Genetic factors in metastatic progression of cutaneous melanoma: the future role of circulating melanoma cells in prognosis and management

The greatest potential for improvement of outcome for patients with Cutaneous Malignant Melanoma lies in the prevention of systemic metastasis. Despite extensive investigation, current prognostic indicators either alone or in combination, although related to melanoma progression, are not sufficient to accurately predict the pattern of progression and outcome for any individual patient. Metastasis related death has been recorded in patients initially diagnosed with early stage tumour as well as in patients many years after initial tumour removal. The trouble finding a predictable pattern in the puzzle of melanoma progression may be linked to the fact that most of the material studied for prognosis is either, cutaneous primaries or metastatic deposits, rather than the melanoma cells in the circulatory system which are responsible for disease progression. In this review article we discuss the potential use of circulating tumour cell (CTC) detection and quantification for identifying patients at risk of metastatic deposits. We also discuss current therapies for the treatment of metastatic melanoma and analyse how CTCs may be used to evaluate the effectiveness of current therapies and to pinpoint patients who require further treatment.     

Cancer Immunotherapy

This review summarises the evolution of recent major advances in cancer immunotherapy, using metastatic melanoma as a model. The first true clinical progress with immunotherapy developed from the application of recombinant DNA technology for the large scale production of immunostimulant cytokines. Clinical trials demonstrated that the systemic administration of recombinant high-dose bolus intravenous interleukin-2 (IL-2; 720 000 IU/kg every 8 hours) mediated objective tumour progression in 20% of patients with metastatic renal cancer and in 17% of patients with metastatic melanoma, with complete responses of 9% and 7%, respectively. The use of adoptive immunotherapy (the transfer of immune cells with anti-tumour activity to the tumour-bearing host) focused interest on T lymphocyte-mediated tumour recognition. Clinical trials described the systemic administration of lymphokine activated killer (LAK) cells and subsequently tumour infiltrating lymphocytes (TIL) to patients with advanced cancer. Although able to kill tumour targets in vitro, LAK cells did not prove useful for the treatment of patients with metastatic melanoma and renal cancer. A randomised trial, in which IL-2 was administered alone or with LAK cells, failed to show a difference in response rate or survival. In contrast, the treatment of 86 patients with metastatic melanoma using TIL plus IL-2 resulted in a 34% objective response rate, which included patients who had previously failed treatment with high-dose IL-2 alone. The focus on cellular immune responses, combined with rapid biotechnological advances, resulted in the identification of tumour specific antigens, such as MART-1 and gp100, that could be recognised by autologous TIL. This provided fundamental evidence of the existence of melanoma-associated antigens that were recognised in vivo by effector cells of the immune system. In vitro studies demonstrated immunodominant epitopes from MART-1 and gp100 that could induce in vitro-specific cytotoxic T lymphocyte reactivity. To enhance in vitro immunogenicity, single amino acid substitutions were made to identify peptides with higher affinity for HLA-A*0201. Modified peptides from gp100 were compared with the parental peptide for increased immunogenicity based on their ability to induce anti-tumour lymphocytes in vitro. From these studies, a candidate peptide was identified (G9-209-2M) which had increased immunogenic reactivity in vitro. Clinical trials demonstrated that the modified G9-209-2M peptide was more effective. Unfortunately, objective tumour regression was still low. However, when high-dose IL-2 was combined with G9-209-2M objective clinical responses increased to 42%. Efforts to find better ways to immunise against self antigens are ongoing and involve further peptide immunisations, as well as recombinant viral vectors, adjuvant cytokine therapy and cellular adjuvants such as dendritic cells.     

Cytokines and Cancer

The relationships between cytokines and cancer are multiple and bidirectional. On the one hand, cytokines may directly influence carcinogenesis and metastasis by modifying the tumor phenotype. On the other hand, during tumor progression, modifications of the cytokine expression in the tumor environment may be induced by the tumor cells, leading to a state of immunosuppression reflected by low cytokine expression in tumor stroma. Cytokines also play a role by stimulating the host immune system to generate anti-tumor specific responses. Finally, the use of cytokines as anti-tumor agents has led to objective clinical responses in about 15–25% of patients with metastatic melanoma or renal cell carcinoma, which presents the basis for the development of promising immunotherapeutic approaches for cancer therapy.     

