells which stably overexpress the PKCeta isoform has been previously described by us. In these cells, overexpression of PKCeta altered the expression of specific cell cycle regulators and promoted differentiation [20]. Since PKC has been implicated in the regulation of gene expression, including that of various cytokines, we examined the production of several cytokines in these cells. We report here that out of the major pro-inflammatory cytokines examined, IL-1alpha, IL-1beta, TNF-alpha and IL-6, only IL-6 was generated and secreted in PKCeta -expressing cells without any additional inducer in serum-supplemented cultures (10% FCS). IL-6 was not detected in the control cell line, transfected with the same vector, but lacking the cDNA coding for PKCeta. Moreover, the production of IL-6 on serum stimulation correlated with the levels of PKCeta expressed in these cells. This implies that factors in the serum activate PKCeta and induce IL-6 production. We have examined several growth factors and cytokines for their ability to induce IL-6 production in our PKCeta-expressing cells. Among the growth factors tested (EGF, PDGF, FGF, insulin, IGF-1 and IL-1), PDGF and FGF were the most potent IL-6 inducers. The effects of FGF and PDGF on IL-6 production were blocked in the presence of PKC inhibitors. We also examined the signaling pathways that mediate production of IL-6 in PKCeta-expressing cells. Using specific inhibitors of the MAPK pathway, we have shown a role for ERK and p38 MAPK in FGF- and serum-stimulated IL-6 production, but only for p38 MAPK in PDGF-stimulated IL-6 production. Our studies provide evidence that PDGF and FGF can serve as upstream regulators of PKCeta and that PKCeta is involved in the expression of IL-6. This suggests that inhibition of PKC may provide a basis for the development of drugs for the treatment of disorders in which IL-6 is pathologically involved.

Antitumor and immunotherapeutic effects of activated invasive T lymphoma cells that display short-term interleukin 1alpha expression.

Expression of cytokines in malignant cells represents a novel approach for therapeutic treatment of tumors. Previously, we demonstrated the immunostimulatory effectiveness of interleukin 1alpha (IL-1alpha) gene transfer in experimental fibrosarcoma tumors. Here, we report the antitumor and immunotherapeutic effects of short-term expression of IL-1alpha by malignant T lymphoma cells. Activation in culture of T lymphoma cells with lipopolysaccharide-stimulated macrophages induces the expression of IL-1alpha. The short-term expression of IL-1alpha persists in the malignant T cells for a few days (approximately 3-6 days) after termination of the in vitro activation procedure and, thus, has the potential to stimulate antitumor immune responses in vivo. As an experimental tumor model, we used the RO1 invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the vertebral column and compress the spinal cord, resulting in hind leg paralysis and death of the mice. Activated RO1 cells, induced to express IL-1alpha in a short-term manner, manifested reduced tumorigenicity: approximately 75% of the mice injected with activated RO1 cells remained tumor free. IL-1 was shown to be essential for the eradication of activated T lymphoma cells because injection of activated RO1 cells together with IL-1-specific inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor, reversed reduced tumorigenicity patterns and led to progressive tumor growth and death of the mice. Furthermore, activated RO1 cells could serve as a treatment by intervening in the growth of violent RO1 cells after tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after the inoculation of violent cells, mortality was significantly reduced. IL-1alpha, in its unique membrane-associated form, in addition to its cytosolic and secreted forms, may represent a focused adjuvant for potentiating antitumor immune responses at low levels of expression, below those that are toxic to the host. Further assessment of the immunotherapeutic potential of short-term expression of IL-1alpha in activated tumor cells may allow its improved application in the treatment of malignancies.

Interleukin-6 release following scorpion sting in children.

Interleukin-6 levels were measured in the serum of ten children following severe scorpion envenomation. Measurements were taken on arrival, at the emergency room, and 12 and 24 hr after arrival. Interleukin-6 was markedly elevated in the serum of eight out of ten children on arrival. Interleukin-6 levels gradually decreased toward normal values on 12 and 24 hr measurements, but remained above control levels on all measurements. These results imply that signs and symptoms following scorpion envenomation may in part be explained by release of cytokines. Human and experimental animal studies are required in order to verify the assumption that interleukin-6 and other cytokines are involved in the pathogenesis of scorpion envenomation.

Interleukin-1 and tumor necrosis factor-alpha in the Reed-Sternberg cells of Hodgkin's disease. Correlation with clinical and morphological "inflammatory" features.

