Murine CD27(?) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages

Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17–deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27⁽⁻⁾ subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17–secreting CD27⁽⁻⁾ Vγ6⁽⁺⁾ γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17–dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.Developing tumors are infiltrated by a variety of leukocyte subsets that can either promote or inhibit inflammation, and thus impact on cancer progression (1). Among such populations are γδ T cells, which are major players in lymphoid stress surveillance likely due to their recognition of stress-inducible molecules independently of MHC-mediated antigen presentation (2). Moreover, abundant IFN-γ secretion and cytotoxic effector functions endow γδ T cells with potent antitumor activity. This has been clearly documented in murine models of spontaneous (3), chemically induced (4), transgenic (5), and transplantable (6, 7) tumors. For example, in the widely used B16 melanoma model, γδ T cells were shown to infiltrate tumors very early and provided a critical source of IFN-γ that significantly delayed tumor growth (6, 7).Human γδ T cells also possess IFN-γ–secreting potential, which is displayed immediately at birth (8) and display cytotoxicity against tumor lines of diverse origin, including epithelial (9, 10) and hematological (11, 12) tumors. This has prompted the development of cancer clinical trials targeting γδ T cells, which have produced encouraging, albeit highly variable, degrees of therapeutic responses (13–15). There is therefore great interest in maximizing the antitumor functions of γδ T cells for cancer immunotherapy.Despite these highly promising reports, a clinical study on breast cancer tissue revealed a surprising inverse correlation between infiltrating γδ T cells and overall patient survival (16). In fact, γδ T cells represented the most significant independent prognostic factor for assessing severity of breast cancer (16). Similarly, a recent report on colorectal cancer showed a positive correlation between clinopathological parameters and the infiltration of γδ T cells specifically producing interleukin-17 (IL-17) (17). A tumor-promoting function of γδ T cells was also suggested in murine fibrosarcoma (18) and hepatocellular carcinoma (19) models, in which γδ T cells were the major cellular source of IL-17, which was required for optimal tumor growth in vivo. These data raise the interesting question as to whether distinct functional attributes of γδ T cells, for example differential cytokine production, may associate with markedly different outcomes for tumor growth.Along these lines, we have pioneered the identification of two distinct functional subsets of murine γδ T cells based on the expression levels of the CD27 coreceptor (20). We showed that robust IFN-γ production is associated with the CD27⁽⁺⁾ phenotype, whereas secretion of IL-17 is restricted to CD27⁽⁻⁾ γδ T cells. This dichotomy of hard-wired commitment to specific cytokine production is established during thymic development and maintained during the immune response to various infection agents (21, 22). Thus, the overall impact of γδ T cells in a given disease may depend on the balance between distinct proinflammatory effector cell subsets.Building on these foundations, we have here analyzed the overall and subset-specific contributions of γδ T cells to a well-established murine syngeneic model of ovarian cancer (ID8; transplantable cell line) that has a strong inflammatory component (23–25), akin to that observed in human patients with high-grade serous ovarian cancer (25, 26). In this murine model, we demonstrate that γδ T cells are major sources of IL-17, and both T cell receptor (TCR)δ-deficient and IL-17–deficient mice display reduced ID8 tumor growth. Interestingly, IL-17 production by γδ T cells in the tumor environment is essentially restricted to a CD27⁽⁻⁾ subset that does not express the commonly used Vγ1 or Vγ4 TCR chains, but rather Vγ6; these Vγ6⁽⁺⁾ cells are highly biased toward IL-17 production, in contrast to their IFN-γ–producing Vγ1⁽⁺⁾ and Vγ4⁽⁺⁾ counterparts. The ID8 tumor environment gets progressively enriched in the IL-17–promoting factor IL-7, whose receptor is highly expressed on Vγ6⁽⁺⁾ cells. This associates with preferential Vγ6⁽⁺⁾ cell proliferation and accumulation of IL-17 in the tumor bed, which in turn induces the mobilization of (recently described) small peritoneal macrophages (SPMs) that are enriched in IL-17 receptor A (IL-17RA) and in protumor and proangiogenic molecular mediators. Importantly, in comparison with large peritoneal macrophages (LPMs), SPMs can strongly and directly promote ovarian cancer cell proliferation. In summary, our work identifies an IL-17–dependent γδ T-cell/SPM axis that promotes tumor cell growth and thus opposes the widely accepted antitumor (and IFN-γ mediated) function of γδ T cells.     