抗OX-40和抗IL-4抗体体外促进Th1细胞的初步探讨

将灭活的自身黑色素瘤细胞MEL 72与BCG混合免疫MEL 72黑色素瘤病人 ,10d后手术方法获取局部引流淋巴结 ,制成淋巴细胞悬液 ,以抗CD3抗体体外培养 ,ELISA方法检测细胞上清中肿瘤特异性Th1、Th2细胞因子的表达。进一步用抗OX 40抗体与CD4+ T淋巴细胞表面OX 40作用或用抗IL 4抗体阻断IL 4的分泌 ,观察肿瘤特异性免疫反应能否向Th1方向转换。结果表明 :用灭活的自体肿瘤细胞免疫机体能诱导出该肿瘤细胞特异性的Th1和Th2。应用抗OX 40抗体与抗IL 4抗体作用后 ,Th1细胞因子分泌水平增加 (TNF β和IFN γ ) ,结果提示 ,应用抗OX 40和抗IL 4抗体可诱导肿瘤特异性Th1反应。     

Apoptotic cell death in conjunction with CD80 costimulation confers uveal melanoma cells with the ability to induce immune responses

Uveal melanoma is a rare malignancy with a poor prognosis despite current therapeutic intervention. The current investigation focuses on the immunogenicity of uveal melanoma cells genetically modified with recombinant adenovirus encoding CD80 (AdCD80) in contrast to their parental counterpart. We demonstrate that costimulation provided by uveal melanoma cells improved immune responses in vitro as determined by mixed lymphocyte tumour cell cultures and cytotoxic T-cell assays using lymphocytes from healthy donors and uveal melanoma patients. Flow cytometry revealed T-cell stimulation by activated CD4+ and CD8+ T cells. Additionally, autologous lymphocytes proliferated in response to CD80-expressing primary uveal melanomas, indicating that this patient group is suitable for immunotherapy. Moreover, this study utilized AdCD80 modified and parental apoptotic tumour cells, loaded onto immature dendritic cells, as a source of tumour antigen. The ability of live or apoptotic tumour cells to stimulate lymphocyte proliferation and activation was determined. Apoptotic uveal melanoma cells expressing CD80 were efficient at inducing an immune response and served as a potent immunogen. The use of apoptotic uveal melanoma cells in combination with expression of costimulatory molecules could prove a novel adjuvant therapy for the treatment of this disease.     

Genetically modified tumour vaccines (GMTV) in melanoma clinical trials

Since melanoma is a model immunogenic malignancy incurable in the disseminated phase of its natural course different immunotherapeutic approaches are tested in clinical trials. A number of tumour vaccines genetically modified (GMTV), with various immunostimulatory factors, are tested in phase I/II clinical trials. These factors include cytokines, tumour antigens (TA), costimulatory molecules or HLA antigens. We have designed a novel, mixed auto/allogeneic cellular melanoma vaccine modified with the IL-6 and the sIL-6R genes. Preclinical studies in a mouse model demonstrated that the IL-6/sIL-6R based vaccine is able to elicit efficient anti-tumour responses, mediated by CD8+ and NK cells, which resulted in inhibition of the tumour growth, metastases formation and prolonged survival of the animals treated. Irradiation of vaccine cells does not only lead to their sterilisation but also causes increased secretion of exogenous IL-6 and sIL-6R. Since January 1996 we have vaccinated more than one hundred metastatic melanoma patients. Promising clinical results (22% CR+PR, 32% SD) and the evidence of immune responses in the vaccinated patients have prompted us to design a phase III clinical trial which is to be open in 2000.     

Serum levels of soluble CD30 are increased in ulcerative colitis (UC) but not in Crohn's disease (CD)

Imbalance in Th1 and Th2 subsets and their derived cytokines seems to be involved in the immune abnormalities underlying UC and CD. CD30 is a member of the tumour necrosis factor/nerve growth receptor superfamily expressed on T cells producing Th2 cytokines and released as a soluble form. In this study high levels of soluble CD30 were found in sera of UC patients independently of disease activity. Furthermore, increased titres of soluble CD30 molecule were shown, in the same patients, by mitogen-stimulated cultures of peripheral blood mononuclear cells. Our data seem to indicate that an activation of Th2 immune response is involved in the pathogenesis of UC, but not of CD. Furthermore, this finding indicates that serum soluble CD30 measurement may be helpful for differentiating these two forms of inflammatory bowel disease.     