We examined paraffin sections for the expression of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha, in 40 cases of Hodgkin's disease. Our purpose was to study the role of these cytokines in the "inflammatory" histological features and "B" symptoms in this disease. Immunohistochemistry with the avidin-biotin-peroxidase complex method was used. The findings were compared with those of 20 cases of non-Hodgkin's lymphomas and of 20 non-neoplastic lymphadenopathies. Evidence for EBV infection and myc and ras oncoproteins expression was also studied in these patients, but no correlation between any of these features and cytokine expression was found. We found a significant correlation between the expression of interleukin-1 beta and several "inflammatory" histological features, as well as between the expression of tumor necrosis factor-alpha and B symptoms and tumor bulk. The differential correlations between these major pro-inflammatory cytokines expression and the "inflammatory" manifestations in Hodgkin's disease are remarkable, considering the complexity of the cytokines composing the cytokine network involved in this disease.

Complementary organ expression of IL-1 vs. IL-6 and CSF-1 activities in normal and LPS-injected mice.

The specific expression of pro-inflammatory cytokines may affect the functioning of organs in different ways. The results of specific cytokine bioassays used in this study show a distinct pattern of tissue expression of IL-1 IL-6 and CSF-1. Cytokine activity was assessed in conditioned media (CM) and lysates (LYS), obtained from different organs of control or lipopolysaccharides (LPS)-injected mice; LPS representing a potent inflammatory stimulus. Low constitutive levels of IL-1 could be demonstrated only in CM/LYS from organs with lymphoreticular function, such as the liver, spleen, intestine and lungs. On the other hand, IL-6 and CSF-1 were mainly detected in the CM (and not in lysates) of organs, such as the heart, kidneys, muscle and brain. LPS injection basically resulted in an accentuated form of the constitutive pattern. CSF-1 displays a similar pattern of expression to that of IL-6, best detected in CM after LPS stimulation. Thus, a mirror-image relationship emerges between the patterns of IL-1 and IL-6/CSF-1 expression in two groups of organs: those with lymphoreticular function, which manifest high IL-1 and low IL-6/CSF-1 activity, as compared to organs characterized by highly specialized and potentially vulnerable functions (such as the heart, brain, muscle and kidney), which exhibit high IL-6/CSF-1 and low IL-1 activity. Due to their defensive functions, lymphoreticular organs, which are in charge of the 'gates of entry' to the body, mount extensive IL-1-mediated inflammatory responses, even at the cost of possible tissue-damage. On the other hand, the more vulnerable internal organs mount IL-6/CSF-1-mediated responses which are milder and bear less potential for tissue damage. The distinct patterns of expression of pro-inflammatory cytokines in different organs, at steady state or under inflammatory conditions, may shed light on tissue characteristic homeostatic and defence mechanisms.

Mechanisms of cytokine production by fibroblasts-implications for normal connective tissue homeostasis and pathological conditions.

Fibroblasts actively participate in cellular immune responses in connective tissues, when activated by signals abundant at inflammatory sites, i.e. cytokines and bacterial products. This is manifested by the generation of proinflammatory cytokines and by presenting antigens to proliferating T cells. The array of cytokines generated by immune-activated fibroblasts is determined by the stimulant and is controlled at multiple regulatory levels, such as transcription, translation, posttranslational modifications, the signal transduction pathways which are activated, the timing of expression as well as compartmentation within the producing cell. In general, cytokines with potential of tissue damage, i.e. IL-1 alpha and, to a lesser extent, IL-6, are more tightly regulated than cytokines with restricted target cell specificity (i.e. CSFs). Deviations in the pattern of expression of IL-1 alpha in pathological conditions affecting connective tissues are described; a long-lasting suppression of IL-1 alpha production was observed in dermal fibroblasts of mice suffering from chronic graft-vs.-host disease (cGVHD), while some oncogene-transformed fibroblastoid cell lines were shown to generate this cytokine in a constitutive manner and as a result expressed reduced tumorigenicity. The latter is due to the adjuvant effects of IL-1 alpha, expressed by the malignant cells, which induce potent antitumor specific immune responses which ultimately lead to its eradication. Understanding the molecular mechanisms which control cytokine production in fibroblasts may enrich our knowledge of connective tissue homeostasis and deviations from it in pathological states. The latter may also lead to the development of novel therapeutical means for controlling chronic inflammatory diseases or malignancies.

Ureaplasma urealyticum in Reiter's syndrome.

