Daniel W. Fowler

Pre-treatment with chemotherapy can enhance the antigenicity and immunogenicity of tumours by promoting adaptive immune responses

Background:Some cancer patients are immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, increasing evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour.Methods:We have assessed the expression of human leucocyte antigen class 1 (HLA1) on tumour cells before and after chemotherapy agents (cyclophosphamide, oxaliplatin or gemcitabine). In addition, we show that chemotherapy-stressed tumour cells may release cytokines that enhance the interactions between dendritic cells (DCs) and T cells into growth media.Results:Here we report that some chemotherapy agents can increase HLA1 expression in tumour cells, even when expression is low. Increases were associated with killing by cytotoxic T cells, which were negated by HLA1-blockade. Furthermore, T-cell function, as indicated by increased proliferation, was enhanced as supernatants derived from tumours treated with chemotherapy augmented DC-maturation and function.Conclusion:There is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may secrete cytokines that can mature DCs, which ultimately enhances T-cell responses.

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the TH1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer.

Cannabis-Derived Substances in Cancer Therapy – An Emerging Anti-Inflammatory Role for the Cannabinoids

Cannabinoids, the active components of the cannabis plant, have some clinical merit both as an anti-emetic and appetite stimulant in cachexic patients. Recently, interest in developing cannabinoids as therapies has increased following reports that they possess anti-tumour properties. Research into cannabinoids as anti-cancer agents is in its infancy, and has mainly focussed on the pro-apoptotic effects of this class of agent. Impressive anti-cancer activities have been reported; actions that are mediated in large part by disruptions to ubiquitous signalling pathways such as ERK and PI3-K. However, recent developments have highlighted a putative role for cannabinoids as anti-inflammatory agents. Chronic inflammation has been associated with neoplasia for sometime, and as a consequence, reducing inflammation as a way of impacting cancer presents a new role for these compounds. This article reviews the ever-changing relationship between cannabinoids and cancer, and updates our understanding of this class of agent. Furthermore, the relationship between chronic inflammation and cancer, and how cannabinoids can impact this relationship will be described.

The gene expression profile of unstimulated dendritic cells can be used as a predictor of function.

Dendritic cells (DCs) represent a subset of professional antigen presenting cell (APC) whose role is to elicit immune responses against harmful antigens. They have been used in DC vaccines to stimulate the immune system to kill cancer cells. However, successes in clinical trials have been limited, which may be attributed to a lack of appreciation of the quality of DCs used. In the present study, whole human genome microarrays were used to examine alterations in gene expression of monocyte‐derived DCs after stimulation with supernatants derived from tumours. Our primary aim was to investigate the possibility of a gene signature for DCs that could be used to forecast responsiveness to tumour stimuli. Results showed that DCs are divided into two groups based on their ability to increase costimulatory markers and to trigger T‐cell responses. The gene profiles of the immature DCs from these two groups were distinct, with particular divergence in genes from the interleukin (IL) 8 and thrombospondin‐1 hubs. A subpanel of genes was identified, whose signature of expression was capable of predicting DC‐stimulatory capacity. Overall, these studies have highlighted a gene‐based screen that predicts DC function, which could be used to guide DC‐vaccine trials.

Supernatants derived from chemotherapy-treated cancer cell lines can modify angiogenesis

Background: There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence. Methods: In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic. Results: Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead. Conclusion: This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.

Zoledronic acid causes γδ T cells to target monocytes and down-modulate inflammatory homing.

Zoledronic acid (ZA) is a potential immunotherapy for cancer because it can induce potent γδ T‐cell‐mediated anti‐tumour responses. Clinical trials are testing the efficacy of intravenous ZA in cancer patients; however, the effects of systemic ZA on the activation and migration of peripheral γδ T cells remain poorly understood. We found that γδ T cells within ZA‐treated peripheral blood mononuclear cells were degranulating, as shown by up‐regulated expression of CD107a/b. Degranulation was monocyte dependent because CD107a/b expression was markedly reduced in the absence of CD14+ cells. Consistent with monocyte‐induced degranulation, we observed γδ T‐cell‐dependent induction of monocyte apoptosis, as shown by phosphatidylserine expression on monocytes and decreased percentages of monocytes in culture. Despite the prevailing paradigm that ZA promotes tumour homing in γδ T cells, we observed down‐modulation of their tumour homing capacity, as shown by decreased expression of the inflammatory chemokine receptors CCR5 and CXCR3, and reduced migration towards the inflammatory chemokine CCL5. Taken together our data suggest that ZA causes γδ T cells to target monocytes and down‐modulate the migratory programme required for inflammatory homing. This study provides novel insight into how γδ T cells interact with monocytes and the possible implications of systemic use of ZA in cancer.