The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines

Immunotherapy has become a novel therapeutic alternative for various kinds of tumours. Recently, we have finalized the first phase I clinical study in Chile for the treatment of advanced malignant melanoma, using dendritic cells (DCs) loaded with allogeneic melanoma cell lysate. This study included 20 patients and the obtained results, pioneer in Latin America, showed that DC-based immunotherapy is innocuous, even provided in combination with IL-2. In addition, immunological responses were detected in 50% of the treated patients, establishing a positive correlation between the delayed type hypersensitivity (DTH) reaction, which indicates induction of in vivo immunological memory, and patients surviving. Nevertheless, objective clinical responses in vaccinated patients are still insufficient. Only sporadic objective metastasis regressions have been registered and an important proportion of the treated patients did not respond, or their responses were weak. Several strategies have been described to be used by tumours to escape from the immune response. Actually, we have demonstrated that IL-10 inhibits antigen presentation in melanoma, reducing tumour sensitivity to melanoma-specific cytotoxic T lymphocytes (CTLs). Regulation of the immunological response by inhibitory cells could be another possible cause of clinical unresponsiveness. Lately, the existence of subpopulations of regulatory T lymphocytes (RTL) able to limit the immune response in a specific form has been established, specially inhibiting the proliferation and activity of CD4+ and CD8+ effector T lymphocytes. These cellular subpopulations, mostly CD4+/CD25+/Foxp3+ T lymphocytes (Treg) of thymic origin, or TR1 lymphocytes able to release IL-10, and tumour growth factor beta (TGF-beta) producing TH3 lymphocytes, would be accumulated in the body during tumour growth, inhibiting the immune response. In relation to RTL and cancer, evidence indicates that Treg cell numbers are increased in blood and other tissues in different types of cancer. Additionally, it has been demonstrated that in patients with refractory metastatic melanoma, the adoptive transference of anti-tumour CD8+ T lymphocytes after non-myeloablative chemotherapy was able to induce important tumour regressions that would be due to elimination of RTL populations. Additionally, chemotherapeutical drugs like decarbazine, besides their effect on tumour proliferation, also have an immunosuppressive effect on T lymphocyte populations, as well as on accumulated RTL. In this article, a novel strategy for the study of RTL is proposed, including potential therapeutic innovations, which is being pioneered in current clinical trials.     

Th17/Treg及其相关细胞因子在结肠癌中的表达并与肿瘤分期的相关性

研究结肠癌患者肿瘤组织中Th17细胞、Treg细胞及患者外周血中相关细胞因子的表达水平,探讨其表达与肿瘤分期的相关性及可能机制。运用流式细胞分析(FACS)技术检测30例结肠癌肿瘤组织及癌旁正常组织中Th17细胞及Treg细胞的比例;采用逆转录聚合酶链反应技术(RT-PCR)检测20例结肠癌患者外周血中Th17、Treg相关细胞因子IL-23和IL-10的表达水平。结果显示结肠癌肿瘤组织中Th17细胞和Treg细胞的比例明显高于癌旁正常组织(P<0.05),进展期肿瘤组织中Treg细胞的比例高于早期(P<0.05),而Th17细胞的比例较早期无明显差异(P>0.05),进展期肿瘤组织中Th17/Treg细胞的比例比早期偏低(P<0.05)。结肠癌患者外周血中IL-23、IL-10的mRNA水平升高,与健康对照组差异明显(P<0.05),且进展期与早期结肠癌IL-10mRNA的表达水平差异显著(P<0.05),而IL-23mRNA在两组间无明显差异(P>0.05)。随着结肠癌病程的进展,肿瘤组织内Th17细胞及Treg细胞的比例逐渐升高,且Treg细胞比Th17细胞升高更加明显。相关细胞因子IL-23和IL-10在患者外周血中的变化趋势和Th17、Treg细胞在肿瘤组织中的变化趋势相一致,提示Th17、Treg细胞在结肠癌的表达可能与肿瘤免疫微环境中相关的细胞因子调节有关。     

Echinococcus multilocularis metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococcosis patients

