Thomas Tursz

Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen–specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming

The initiation of T-cell–mediated antitumor immune responses requires the uptake and processing of tumor antigens by dendritic cells and their presentation on MHC-I molecules. Here we show in a human in vitro model system that exosomes, a population of small membrane vesicles secreted by living tumor cells, contain and transfer tumor antigens to dendritic cells. After mouse tumor exosome uptake, dendritic cells induce potent CD8+ T-cell–dependent antitumor effects on syngeneic and allogeneic established mouse tumors. Therefore, exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointerventions.

Recombinant Human Interleukin-2, Recombinant Human Interferon Alfa-2a, or Both in Metastatic Renal-Cell Carcinoma

Background Recombinant human interleukin-2 (aldesleukin) and recombinant human interferon alfa can induce notable tumor regression in a limited number of patients with metastatic renal-cell carcinoma. We conducted a multicenter, randomized trial to determine the effect of each cytokine independently and in combination, and to identify patients who are best suited for this treatment. Methods Four hundred twenty-five patients with metastatic renal-cell carcinoma were randomly assigned to receive either a continuous intravenous infusion of interleukin-2, subcutaneous injections of interferon alfa-2a, or both. The main outcome measure was the response rate; secondary outcomes were the rates of event-free and overall survival. Predictive factors for response and rapid progression were identified by multivariate analysis. Results Response rates were 6.5 percent, 7.5 percent, and 18.6 percent (P<0.01) for the groups receiving interleukin-2, interferon alfa-2a, and interleukin-2 plus interferon alfa-2a, respectiv...

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell–mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)–β, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-β −/− T reg cells into nude mice suppressed NK cell–mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell–mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.

Malignant effusions and immunogenic tumour-derived exosomes.

BACKGROUND Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supernatants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells. METHODS We isolated exosomes by ultracentrifugation on sucrose and D(2)O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes. FINDINGS Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules. INTERPRETATION Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

BackgroundDC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome.Patients and methodsExosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 × 1014 molecules) or peptides (10 versus 100 μg/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression.ResultsThe GMP process allowed to harvest about 5 × 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood.ConclusionThe first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration.

Prognostic Factors for the Outcome of Chemotherapy in Advanced Soft Tissue Sarcoma: An Analysis of 2,185 Patients Treated With Anthracycline-Containing First-Line Regimens—A European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group Study

PURPOSE A total of 2,185 patients with advanced soft tissue sarcomas who had been treated in seven clinical trials investigating the use of doxorubicin- or epirubicin-containing regimens as first-line chemotherapy were studied in this prognostic-factor analysis. PATIENTS AND METHODS Overall survival time (median, 51 weeks) and response to chemotherapy (26% complete response or partial response) were the two end points. The cofactors were sex; age; performance status; prior therapies; the presence of locoregional or recurrent disease; lung, liver, and bone metastases at the time of entry onto the trial; long time period between the initial diagnosis of sarcoma and entry onto the study; and histologic type and grade. RESULTS Univariate analyses showed (a) a significant, favorable influence of good performance status, young age, and absence of liver metastases on both survival time and response rate, (b) a significant, favorable influence of low histopathologic disease grade on survival time, despite a significantly lower response rate, (c) increased survival time for patients with a long time period between the initial diagnosis of sarcoma and entry onto the study, despite equivalent response rates, and (d) increased survival time with liposarcoma or synovial sarcoma, a decreased survival time with malignant fibrous histiocytoma, a lower response rate with leiomyosarcoma, and a higher response rate with liposarcoma (P < .05 for all log-rank and chi2 tests). The Cox model selected good performance status (P < .0001), absence of liver metastases (P = .0001), low histopathologic grade (P = .0002), long time lapse since initial diagnosis (P = .0004), and young age (P = .0045) as favorable prognostic factors of survival time. The logistic model selected absence of liver metastases (P < .0001), young age (P = .0024), high histopathologic grade (P = .0051), and liposarcoma (P = .0065) as favorable prognostic factors of response rate. CONCLUSION This analysis demonstrates that for advanced soft tissue sarcoma, response to chemotherapy is not predicted by the same factors as is overall survival time. This needs to be taken into account in the interpretation of trials assessing the value of new agents for this disease on the basis of response to treatment.

Epstein—Barr Virus Gene Expression in Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV), an agent with growth transforming potential for human B cells, is associated with certain B cell lymphomas in man and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma (NPC). Since B cell growth transformation is associated with the constitutive expression of a small number of EBV-coded latent proteins, the nuclear antigens EBNA 1, EBNA 2, EBNA 3 and EBNA-LP and the latent membrane protein (LMP), the present work sought to determine whether this same pattern of virus gene expression occurred in NPC. Tumour biopsies were taken from NPC patients from three areas of differing tumour incidence (Kenya, Algeria, Britain) and immediately snap-frozen, as were biopsies of non-EBV-related carcinomas for controls. Immunoblotting of PAGE-separated proteins with selected human sera identified 24 NPC biopsies clearly expressing EBNA 1. When the analysis was extended using selected human sera with antibodies against the other EBNAs, there was no detectable expression of EBNA 2, EBNA 3 or EBNA-LP in any of these 24 biopsies; their EBNA 2-negative status was confirmed using a monoclonal antibody (MAb) PE2 which was reactive in immunoblotting and in immunoprecipitation with EBNA 2A and EBNA 2B proteins. Similar experiments with two different LMP-specific MAbs, CS1 to 4 and S12, revealed heterogeneity between NPC biopsies; 9/24 biopsies were demonstrably LMP-positive, the degree of expression varying considerably between individual tumours in a manner which was not related to the level of EBNA 1 expression. None of the 24 NPC biopsies expressed detectable amounts of EBV lytic cycle antigens. A nude mouse-passaged NPC cell line, C15, likewise expressed EBNA 1 and LMP but none of the other EBV latent proteins nor lytic cycle antigens. This work identifies a novel type of EBV-cell interaction in NPC cells which is distinct from that seen in in vitro transformed B cell lines and from that seen to date in EBV-positive B cell lymphomas.

A novel dendritic cell subset involved in tumor immunosurveillance

The interferon (IFN)-γ–induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-γ is not the conventional NK cell but a subset of B220+Ly6C− dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220+NK1.1+ dendritic cells secrete high levels of IFN-γ and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2−/−Il2rg−/− mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.

Identical reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes

NC-1 and HNK-1, two mouse monoclonal antibodies raised against quail ciliary ganglion and a human leukemic cell-line, respectively, were found to display the same pattern of reactivity. Species investigated included human, rodents, birds and amphibians. In 1- to 3-day-old avian embryos, migrating crest cells are stained, whereas in older animals neuroepithelial cells and neurons are labelled. Staining with fluorescein isothiocyanate-conjugated NC-1 is blocked by preincubation with HNK-1. In immunoblot analyses both antibodies recognize the same pattern of bands which are different in central and peripheral nervous systems and vary during development. Thus, HNK-1/NC-1 provides a useful tool for investigating the ontogeny of neural and lymphocytic cells carrying this determinant, which, in view of its high degree of conservation during vertebrate evolution, may play an important part within the haematopoietic and nervous systems.