Supernatants from lymphocytes stimulated with Bacillus Calmette-Guerin can modify the antigenicity of tumours and stimulate allogeneic T-cell responses

Background:Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis.Methods:In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines.Results:BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th1-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th1-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement.Conclusion:These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.

Tripartite immune cell co-operation in the Bacillus Calmette Guérin-induced activation of γδ T cells

γδ T cells contribute to immunosurveillance of pathogenic infections and malignant transformations; however, mechanisms of activation have yet to be fully defined. In this study we demonstrate a novel mechanism by which human Vδ2+ γδ T cells are activated by the model pathogen Bacillus Calmette Guérin (BCG). We show in vitro that Vδ2 cell cytokine production and cytotoxic activity in response to BCG are dependent on both dendritic cells (DCs) and memory CD4+ αβ T cells (CD4 T cells). We found that Vδ2 cells are indirectly activated by BCG in an interleukin (IL)‐12p70‐dependent manner, and that DC production of the IL‐12p70 responsible for Vδ2 cell activation requires Toll‐like receptor 2/4 ligands from BCG and interferon (IFN)‐γ from memory CD4 T cells. Our data suggest that Vδ2 cell responses to BCG are dependent on the activation of IFN‐γ‐producing memory CD4 T cells, and provide novel insight into the complex interplay between cells of the innate and adaptive immune response.

Abstract 5612: Pretreatment with chemotherapy can enhance the immunogenicity of tumors by promoting adaptive immune responses

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cancer patients are typically immuno-compromised, and it has been long felt that immune-intervention is not compatible with standard chemotherapies. However, evidence suggests that standard chemotherapy drugs may stimulate beneficial changes in both the immune system and tumour. We have assessed the impact of chemotherapy agents (cyclophosphamide, oxaliplatin and gemcitabine (GEM)) on the potential immunogenicity of tumour cells (A549, Caki2, HCT116, MCF7 and PC3), and investigated the interactions between supernatants derived from these stressed cells and cells of the immune system, by studying the activities between dendritic cells and T-cells. Results showed that some chemotherapy agents can increase total MHC class I expression on tumour cells, even when expression is low. For example, expression on MCF7 cells was increased by ∼4-fold upon treatment with GEM (133 ± 31 vs. 33 ± 7.8 in untreated cells; p<0.001). The increases in expression were associated with enhanced killing by cytotoxic T-cells (e.g. in HCT116, the MTT reading for GEM pre-treated tumour cells cultured with T-cells was 0.17 ± 0.011 vs. 0.12 ± 0.0096 in untreated tumour cells with T-cells added; p=0.007), which were negated by HLA1-blockade. Results also showed that the supernatants derived from tumours treated with chemotherapy augmented DC-maturation as indicated by increases in the maturation markers CD80/CD83/CD86. Furthermore, DC function was enhanced as confirmed by an increase in DC-mediated proliferation of CFSE-loaded T-cells. For example, the percentage of CD3+/CD4+ cells with a reduced CFSE content (indicating proliferation) was higher in cells exposed to supernatants derived from GEM treated A549 cells (12 ± 2.1% vs. 5.2 ± 1.4% in controls where the supernatant was from untreated tumours; p=0.028). In summary, there is evidence that a facet of immune surveillance can be restored by appropriate chemotherapy agents. Also, tumours exposed to some chemotherapy may deliver signals that can mature DCs, which ultimately enhances T-cell responses. This work was supported by the Cancer Vaccine Institute. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5612.