OBJECTIVE: To evaluate the role of Ureaplasma urealyticum (Uu) in the pathogenesis of Reiter's syndrome (RS). METHODS: Infection with Uu was determined in 31 patients with RS and 28 patients with other arthritides by urethral, cervical and synovial fluid (SF) culture and by measuring anti-Uu serum antibody. Infection with Chlamydia trachomatis was determined by examining SF by a direct immunofluorescence technique, by a polymerase chain reaction and by measuring anti-C. trachomatis serum antibody. The proliferative response of SF and peripheral blood mononuclear cells (PBMC) to Uu antigens in patients with RS was compared to that of a control group. The effect that treatment of 6 patients with RS with ciprofloxacin had on repeated cultures, on titer of anti-Uu antibody and on mononuclear cell reactivity was measured sequentially. RESULTS: The colonization rate of Uu in patients with RS (74%) was significantly greater than in patients with other arthritides (14%). Genital C. trachomatis isolation and serum anti-C. trachomatis antibody were uncommon in both groups (11 and 13%, respectively). SF mononuclear cells of the patients with RS proliferated specifically in response to Uu antigens [up to 6.9 stimulation index (SI)], as did their PBMC (up to 14.5 SI). In some patients, high anti-Uu antibody titers were measured in the serum. Clinical remission was observed in 4 of 6 patients and correlated with eradication of Uu, decrease in antibody titers and disappearance of mononuclear cell reactivity to Uu antigens. CONCLUSION: Our findings suggest that Uu might be a causative agent or a trigger in the development of sexually acquired RS.

Involvement of immune responses in the eradication of IL-1 alpha gene-transduced tumour cells: mechanisms of tumour rejection and immunotherapeutical implications.

We detected a strong correlation between the constitutive expression of IL-1 alpha and reduced tumorigenicity, using fibrosarcomas which produce the cytokine spontaneously (as an aberration of the transformation process) or upon gene transfer. In fibroblasts intracellular or membrane-associated IL-1 alpha is expressed, whereas the secreted form of the cytokine (IL-1 beta) is absent. Studies on the mechanisms of tumour regression of the IL-1 alpha producing fibroblastoid cell lines indicated that IL-1 alpha potentiates the development of tumour cell-specific CTLs, which are of importance for tumour eradication. It also appears that IL-1 alpha-induced enhanced helper T-cell activity provides auxiliary signals for the growth/development of CTLs. In addition, we observed a massive lymphocytic infiltrate in IL-1 alpha producing regressing tumours which ultimately replaces the tumour's mass. Non-adaptive effector cells, activated locally by IL-1 alpha expressing fibrosarcoma cells, were also shown to contribute, to some extent, to the eradication of IL-1 alpha expressing fibrosarcomas. Local IL-1 alpha expression potentiated antigen presentation, by the malignant fibroblasts as well as by tissue-resident antigen-presenting cells, and by this anti-tumour immune responses were further potentiated. Mice, in which IL-1 alpha producing tumours regressed, developed systemic immunity and rejected a challenge with a non IL-1 producing violent tumour cell line. It appears that endogenous IL-1 alpha, being a strong inducer of cytokine production, operates a whole cytokine cascade (such as IL-6, CSFs and prostaglandins). However, studies using clonal populations have indicated that IL-1 alpha is essential for fibrosarcoma eradication, whereas the other cytokines possibly amplify and sustain its action. We assume that most naturally occurring tumours are not constitutive IL-1 alpha producers, as it would be disadvantageous for the tumour to express a cytokine which increases its immunogenicity. However, IL-1 non-producing fibrosarcomas can be induced easily to express IL-1 transiently, by treatment with cytokines/LPS, and upon the induction of the cytokine they shift from progressor to regressor tumours. We also obtained positive immunotherapeutical effects when treating mice bearing IL-1 non-producing fibrosarcomas with cells from the same line induced in vitro to express IL-1 alpha. The results may shed light on a novel parameter affecting tumour-host interactions, namely cytokine expression by the tumorous cells, and may provide the basis for new immunotherapy protocols for fibrosarcoma management.

Differential Proliferative Characteristics of Alveolar Fibroblasts in Interstitial Lung Diseases: Regulative Role of IL-1 and PGE(2).