The cytokine network

The availability of pure recombinant cytokines and molecular probes for their genes has generated an avalanche of scientific information. These data show that cytokines have a broad and overlapping range of cell regulatory activity both in vitro and in vivo. New factors are added to the cytokine list, and new functions reported for existing cytokines, with such frequency that it is difficult to retain an overall picture. With this problem in mind, a large wallchart was designed and was displayed at the second meeting of the British Cytokine Group* whose members pooled their collective knowledge, to list the known biological activities of these cytokines. This wallchart of cytokine activity, now referenced, is reproduced for Immunology Today. It is not a final list: new information and cytokines are continually reported and space has been left for readers to make their own additions. A neutrophil-activating peptide variously named monocyte-derived neutrophil chemotactic factor (MDNCF), neutrophil-activating factor (NAF), lymphocyte-derived neutrophil-activating peptide (LYNAP), which has been suggested as a candidate for interleukin 8 (IL-8), is included.     

Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events.     

A clinical study assessing the tolerability and biological effects of infliximab, a TNF-alpha inhibitor, in patients with advanced cancer

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective. PATIENTS AND METHODS: Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = 20) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, IL-6 and C-reactive protein (CRP). RESULTS: Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient. CONCLUSIONS: Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.     

Differential action of six human interferons against two human carcinomas growing in nude mice

Six different pure human interferons (IFNs), were tested for anti-tumour effect against two human carcinomas, breast and bowel, growing in nude mice, in a total of 36 experiments. The IFN-alpha mixture, analogue and subtypes showed the greatest activity, particularly against the breast cancer xenograft, whereas IFN-beta and IFN-gamma had little effect. However, circulating IFN could be found in the sera of mice treated with all 3 IFN types. In terms of amount of IFN protein, IFN-alpha Con1, an IFN-alpha analogue, was the most effective, a dose of 0.1 micrograms/mouse/day being sufficient to induce breast tumour regression, and IFN-gamma the least effective, a dose of 10 micrograms/mouse/day having no effect on the same tumour. A more detailed comparison of 2 IFN-alpha subtypes showed that a daily dose of 1 microgram IFN-alpha A was more effective than the same dose of IFN-alpha D, but as this IFN had approximately 30 times less antiviral activity on human cells than IFN-alpha D, these IFNs were probably at least equally effective in terms of human cell units. IFN-alpha D stimulated mouse spleen natural killer cell activity but it was not clear whether this stimulation was involved in anti-tumour activity. We conclude that this model system is useful for investigating direct anti-tumour activity of a wide range of IFN types and subtypes.     

Involvement of both intrinsic and extrinsic pathways in IFN-gamma-induced apoptosis that are enhanced with cisplatin

IFN-gamma has direct anti-proliferative effects on ovarian cancer cell lines and tumour cells isolated from ovarian cancer ascites. The aim of this study was to further elucidate the mechanisms involved. An IFN-gamma-mediated cell cycle blockade was detectable in synchronised cell populations. Apoptosis, which was caspase dependent, was also induced. When caspase activity was blocked, the anti-proliferative effect of IFN-gamma was only partially reduced indicating independent roles for both growth inhibition and apoptosis in its actions. We have demonstrated involvement of the intrinsic apoptotic pathway; IFN-gamma treatment resulted in mitochondrial membrane depolarisation, cytochrome c release into the cytosol and activation of caspase 9. Cytochrome c release was blocked by the presence of a general caspase inhibitor, suggesting a role for caspases upstream of the mitochondria. One candidate is caspase 8, which was also activated in cells treated with IFN-gamma. Levels of Bid, a pro-apoptotic molecule that can mediate mitochondrial membrane permeabilisation when cleaved by caspase 8, were also decreased and indicated a potential link between these two pathways in IFN-gamma-induced apoptosis. Furthermore, together with cisplatin, IFN-gamma exerted a more powerful anti-proliferative effect.     

Combination antiangiogenesis therapy with marimastat, captopril and fragmin in patients with advanced cancer

Marimastat, low molecular weight heparins and captopril have antiangiogenic activity in vitro and in animal models. We studied the safety and efficacy of the combination of these drugs in patients with advanced cancer. In all, 50 patients were enrolled. Captopril was given orally at a dose of 50 mg bd daily. Fragmin was administered as a daily subcutaneous injection of 200 units kg(-1) for the first 28 days and 5000 units thereafter. Marimastat was given at 10 mg bd orally. Serum, plasma and urinary angiogenic factors were measured at baseline and after 1 month of treatment. Inhibition of release of tumour necrosis factor alpha (TNF-alpha) from peripheral lymphocytes was used as a surrogate pharmacodynamic end point. There was one case of haemorrhagic stroke and one upper gastrointestinal haemorrhage. The commonest toxicity was myalgia. One of 10 patients with renal cancer had a partial response, and three patients had a prolonged period of stable disease. The treatment significantly inhibited phytohaemagglutinin (PHA)-stimulated TNF-alpha release from patient's lymphocytes. The combination of marimastat, fragmin and captopril is well tolerated and has in vivo activity. Inhibition of PHA-stimulated TNF-alpha release from lymphocytes is a surrogate pharmacodynamic marker of metalloprotease inhibition.