Infection with the cestode Echinococcus multilocularis causes human alveolar echinococcosis (AE), a life-threatening disease affecting primarily the liver. Despite the severity of AE, clinical symptoms often develop only many years after infection, which suggests that E. multilocularis has developed mechanisms which depress anti-parasite immune response, thus favouring immune evasion. In this study we examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable E. multilocularis vesicles and E. multilocularis vesicle fluid antigen in vitro. After 48 h of co-culture, E. multilocularis metacestode culture supernatant and E. multilocularis vesicles depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-alpha and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-alpha was reduced in AE patients, which was accompanied by an increased number of CD4+ CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to E. multilocularis metacestode in AE patients. Instead the production of interferon (IFN)-gamma and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both regulatory and inflammatory Th1 and Th2 cytokines and chemokines. Such a mixed profile might be required for limiting parasite growth but also for reducing periparasitic tissue and organ damage in the host.     

Downregulation of CXCR6 and CXCR3 in lymphocytes from birch-allergic patients

Preferential expression of chemokine receptors on Th1 or Th2 T-helper cells has mostly been studied in cell lines generated in vitro or in animal models; however, results are less well characterized in humans. We determined T-cell responses through chemokine receptor expression on lymphocytes, and cytokine secretion in plasma from birch-allergic and healthy subjects. The expression of CCR2, CCR3, CCR4, CCR5, CCR7, CXCR3, CXCR4, CXCR6, IL-12 and IL-18R receptors was studied on CD4⁺ and CD8⁺ cells from birch-allergic ( n  = 14) and healthy ( n  = 14) subjects by flow cytometry. The concentration of IL-4, IL-5, IL-10, IL-12, IFN-γ and TNF-α cytokines was measured in plasma from the same individuals using a cytometric bead array human cytokines kit. The similar expression of CCR4 in T cells from atopic and healthy individuals argues against the use of the receptor as an in vivo marker of Th2 immune responses. Reduced percentages of CD4⁺ cells expressing IL-18R, CXCR6 and CXCR3 were found in the same group of samples. TNF-α, IFN-γ, IL-10, IL-5, IL-4 and IL-12 cytokines were elevated in samples from allergic individuals. Reduced expression of Th1-associated chemokine receptors together with higher levels of Th1, Th2 and anti-inflammatory cytokines in samples from allergic patients indicate that immune responses in peripheral blood in atopic diseases are complex and cannot be simplified to the Th1/Th2 paradigm. Not only the clinical picture of atopic diseases but also the clinical state at different time points of the disease might influence the results of studies including immunological markers associated with Th1- or Th2-type immune responses.     

Pathogenic immunity in Theiler's virus-induced demyelinating disease: a viral model for multiple sclerosis

Multiple sclerosis involves inflammatory immune responses in the central nervous system (CNS) and is considered as an autoimmune disease potentially associated with viral infection. The majority of experimental models rely heavily on the autoimmune components since similar diseases can be induced following immunization with various myelin antigens. A very attractive alternative model is the Theiler's murine encephalomyelitis virus-induced demyelinating disease. This disease is primarily a CD4+ T cell-mediated, inflammatory demyelinating disease induced following viral infection. Virus-specific inflammatory Th1 cell responses, rather than cytotoxic T lymphocyte response, play a critical role in the pathogenic immune responses. The major pathogenic epitopes have been identified and these are correlated with a Th1 type response to the epitopes following viral infection. In addition, the initial virus-specific immune response is followed by the autoimmune responses to myelin antigens. Assessment of cytokines produced locally in the CNS during the course of disease suggests involvement of inflammatory cytokines in the disease. Furthermore, the manipulation of inflammatory cytokine levels by administration of either recombinant cytokines or antibodies to the cytokines strongly influences the induction and/or progression of disease, supporting the importance of these inflammatory cytokines in this virus-induced demyelinating disease.     

Interleukin 10 in the tumor microenvironment: a target for anticancer immunotherapy

IL-10 is an immunomodulatory cytokine that is frequently upregulated in various types of cancer. The biological role of IL-10 in cancer is quite complex; however, the presence of IL-10 in advanced metastases and the positive correlation between serum IL-10 levels and progression of disease indicates a critical role of IL-10 in the tumor microenvironment. IL-10 has been shown to directly affect the function of antigen-presenting cells by inhibiting the expression of MHC and costimulatory molecules, which in turn induces immune suppression or tolerance. Additionally, IL-10 downregulates the expression of Th1 cytokines and induces T-regulatory responses. Taken together, a combination of IL-10 antagonism and immunostimulatory treatments such as cancer vaccines, Toll-like receptor agonists, Th1 cytokines, and chemokines would be a logical approach to enhance an antitumor immune response.     