Fibroblasts (Fb) from patients with sarcoidosis (SA) and hypersensitivity pneumonitis (HP) exhibited a lower proliferative capacity compared with Fb obtained from control (CO) and diffuse interstitial fibrosis patients (DIF). Proliferation of Fb from SA or lip patients was suppressed by autologous LPS-stimulated alveolar macrophages (AM) supernatants but not by those from CO patients. Similarly, alveolar macrophages (AM) derived supernatant, obtained from CO, did not suppress the proliferation of SA and HP Fb. AM from SA and HP patients secreted higher amounts of IL-1alpha and beta compared with controls and compared with Fb from SA and HP patients. Steady levels of IL-1alpha and betamRNA were expressed in unstimulated and stimulated cultures. Fb from SA and HP patients could be stimulated by LPS to secrete significantly higher levels of PGE(2) than those detected in supernatants from LPS stimulated Fb of DIF patients. Only the proliferation of Fb from SA and HP patients was sensitive to amounts of IL-1 equivalent to those detected in the lung of these diseases. As SA and HP are two diseases where irreversible deterioration occurs in only 20% of the patients, we hypothesize that mediators in the lung may modulate Fb proliferation. IL-1 of AM origin and PGE(2) of Fb origin secreted at high levels, may be candidates for this suppression because it was abrogated by anti IL-1beta and indomethacin.

Cytokine-induced tumor immunogenicity: endogenous interleukin-1 alpha expressed by fibrosarcoma cells confers reduced tumorigenicity.

A direct correlation between the constitutive expression of IL-1 alpha and reduced tumorigenicity of fibrosarcomas was observed. This was established in fibrosarcoma cell lines which produce IL-1 alpha 'spontaneously', possibly as an aberration of oncogene-mediated transformation or upon IL-1 alpha gene transfer. In fibroblasts intracellular or membrane-associated IL-1 alpha is expressed, whereas the secreted form of the cytokine (IL-1 beta) is absent. Studies on the mechanisms of tumor regression of the IL-1 alpha-positive fibroblastoid cell lines indicated that IL-1 alpha potentiates the development of tumor cell-specific CTLs, which are of importance for tumor eradication. Thus, IL-1 alpha induces enhanced helper T cell activity which provides auxiliary signals for the growth/development of CTLs. Non-adaptive effector cells, activated locally by IL-1 alpha-expressing fibrosarcoma cells, also contribute to the eradication of IL-1 alpha-expressing fibrosarcomas. Local IL-1 alpha expression potentiated antigen presentation, by the malignant fibroblasts as well as by tissue-resident antigen-presenting cells, thus further potentiating anti-tumor immune responses. Mice, in which IL-1 alpha-producing tumors were regressed, developed an immune memory and rejected a challenge with an IL-1 non-producing violent tumor cell line. Endogenous IL-1 alpha activates a cytokine cascade (i.e., IL-6, CSF), produced by the malignant cells and possibly also by stromal cells. However, IL-1 alpha expression is essential for fibrosarcoma eradication, while other cytokines possibly amplify and sustain its action.(ABSTRACT TRUNCATED AT 250 WORDS)

Different regulatory levels are involved in the generation of hemopoietic cytokines (CSFs and IL-6) in fibroblasts stimulated by inflammatory products.

In this present study we have characterized the array of hemopoietic cytokines generated by fibroblasts in response to inflammatory signals. It was shown that murine embryo fibroblasts (MEF) are able to generate colony stimulating factors (CSFs) [granulocyte-macrophage (GM-CSF), macrophage-CSF (CSF-1) and granulocyte-CSF (G-CSF)] as well as the hemopoietin interleukin 6 (IL-6), while the production of IL-3, IL-4 or tumor necrosis factor (TNF) could not be detected in MEF, as assessed by bioassays or expression of specific mRNA. The production of colony promoting activity was observed when fibroblasts were stimulated by lipopolysaccharide (LPS) or individual cytokines [IL-1, interferon-gamma (IFN-gamma), IL-2, IL-4 or TNF] in serum-free conditions as well as by serum itself. These inducers differentially stimulated in MEF the production of various CSFs; LPS induced mainly CSF-1, while cytokines or serum induced equivalent amounts of GM-CSF and CSF-1. The production of IL-6 was induced by LPS in serum-free conditions, while stimulation by cytokines (IL-1 or IFN-gamma) resulted in IL-6 production only in serum-supplemented cultures. Serum by itself did not induce IL-6 production by MEF. The secretion of IL-6 by fibroblasts was detected early and peaked after 6 hours, while CSF activity peaked after 24-72 hours, depending on the inducer. Constitutive mRNA expression of CSF-1 was detected in serum-free conditions in unstimulated MEF, however colony-promoting activity was detected only upon stimulation with cytokines, LPS or serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced tumorigenicity of fibrosarcomas which constitutively generate IL-1 alpha either spontaneously or following IL-1 alpha gene transfer.

Interleukin-1 (IL-1) is a major immunoregulatory/proinflammatory cytokine which also affects fibroblast proliferation and function and therefore it was of interest to investigate whether its constitutive expression influences the in vivo tumorigenic potential of transformed fibroblastoid cell lines. Here we report on a strong correlation between the constitutive expression of IL-1 alpha and reduced tumorigenicity, using various series of oncogene-transformed NIH/3T3-derived cell lines which produce the cytokine spontaneously or upon gene transfer, following transfection with the IL-1 alpha cDNA. Reduced tumorigenicity of the constitutive IL-1 alpha producing cell lines was manifested either by inability to grow in animals or by regressions of initially growing tumors, within 2 to 3 weeks from cell inoculation. In contrast, mice inoculated with non-IL-1-producing cell lines developed progressive tumors which ultimately killed the animals. Clones obtained from a non-IL-1-producing met-transformed cell line shifted from a progressive to a regressive phenotype, following transfection with an IL-1 alpha-encoding gene, inserted into an appropriate expression vector, resulting in constitutive expression of the cytokine. The effects of constitutive IL-1 expression on tumor development were observed both in histocompatible (NFS/N) and partially allogeneic (BALB/c) mice; however, they were more pronounced in the allogeneic environment. Fibrosarcomas which are non-IL-1 producers induced progressive tumors in both strains of mice at the same growth rate. The differences between the growth characteristics of the fibrosarcomas in histocompatible vs. partially allogenic mice suggest that IL-1 exerts adjuvant-like effects which increase the immunogenicity of tumor-cell antigens, and they also argue against the possibility that an IL-1-mediated local non-specific inflammatory response is the major effector mechanism of tumor rejection. Indeed, in subsequent studies we shall report on the importance of specific cellular immune responses, especially cytotoxic T lymphocytes (CTLs), in the eradication of constitutive IL-1-producing fibrosarcomas. Thus, our findings may serve as the basis for novel immunotherapy strategies aimed at the induction of IL-1 expression by cells comprising the neoplasm or alternatively by local application of the cytokine in the vicinity of the tumor.

Interleukin-1 production by transformed fibroblasts. II. Influence on antigen presentation and T-cell-mediated anti-tumor response.

Oncogene-transformed fibroblasts which expressed IL-1, spontaneously or after activation with conditioned medium (CM) and lipopolysaccharide (LPS), regressed in the syngeneic host. Since regression was significantly influenced by the immune competence of the host (see companion report), we speculated that regression was T-cell-mediated. Frequencies of cytotoxic T-cell precursors (CTLp) were in the same range for activated and non-activated, transformed fibroblasts. Furthermore, it was found that lysability of transformed fibroblasts was not influenced by expression of IL-1. These findings exclude the possibility that regression of CM- and LPS-treated transformed fibroblasts may have been due to the appearance of new, strongly immunogenic epitopes. On the other hand, frequencies of CTL were significantly increased after in vivo immunization with IL-1-expressing as compared to IL-1-non-expressing transformed fibroblasts. The in vivo maturation/expansion of CTL could have been the consequence of activation of helper T cells (TH), transformed fibroblast-associated IL-1 delivering the costimulatory signal. Analysis of frequencies and proliferation rates of TH confirmed this assumption. Both parameters were significantly increased after stimulation with transformed fibroblasts expressing IL-1 in comparison to transformed fibroblasts not expressing IL-1. Furthermore, purified T cells apparently depleted of cells expressing MHC class-II antigens, i.e. antigen-presenting cells, proliferated in the presence of transformed fibroblasts expressing IL-1. Since IL-1 rather than MHC class-II antigen expression was the limiting factor, antigen presentation by IL-1-expressing transformed fibroblasts appears unlikely. Instead, maturation of antigen-presenting cells could well have been initiated by tumor-associated IL-1. We conclude that IL-1 expression of transformed fibroblasts plays an important role in the induction of a T-cell-mediated anti-tumor response. The effect is due to increased efficiency in the activation of helper T cells and may be supported by activation of antigen-presenting cells.