Association between HIV-specific T helper responses and CTL activities in pediatric AIDS

We examined the relationship between the profile of HIV-specific T helper (Th) cell responses, cytotoxic T lymphocyte (CTL) activity, HIV viral load, and CD4(+) T cell counts during longitudinal studies in children with perinatal HIV infection. Patients with AIDS demonstrated undetectable or low levels of HIV-specific Th and CTL activities, and exhibited almost exclusively Th0 type of responses with low IFN-gamma and IL-4 production. The levels of IL-2 expression in the envelope (env) peptide-stimulated peripheral blood mononuclear cells were increased in children with a slowly progressive disease, concomitant with higher numbers of CD45RO(+) memory T cells and increased proportions of Th1 clones. In these patients, high levels of env peptide-specific IL-2 expression correlated with increases in HIV-specific CTL responses, whereas a delay in the generation of HIV-specific CTL activity was associated with lower IL-2 production and elevated Th2 responses. Patients with slow disease progression produced higher levels of beta-chemokines than those detected in children with AIDS. These results suggest that an impaired development of HIV-specific cellular responses and inhibition of T cell differentiation during infancy are associated with fast disease progression. They also point to a protective role of noncytotoxic antiviral activity that might complement HIV-specific CTL responses in children with a slowly progressive disease.     

Metastatic melanoma secreted IL-10 down-regulates CD1 molecules on dendritic cells in metastatic tumor lesions

CD1 molecules are expressed by antigen-presenting cells such as dendritic cells and mediate primary immune responses to lipids and glycolipids which have been shown to be expressed by various tumors. Glycolipids are expressed by melanoma cells but, despite their immunogenicity, no efficient spontaneous immune responses are elicited. As IL-10 has previously been shown to down-regulate CD1a on dendritic cells and is known to be expressed by various melanoma cell lines, we investigated if melanoma-derived IL-10 could down-regulate CD1 molecule expression on dendritic cells as a possible way to circumvent immune recognition. We found that CD1a, CD1b, CD1c, and CD1d were significantly down-regulated on dendritic cells in metastatic (n = 10) but not in primary melanoma lesions (n = 10). We further detected significantly higher IL-10 protein levels in metastatic than in primary melanomas. Moreover, supernatants from metastatic melanomas were significantly more effective in down-regulating CD1 molecules on dendritic cells than supernatants from primary melanoma cultures. This effect was blocked using a neutralizing IL-10 antibody in a dose dependent manner. Our findings suggest that metastatic but not primary melanomas can down-regulate CD1 molecules on infiltrating dendritic cells by secreting IL-10 which may represent a novel way to escape the immune response directed against the tumor.     

Interleukin-13 induces T helper type 2 immune responses in OVA-immunized BALB/c mice bearing a T cell lymphoma

T lymphocytes play a crucial role in the regulation of immune responses against the tumour cells. Tumour progression results in dysfunction and inhibition of T cells, which ultimately leads to impairment in the antitumour immune response. The impaired antitumour immune response in the host is represented by the decreased number of T cells and their incomplete and improper function. The immunosuppressive network in tumour-bearing host mediated by tumour cells also leads to the inequities of T cell subsets and imbalance of Th1/Th2 dichotomy. Therefore, in the present study, we sought to investigate the role of tumour progression in the development of T cell phenotype and the involvement of interleukin-13 thereof selecting Dalton's lymphoma (DL) as a tumour model. It was observed that a significant increase in the number of CD4(+) T cell population, whereas a significant decline in the CD8(+) T cells among lymphoid cell population of OVA-immunized DL-bearing BALB/c mice occurs. Similar observation was found following the administration of IL-13 to the normal healthy mice. It was further confirmed that expansion in Th2 type cells among CD4(+) T cell population occurs following the progression of tumour and administration of IL-13 to normal healthy mice by an yet to define mechanism. Therefore, it can be concluded that IL-13 has immense role in polarizing the immune responses by inducing the differentiation of Th2 type of cells.     

Modulation of immune responses and suppression of experimental autoimmune myasthenia gravis by surgical denervation of the spleen

Critical interactions between the nervous system and the immune system during experimental autoimmune myasthenia gravis (EAMG) were examined in an animal model for human MG after immunization of adult female Lewis rats with Torpedo acetylcholine receptor (AChR) and complete Freund's adjuvant. Immunized rats depicted marked clinical severity of the disease. Using enzyme-linked immunospot (ELISPOT) assay and in situ hybridization techniques, immune responses in these animals were examined and showed elevated numbers of anti-AChR IgG secreting B cells and AChR reactive interferon (IFN)-gamma-secreting cells, enhanced mRNA expression of the proinflammatory cytokines IFN-gamma and tumour necrosis factor (TNF)-alpha as Th1 subset and the anti-inflammatory cytokines interleukin (IL)-4 and IL-10 as a Th2 subset, and transforming growth factor (TGF)-beta as a Th3 cytokine. Corticosterone and prostaglandin E(2) (PGE(2)) levels were measured by radioimmunoassay and illustrated increased production after immunization. Surgical denervation of the spleen reduced significantly the clinical severity of the disease, suppressed the numbers of IgG and IFN-gamma-secreting cells, down-regulated the mRNA expression for cytokines and reduced corticosterone and PGE(2) production. As controls, sham-operated rats were used and showed results as the EAMG non-denervated control rats. The data present herein, and for the first time, substantial effects of the nervous system on immune responses that may influence the outcome of EAMG. These effects were not dependent on cytokine inhibitory mediators such as prostaglandins or stress hormones. IL-10 and TGF-beta, the two potent immunosuppressive cytokines, were also suppressed, indicating a general suppression by splenic denervation. More investigations are initiated at our laboratories to understand the evident neural control over the immune system during challenges leading to the break of tolerance and development of autoimmunity, which may assist in innovative therapeutic approaches.     

Th1 cytokines are more effective than Th2 cytokines at licensing anti‐tumour functions in CD40‐activated human macrophages in vitro

CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T‐cell‐activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T‐cell‐derived cytokines license anti‐tumour functions in CD40‐activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL‐10, in a similar fashion to bacterial LPS, but failed to promote anti‐tumour functions. The Th1 cytokine IFN‐γ optimally licensed CD40L‐induced macrophage anti‐tumour functions, inducing a switch from IL‐10 to IL‐12p70 production, promoting macrophage‐mediated Th1 T‐cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL‐4 and IL‐13 promoted IL‐12p70 production (albeit without inhibiting IL‐10 production) and enhanced Th1 T‐cell skewing by CD40L‐activated macrophages. However, IL‐4 and IL‐13 did not enhance tumouricidal activity in CD40L‐activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L‐activated macrophages. The presence of tumour‐infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.     

Local expression of interleukin‐2 by B16 melanoma cells results in decreased tumour growth and long‐term tumour dormancy

The tumour microenvironment is complex containing not only neoplastic cells but also a variety of host cells. The heterogeneous infiltrating immune cells include subsets of cells with opposing functions, whose activities are mediated either directly or through the cytokines they produce. Systemic delivery of cytokines such as interleukin‐2 ( IL‐2) has been used clinically to enhance anti‐tumour responses, but these molecules are generally thought to have evolved to act locally in a paracrine fashion. In this study we examined the effect of local production of IL‐2 on the growth and the immune response to B16 melanoma cells. We found that the local production of IL‐2 enhances the number of interferon‐γ‐expressing CD8 T and natural killer cells in the tumour, as well as inducing expression of vascular cell adhesion molecule 1 on tumour vessels. These responses were largely absent in interferon‐γ knockout mice. The expression of IL‐2 in the tumour microenvironment decreases tumour growth despite also enhancing Foxp3⁺ CD4⁺ regulatory T cells and anti‐inflammatory cytokines such as IL‐10. Higher levels of IL‐2 in the tumour microenvironment eliminated the progressive growth of the B16 cells in vivo, and this inhibition was dependent on the presence of either T cells or, to a lesser extent, natural killer cells. Surprisingly however, the B16 tumours were not completely eliminated but instead were controlled for an extended period of time, suggesting that a form of tumour dormancy was established.